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JOURNAL OF SHELLFISH RESEARc

VOLUME 19, NUMBER 1

JUNE ^000

The Journal of Shellfish Research (formerly Proceedings of the

National Shellfisheries Association) is the official publication

of the National Shellfisheries Association

Editor

Dr. Sandra E. Shumway

Natural Science Division

Southampton College, Long Island University

Southampton, NY 11968

Dr. Standish K. Allen, Jr. (2000)
School of Marine Science
Virginia Institute of Marine Science
Gloucester Point, VA 23062-1 1346

Dr. Peter Beninger (2001)

Laboratoire de Biologic Marine

Faculte des Sciences

Universite de Nantes

BP 92208

44322 Nantes Cedex 3

France

Dr. Andrew Boghen (2001)
Department of Biology
University of Moncton
Moncton, New Brunswick
Canada El A 3E9

Dr. Neil Bourne (2001)
Fisheries and Oceans
Pacific Biological Station
Nanaimo, British Columbia
Canada V9R 5K6

Dr. Andrew Brand (2001)
University of Liverpool
Marine Biological Station
Port Erin, Isle of Man

Dr. Eugene Burreson (2001)
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062

Dr. Peter Cook (2000)
Department of Zoology
University of Cape Town
Rondebosch 7700
Cape Town, South Africa

EDITORIAL BOARD

Dr. Simon Cragg (2000)
Institute of Marine Sciences
University of Portsmouth
Ferry Road
Portsmouth P04 9LY
United Kingdom

Dr. Leroy Crcswell (2001)
Harbor Branch Oceanographic

Institute
US Highway 1 North
Fort Pierce, Florida 34946

Dr. Lou D'Abramo (2000)
Mississippi State University
Dept of Wildlife and Fisheries
Box 9690
Mississippi State, Mississippi 39762

Dr. Ralph Elston (2001)
Battelle Northwest
Marine Sciences Laboratory
439 West Sequim Bay Road
Sequim, Washington 98382

Dr. Susan Ford (2000)

Rutgers University

Haskin Laboratory for Shellfish

Research
P.O. Box 687
Port Norris, New Jersey 08349

Dr. Raymond Grizzle (2001)
Randall Environmental Studies Center
Taylor University
Upland, Indiana 46989

Dr. Mark Luckenbach (2001)
Virginia Institute of Marine Science
Wachapreague, Virginia 23480

Dr. Bruce MacDonald (2000)
Department of Biology
University of New Brunswick
P.O. Box 5050
Saint John, New Brunswick
Canada E2L 4L5

Dr. Roger Mann (2000)

Virginia Institute of Marine Science

Gloucester Point, Virginia 23062

Dr. Islay D. Marsden (2000)
Department of Zoology
Canterbury University
Christchurch, New Zealand

Dr. Tom Soniat (2000)
Biology Department
Nicholls Slate Uni\ersity
Thibodaux, Louisiana 70310

Dr. J. Evan Ward (2001)
Dcpt. of Marine Sciences
University of Connecticut
Grolon, CT 06340-6097

Dr. Gary Wikfors (2()()())

NOAA/NMFS

Rogers Avenue

Milford, Connecticut 06460

Journal of Shellfish Research

Volume 19, Number 1

ISSN: 00775711

June 2000

Journal of ShcUfish Research. Vol. 19. No. 1. 1-?. 2000.

^O'L 1 (j

^mQ

IN MEMORIUM

JOHN CARL MEDCOF

1911-1997

J. Carl Medcof, a well-known and highly respected authority in the field of molluscan biology, oyster biology, and shellfish
management in Atlantic Canada, died on 28 February 1997, in St. Andrews. New Brunswick. He was 86 years old.

Carl was bom into a family with a strong academic background in Ruthven. Ontario in 1911. and the family moved soon after his
birth to Toronto. His father, stepmother (his mother died when he was quite young), and uncles were all teachers. He received his
elementary and secondary education mostly in Toronto and enrolled in the University of Toronto, where he received his B.A. in biology
in 1932. He received an M.A. degree from the University of Western Ontario in 1934, undertaking a study of a snail, Campeloma, in
a small river in southern Ontario as his thesis topic. Thus, began his lifelong interest in the field of molluscan biology. He received his
Ph.D. degree from the University of Illinois in 1938. studying under Dr. H. J. Van Cleave, who was very influential in his early career.
While at the University of Illinois, he was elected to the Phi Beta Kappa Society.

During his senior year at the University of Toronto and throughout his graduate years, Carl served as a teaching assistant. He
maintained a keen interest in young people and always welcomed opportunities to talk with students and young scientists and encourage
them in their work. He was a lifelong teacher, and many summer students benefited from his store of knowledge.

While an undergraduate, Carl began work as a summer student with the Biological Board of Canada, later the Fisheries Research
Board of Canada. His first job was with investigations of Atlantic salmon. Subsequently, he worked as a summer student at Ellerslie,
Prince Edward Island, where he came under the influence of Drs. Alfreda B. Needier for "red tides" and Alfred W. H. Needier, the
director of the Station. Ellerslie was established to study oyster culture and foster development of the industry in the Canadian Maritimes.
Thus, began Carl's enduring association with oyster research and the oyster industry of the Canadian East Coast. The title of his Ph.D.
dissertation was "Studies on the larva of the Canadian oyster" and was focused on experiments with Ostrea (Cnis.wslreal virginica in
the Bideford River. Prince Edward Island. Canada.

On completion of his Ph.D.. Carl joined the staff of the Fisheries Research Board of Canada and was employed first at the Ellerslie
Station, where he assumed responsibility for oyster research. He moved to the Biological Station in St. Andrews. New Brunswick in
1940, where he continued his work with oysters and also assumed responsibility for research work on all molluscan species including
soft-shell clams, Mya arenaria, quahaugs, Mercenaria mercenaria, and sea scallops Placopecten magellaniciis with his technician, Mrs.
Esther Lord. Up until 1944. he moved with his family in the summers to Ellerslie and to St. Andrews in the winters.

Carl undertook a wide range of studies on oysters, including investigations on breeding to ensure a supply of juveniles for the
industry, work to improve culture methods, and studies to improve harvesting and marketing. He maintained a close working relationship
with the industry during his career, and much of his work focused on developing methods to improve it. He enjoyed working with people
in industry and had their complete trust. Much of his work with oysters culminated with the publication. Oyster Farming in The
Maritimes. which became a standard text for oyster culture in eastern Canada. This expertise took him to Cape Breton. Nova Scotia,
where he worked with natives from the Eskasone Indian Band to grow oysters. He was also involved in experiments in the mid-1950s
to transplant European oysters {Ostrea edulis) from France to the Bay of Fundy.

Carl worked extensively on the soft-shell clam with Mr. Stuart MacPhail from the Fisheries Research Board as well as with colleagues

Bourne and Robinson

from New England such as Mr. Dana Wallace. His clam work took him to eastern Nova Scotia, the Bay of Fundy. and the Gulf of St.
Lawrence, where he worked with local harvesters to survey their areas and methods to increase production. He and Stuart MacPhail
introduced the first water jet harvesters for soft-shell clams to the local industry and built prototypes for hand-held models as well as
a vessel-equipped escalator harvester. Other clam species were studied as well. He and Ross Chandler from the St. Andrews Biological
Station did the early biological work for the ocean quahaug clam fishery in southeast Nova Scotia.

Another major scientific contribution Carl made was in the field of shellfish toxins, particularly paralytic shellfish poisoning (PSP).
As a result of his work and leadership, much of the history of PSP in the Canadian Maritimes was recorded and the etiology of outbreaks
established. Results of this work led to establishment of a monitoring system for PSP to ensure that safe shellfish were marketed. Carl
was the guest of honor at the Third International Conference on Toxic Dinoflagellates in 1985 and was awarded with a plaque in
recognition of his contribution in this field. Later in his life, Carl said, "The most important work I did as a scientist was on resolving
many paralytic shellfish problems. In addition I was able to work on methods of producing reliable forecasts about the expectancy of
one of the major poisonous agents."

Carl also had a vision for the future. As early as the 1950s he was promoting the concept of aquaculture in marine production and
was actively working on developing the protocols for producing soft-shell clams. He gave several media (including television) interviews
and lectures on this topic. In addition to clams, he also predicted the development of the mussel culture industry in the Maritimes and
an industry for sea urchin roe; something that is only just developing 25 years later.

Because of his expertise in shellfish, Carl was seconded to the Colombo Plan for 2 years in 1953 and 1955 and worked in Sri Lanka,
assisting with development of invertebrate and other fisheries there. In 1955. he went to Europe to tour the various shellfish industries
for information exchange with colleagues and for technology transfer. In the late 1960s, he undertook a similar trip to Japan with a group
of Canadian scientists.

Carl retired from the Fisheries Research Board of Canada in 1973 and then spent a year in Australia, where he worked as a consultant
to the shellfish industry through the University of Southern Australia in New South Wales. One project he undertook there was an
investigation of the introduction of organisms via ballast water in ships, a subject that has become of great interest recently interna-
tionally.

Carl received numerous awards, both scientific and nonscientific, for the contributions he made during his lifetime. He was a
long-standing member of the National Shellfisheries Association and served on the Editorial Board for the Proceedings and the Journal
for many years. He was made an Honorary Member of the Association in 1973. He received a Centennial Medal from the Canadian
Federal government in 1967 for his contributions to the Fisheries Research Board of Canada.

During his retirement, he taught a course in Marine Ecology at the Huntsman Marine Science Centre in St. Andrews. The course
involved a rich mixture of basic biology, natural ecology, and the geological and paleological history of the Charlotte County area of
New Brunswick.

Cari had a multitude of other interests and on his retirement began another career, recording the history of Charlotte County, New
Brunswick, an offshoot of his previous hobby. He had long had an interest in the history of the area and in 1961 was a co-founder of
the Charlotte County Historical Society, serving as its first president. He encouraged people to record what information they possessed
of the area, and it was through his efforts as editor over a period of 21 years that an 12-volume collection of papers was published as
Contributions From Tlic Charlotte County Historical Society. As a result of his work with the Historical Society. Carl was presented with
an Award of Merit from the Canadian Museums Association in 1981.

He was a devoted citizen of St. Andrews and took an active part in the affairs of the town, contributing to it in many ways over the
years. During his lifetime, he was a Scoutmaster, Chairman of the Board of School of Trustees, a member of the local Kiwanis Club,
and served as president. He was a member of the St. Andrews Anglican Church and published a history of that church. He served as
vestryman, was an honorary church warden, and with his joy of singing, was a member of the church choir for many years. In 1987,
the local Kiwanis Club selected him as their Man of the Year in recognition of his numerous contributions to the town.

Carl was a kind, gentle, humanitarian who brought out the best in people. He enjoyed working quietly, smoking his pipe (although
more matches were burned than tobacco!), and he always had a package of dulse in his pocket, which he chewed and offered to anyone
he met; he was one of the few people in the world who actually enjoyed chewing dulse! Those of us who were privileged to work under
his direction will always remember the kind, thoughtful guidance and encouragement he gave us in our careers and his kind advice not
only to be good scientists but to be good citizens. It was a joy to work and go on field trips with him, where we could enjoy his
companionship and take part in long philosophical discussions on a wide range of biological and other topics.

Cari is survived by his wife of 55 years, Bessie, their three children. Susan, John, and Ranby. by three grandchildren, and by a great
many people whose lives have been made more meaningful through their association with him.

ACKNOWLEDGMENTS

NEIL F. BOURNE' AND SHAWN M.C. ROBINSON'

We thank Mrs. Lsiher Lord and Mr. Ross Chandler lor reading
an eariier draft of the manuscript and sharing their insights on
Cari's life. We also thank Mrs. Bessie Medcof for helping us with
the chronology of the events and her perspective on her husband.
Marilynn Rudi of the St. Andrews Biological Station librarv kindly
provided some of the historical information.

'Pacific Hiolofiical Station

Nanaimo, British Columbia

V9R 5K6. Canada

'Biological Station

St. Andrews. New Brunswick

E5B 2L9, Canada

John Carl Medcof 3

publications

Medcof. J. C. 1935. Margaree salmon investigations 1935. Scotsville: notes on the various marine species of animals. Manuscript Reports of the

Biological Stations 1 17:89 pp.
Smallman. B. N.. J. C. Medcof. 1935. Margaree salmon investigation. 1935. Manuscript Reports of the Biological Stations No. 116.
Medcof J C. 1936. Report of oyster studies at the P.E.I. Biological Station, June to August. Manuscript Reports of the Biological Stations 143:23 pp.
Medcof J. C. 1937. Report of oyster studies at the P.E.I. Biological Station June to September. 1937. Manuscript Reports of the Biological Stations 160:63 pp.
Medcof. J. C. 1938. Excerpts from "Studies on the larvae of the Canadian oyster." Manuscript Reports of the Biological Stations 292:27 pp.
Medcof, J. C. 1938. Oyster investigations in Bras d'Or Lakes. 1938. Manuscript Reports of the Biological Stations 159:64 pp.
Medcof. J. C. 1938. Studies on the Larva of the Canadian oyster. Ph.D. Dissertation, University of Illinois. Champaign-Urbana. IL. 74 pp.
Medcof. J. C. 1939. Additional records of the terrestrial amphipod, Talirrus allaudi Chevreux, in North America. Am. Midland Naturalist 22:216-217.
Medcof J. C. 1939. Larval life of the oyster iOstrea virginica) in Bideford River. / Fisheries Res. Board Can. 4:287-301.
Medcof J. C. 1939. Oyster investigations in the Bras d'Or lake and studies on the condition factor of oy.sters. Manuscript Reports of the Biological

Stations 162:92 pp.
Medcof. J. C. 1940. On the life cycle and other aspects of the snail, Campeloma. in the Speed River. Can. J. Res. 18:165-172.
Medcof. J. C. 1940. Oyster investigations in 1940. Fisheries Research Board of Canada. Manuscript Report 184. Biological Station. St. Andrews. NB, 1940.
Medcof. J. C. 1940. Variations in the pleopod structure of the tertestrial amphipod Talitnis allaudi Chevreux. Lloydia 3:79-80.
Medcof J. C. 1 94 1 . Examination of oyster areas in Richibucto. Little Shemogue, and Big Shemogue Rivers, 1 95 1 . Fisheries Research Board of Canada,

Original manuscript of the Biological Station. St. Andrews. No.686.
Medcof J. C. 1941. Oyster investigations in 1941. Manuscript Reports of the Biological Stations 239:50 pp.
Medcof J. C. & A. W. H. Needier. 1941. The influence of temperature and salinity on the condition of oyster (Oilrea virginica). J. Fisheries Res. Board

Can. 5:253-257.
Gibbard. J. A. G. Campbell, A. W. H. Needier & J. C. Medcof 1942. Effect of hibernation on content of coliform bacteria in oysters. Am. J. Puhl. Health

32:979-986.
Medcof, J, C. 1942. Report on 1942 investigations: principally oysters, with small note on Irish moss. Manuscript Reports of the Biological Stations

331:102 pp.
Medcof. J. C. 1943. La besoin d'une production d'huitres d'ensemencement("seed oysters") a Shippegan. Fisheries Research Board of Canada. Oyster

Farming Circular 17:1 p.
Medcof. J. C. 1943. Memorandum of oyster problems deserving biological investigations. Fisheries Research Board of Canada, original manuscript 749,

Atlantic Biological Station, December 1943.
Medcof J. C. 1943. Need for production of seed oyster at Shippegan. Fisheries Research Board of Canada, Oyster Farming Circular No. 17, May 1943.
Medcof J. C. 1944. Structure, deposition, and quality of oyster shell iO.strea virginica Gmelin). / Fisheries Res. Board Can. 6:209-216.
Medcof J. C. & E. I. Morrison. 1943. Report on 1943 shellfish investigations. Manuscript Reports of the Biological Stations 370:65 pp.
Medcof J. C. 1944. 1944 investigations: oysters and clams. Manuscript Reports of the Biological Stations 378:90 pp.

Medcof J. C. 1944. How relaying and transferring at different seasons affects the famess of oysters. Fisheries Res. Board Prog. Repts. 35:1 1-14.
Medcof J. C. 1944. Report of information on shellfish gathered during the Massachusetts trip April 10-17, 1944. Original manuscript 780. St. Andrews

Biological Station. New Brunswick. Canada.
Medcof. J. C. 1945. Green oysters from New Brunswick. Acadian Naturalist 2:40^3.

Medcof J. C. 1945. The mud-blister worm. Polydora. in Canadian oysters. J. Fisheries Res. Board Can. 7:498-505.
Medcof J. C. & R. J. Gibbons. 1945. Paralytic shellfish poisoning in Nova Scotia and New Brunswick. Manuscript Reports of the Biological Stations

376:39 pp.
Medcof J. C. 1946. Effect of relaying and transferring on fatness of oysters. / Fisheries Res. Board Can. 6:449—155.
Medcof J. C. 1946. More reversed winter flounder. Science 103:488.

Stinson, R. H. & J. C. Medcof 1946. Observations on the natural history of clam drills {Polinices). Manuscript Reports of the Biological Stations 383:63 pp
MacPhail. J. S. & J. C. Medcof 1947. 1947 Clam investigations. Fisheries Research Board of Canada, Atlantic Biological Station, original manu-
script 855.
MacPhail. J. S. & J. C. Medcof 1947. Report on information of the bar-clam {Mactra) gathered during a trip to New York state in August. 1946. Fisheries

Research Board of Canada, original manuscript 698. December. 1947.
Medcof. J. C. 1947. Clam farming in the Maritimes — preliminary information. Fisheries Research Board of Canada. General Series,

No.9. Circular. Atlantic Biological Station. St. Andrews. N.B.. May. 1947.
Medcof, J. C. A. H. Leim. A. B. Needier. A. W. H. Needier. J. Gibbard & J. Naubert. 1947. Paralytic shellfish poisoning on the Canadian Atlantic Coast.

Fisheries Research Board of Canada. Bulletin No.75. Ottawa, 1947.
Medcof J. C. & F. S. Schiffman. 1947. Recent records of the sea sunfish {Mola mola L.) in the Gulf of St. Lawrence. Acadian Naturalist 2:63-66.
Medcof J. C. 1948. A snail commensal with the soft-shell clam. J. Fisheries Res. Board Can. 7:219-220.

Medcof J. C. 1949. Dark-meat and the shell disease of scallops. Fisheries Research Board of Canada, Atlantic Biological Station Progress Report 45.
Medcof. J. C. 1949. Meat yield from Digby scallops of different sizes. Fisheries Res. Board Prog. Repts. 44:6-9.
Medcof, J, C, 1949. "Puddling" — a method of feeding by herring gulls. The Auk 66:204-205.
Thurber. L. W. & J. C. Medcof 1949. Meat yield of clams (-) and percentage total dry solids of clam meats. Manuscript Reports of the Biological Stations

399:.30 pp.
Medcof J. C. 1950. Burtowing habits and movements of soft-shelled clams. Fisheries Res. Board Prog. Repts. 50:17-22.
Medcof, J. C. & J. S. MacPhail. 1951. 1945 Investigations — clams and oysters. Manuscript Reports of the Biological Stations 414:92 pp.
Medcof. J. C. & J. S. MacPhail. 1952. The winter flounder — a clam enemy. Fisheries Res. Board Prog. Repts. 52:3-8.
Mullan, M. W., A. B. Williams, A, D, Tennant, 1. E. Erdman. V. C. Dohaney & J. C. Medcof 1953. 1952 clam cleansing studies (Wvo) — combined

reports. Manuscript Reports of the Biological Stations 503:65 pp.
Durairatnam. M. & J. C. Medcof 1954. Ceylon's red seaweed resources. Ceylon Trade J. 19:1-6.
Medcof. J. C. 19.54. How to improve Ceylon's small-boat fisheries. Paper delivered at 1954 meeting of Ceylon Association for Advancement of Science.

4 Bourne and Robinson

Medcof, J. C. 1955. Day and night characteristics of spatfall and of behaviour of oyster larvae. J. Fisheries Res. Board Can. 12:270-286.

Medcof. J. C. & L. M. Dickie. 1955. Watch for the green crab — new clam enemy. Fisheries Research Board of Canada. General Series Circular 26.

Atlantic Biological Station. St. Andrews, N.B., July. 1955.
Medcof. J. C. & J. S. MacPhail. 1955. Survey of bar clam resources of the Maritime Provinces. Fisheries Research Board of Canada, Bulletin 102, Atlantic

Biological Station, St. Andrews, N.B.
Canagaratnam, P. & J. C. Medcof. 1956. Ceylon's beach seine fishery. Fisheries Research Station. Dept. of Fisheries, Ceylon. Bulletin 4.
Dickie, L. M. & J. C. Medcof. 1956. Environment and the scallop fishery. Can. Fisherm. 7:9,

Bond. R. M. & J. C. Medcof. 1957. Epidemic shellfish poisoning in New Brunswick, 1957. Dept. of Fisheries, Fish Inspection Laboratory, St. Andrews, N,B.
Medcof, J, C. 1957. Nuptial or prenuptial behaviour of the shad. Alusa sapidissinui (Wilson). Copeia 1957:252-253.
Bond, R. M. & J. C. Medcof. 1958. Epidemic shellfish poisoning in New Brunswick, 1957. Can. Med. Assoc. J. 79:19-24.
Medcof, J. C. 1958. Mechanized gear for shellfish harvesting and shellfish culture. Manuscript Reports of the Biological Stations 644:16 pp.
Medcof, J. C. 1958. Stock-taking of molluscan shellfish resources and prospects for improvement. Reprinted from Progress Reports of the Atlantic Coast

Stations of the Fisheries Research Board of Canada. Issue 71, pp. 21-26, December, 1958.
Medcof, J. C. 1958. Studies on stored oysters {Crassosuea virginica). Proc. Nail. Shellfisli. Assoc. 47:13-28.
Medcof, J. C. 1958. Useful publications for oyster farmers of the Maritimes. Fisheries Research Board of Canada. General Series Circular 32. Biological

Station. St. Andrews. N.B.. October.l95S.
Medcof. J. C. & J. E. Mortimer. 1958. Introducing European oysters to the Maritimes. Reprinted from Progress Reports of the Atlantic Coast Stations

of the Fisheries Research Board of Canada. Issue 71. pp. 27-29. December. 1958.
Medcof. J. C. & L. W. Thurber. 1958. Trial control of the greater clam drill (Lunatia heros) by manual collection. J. Fisheries Res. Board Can.

15:1355-1369.
Bell, M. C. V. M. Brawn. C. H. Clay. C. J. Kerswell. J. E. H. Legare.D. C. Maclellan. W. C. Martin, F. D. McCracken, R. A. McKenzie,

J. C. Medcof, L. W. Scattergood, R. F. Temple, S. N. Tibbo & D. G. Wilder. 1959. Report to International Joint Comission, Ottawa, Ontario.

Washington DC. Appendi.x II. Biology — Canada: studies in fisheries biology for the Passamaquoddy Power Project.
MacPhail. J. S. & J. C. Medcof. 1959. Ocean quahog explorations. Trade News -6.
Medcof, J. C. 1959. Report on visits to British Columbia and Washington state shellfish industrial and research centres. Available from: Library,

Biological Station, St. Andrews, N.B., E5B-2L9.
Medcof, J. C. I960. Shellfish poisoning — another North American ghost. Can. Med. Assoc. J. 82:87-90.
Drinnan, R. E. & J. R. Medcof. 1961, Progres du Reetablissement des Stocks d'Huitres Decimes par la Maladie. Office des Recherches sur les Pecheries

du Canada, Circulaire No 34 de la Serie Generale, Octobre 1961 (version Fran^aise), Station Biologique. St. Andrews.N.B.
Drinnan. R. E. & J. C. Medcof. 1961. Progress in rehabilitating di.sease-affected oyster stocks. Fisheries Research Board of Canada. General Series

Circular 34. Biological Station, St. Andrews, N.B., October, 1961.
Medcof, J. C. 1961. Effect of hydraulic escalator harvester on undersize soft-shell clams. Proc. Natl. Sliellfish. Assoc. 50:151-161.
Medcof, J. C. 1961. Fuller exploitation of natural spatfall. Fisheries Research Board of Canada, original manuscript of the Biological Station. St. Andrews.

No.932. April 10. 1961.
Medcof. J. C. 1961. Oyster farming in the Maritimes. Fisheries Research Board of Canada. Bulletin 131. Biological Station. St. Andrews, N.B., 1961.
Medcof, J. C. 1961. Present shellfish fishery needs for oceanographic and biological research on Canadian Atlantic. Conference of A.O.G. and St.

Andrews Staff at St. Andrews, October 19, 1961.
Medcof, J. C. 1961. Trial introduction of European oysters (Ostrea ediilis) to Canadian East Coast. Proc. Natl. Shellfish. As.soc. 50:1 13-124.
Medcof, J. C. 1961. Loss of the barque James W. Elwell and tragic experiences of a St. Andrews sailing captain 1872. Charlotte County Archives. St.

Andrews. New Brunswick. BOG 2X0. 18 pp.
MacPhail. J. S. & J. C. Medcof. 1962. Fishing Efficiency Trials with a hydraulic clam [Mya) Rake — 1961. Fisheries Research Board of Canada.

Manuscript Report Series 724. Biological Station. St. Andrews. N.B.. July. 1962.
Medcof. J. C. 1962. 1961 Tests of spat collection in the Intertidal /one. Fisheries Research Board of Canada, original manuscript of the Biological Station.

St. Andrews. No.943. June, 1962.
Medcof, J. C. 1962. Collecting spat and producing bedding oy.sters on shell strings. Fisheries Research Board of Canada. General Series Circular 36.

Biological Station, St. Andrews, N.B., July, 1962.
Medcof, J. C. 1962. Hydraulic escalator oyster harvesters. Address by J.C. Medcof at the 1962 July-August Convention of the Oyster Institute and the

National Shellfisheries Association at Baltimore, Maryland.
Medcof, J. C. & J. S. MacPhail. 1962. Fishing efficiency of clam hacks and morlalities incidental to fishing. Fisheries Research Board of Canada.

Manuscript Report Series 784.
Medcof. J. C. 1962. Land reconnaissance of Darnley-New London. P.E.I.. region to judge its oyster spat rearing potenlial. Fisheries Research Board of

Canada, original manuscript of (he Biological Station. St, Andrews. N.B.. No.944. June 1962,
Medcof. J. C. 1962. Possible elfecls of Passamaquoddy power project on clams, scallops, and shipvvorms in Canadian w;ilers. J. Fisheries Res. Board

Can. 19:877-889.
Prakash. A. & J. C. Medcof. 1962. Hydrographic and meteorological factors affecting shellfish toxicity at Head Harbour. New Brunswick. J. Fisheries

Res. Board Can. 1 9: 1 0 1 - 1 1 2.
Dickie, L. M, & J. C. Medcof. 1963. Causes of mass mortalities of scallops [Placopeclcn ma.t;ellaniciis) in the Southwestern Gulf of St. Lawrence. J.

Fisheries Res. Board Can. 20:451-^82.
MacPhail, J. S, & J. C. Medcof. 1963. A new digger for sofl-shcll clams, Reprlmcd liom March. I9(i3, issue of "Trade News" of the Dcpl. of Fisheries

of Canada.
Medcof, J C. 1963. Molluscan shellfish. Shellfish course, given by Dr. J. C. Medcof. 1-ebruary 12. 1963, Si. .Andrews, N.B. .'Available from: Library,

Biological Station. St. Andrews. N.B., H5B-2L9.
Medcof, J, C. 1963. Partial survey and critique of Ceylon's marine fisheries, 195.3-55. Bulletin of the Fisheries Research Station. Ceylon 16:29-1 18.
Medcof, J. C. 1963. Puzzling clay tubes from the sea bottom. Can. Field-Namrulist 77:179-242.
Medcof, J. C. 1963. Shell strings for collecting spat and rearing bedding oysters — 1962 tests. Fisheries Research Hoard of Canada, original manuscripi

of the Biological Station, St. Andrews, No.962, April, 1963.
Medcof, J. C. 1964. Subareas 4 and 5. Reproduced by permission Ironi ICNAI Kcdhook. I4h4. Pan II. pp. 21-34.

John Carl Medcof 5

Medcof. J. C. & N. Bourne. 1964. Causes of mortality of the sea scallop, Placopecten magellanicus. Proc. Natl. Shellfish. Assoc. 53:33-50.
Bearisto. F. 1.. J. C. Medcof & E. I. Lord. 1965. Clam drill (Polinices) investigations at St. Andrews. 1948. Manuscript Reports of the Biological Stations

845:11 pp.
Medcof. J. C. 1965. .\ recording caliper for measuring oysters and clams. Fisheries Research Board of Canada, original manuscript of the Biological

Station. St. Andrews. No. 1001.
Medcof J. C. 1965. Study of Lower Oyster Pond at Pleasant Point, Halifax County, N.S. Fisheries Research Board of Canada. Manuscript Report Series

829, Biological Station. St. Andrews. N.B., August 27, 1965.
Medcof, J. C. A. H. Clarke & J. Erskine. 1965. Ancient Canadian East Coast oyster and quahaug shells. J. Fisheries Res. Board Can. 22:631-634.
Medcof, J. C. & C. J. Kerswill. 1965. Effects of light on growth of oysters, mussels, and quahogs. J. Fisheries Res. Board Can. 22:281-288.
White. H. C, J. C. Medcof & L. R. Day. 1965. Are killifish poisonous? J. Fisheries Res. Board Can. 22:635-638.
Medcof, J. C. 1966. Incidental records on behaviour of eels in Lake Ainslie, Nova Scotia. J. Fisheries Res. Board Can. 23:1 101-1 105.
Medcof J. C. 1966. The rough whelk fishery at Godbout, P.Q. Fisheries Research Board of Canada, original manuscript of the Biological Station, St.

Andrews. No.l038. July 19. 1966.
Medcof, J. C, N. Morin, A. Nadeau & A. Lachance. 1966. Survey of incidence and risks of paralytic shellfish poisoning in the province of Quebec.

Fisheries Research Board of Canada. Manuscript Report Series 886, Biological Station. St. Andrews. N.B.. December. 1966.
Clarke. A. H.. D. J. Stanley. J. C. Medcof. & R. E. Drinnan. 1967. Ancient oyster and bay scallop shells from Sable Island. Nature 215:1146-1148.
Medcof, J. C. 1967. Third survey of Eel River Cove, N.B., soft-shell clam (Mvn arenaria) population. Manuscript Report Fisheries Research Board of

Canada 941:57 pp.
Medcof, J. C. 1968. L'ostreiculture dans les provinces Maritimes. Bulletin Office des Recherches sur les Pecheries du Canada 131:178 pp.
Medcof J. C. 1968. Medcof s visits to European molluscan shellfish industrial and research centres 1955. Fisheries Research Board of Canada,

Manuscript Report Series 988, Biological Station. St. Andrews, N.B.. June. 1968.
Medcof J. C. & R. A. Chandler. 1968. Exploring for uses of ocean quahogs obstacles and opportunities. Fisheries Research Board of Canada. Technical

Report 101. 1968.
Medcof, J. C. & E. I. Lord. 1968. Strange catch — a walrus tusk. Notes from the Fisheries Research Board of Canada 21:19-20.
White, H. C. & J. C. Medcof 1968. Atlantic salmon scales as records of spawning history. J. Fisheries Res. Board Can. 25:2439-2441.
Chandler. R. A. & J. C. Medcof 1969. A "catch" 20 million years old. Notes from the Fisheries Research Board of Canada 21:15-16.
Medcof J. C. 1969. Fishermen's reports of freshwater and saltwater migrations of Nova Scotia eels (Anguilla rostratu). Can. Field-Naturalist 83:132-138.
Medcof J. C. D. F. Alexander & R. A. Chandler. 1969. Promising places to look for ocean Quahogs and bar clams and trial fishing with a rocker dredge

off Richibucto. N.B.. and Clark's Harbour, N.S. Fisheries Research Board of Canada. Manuscript Report Series 1068. Biological Station, St. Andrews.

N.B.. December. 1969.
Medcof J. C. & M. L. H. Thomas. 1969. Canadian Atlantic oyster drills (Urosalpin.x) — distribution and industrial importance. J. Fisheries Res. Board

Can. 26:1121-1131.
MacPhail. J. S. & J. C. Medcof 1970. Observations on marine bait worm fisheries in the state of Maine. U.S.A.. June 1950. Fisheries Research Board

of Canada, original manu.scnpt of the Biological Station. St. Andrews, No. 1095. December 1970.
Medcof J. C. 1971. Winter variability in paralytic shellfish poison scores for Crow Harbour, New Brunswick. Fisheries Research Board of Canada,

Manuscript Report Series 1163, Biological Station, St. Andrews, N.B., December, 1971.
Medcof J. C. & J. F. Caddy. 1971. Underwater observations on performance of clam dredges of three types. International Council for the Exploration

of the Sea. Gear and Behaviour Committee. 1971.
Prakash, A., J. C. Medcof & A. D. Tennant. 1971. Paralytic shellfish poisoning in eastern Canada. Manuscript of Fisheries Research Board of Canada

177:10 pp.
Lister. D. B., J. C. Medcof & T. W. Rowell. 1972. Maritimes Region Task Force position paper on aquaculture. Canada. Fisheries Service (Maritimes

Region), 24 pp.
Medcof J. C. 1972. The St. Lawrence rough whelk fishery and its paralytic shellfish poison problems. Fisheries Research Board of Canada. Manuscript

Report Series 1201:26 pp.
Medcof J. C. 1973. Pacific oyster industries in Tasmania and South Australia and potential problems in oyster-pest and disease control. Manuscript

Report to New South Wales State Fisheries, 12 January 1973.
Medcof J. C. 1973. Some thoughts on the New South Wales oyster industry and New South Wales State Fisheries Research and Development

Programmes. New South Wales Fisheries. Sydney Laboratory, October 15, 1973.
Medcof J. C. & W. B. Malcolm. 1973. Oyster culture in New South Wales. The Fishennan 4:1-2:22-23.
Medcof J. C. & P. H. Wolf 1973. Possibilities of oyster culture in the Northern Ten-itory. Depl. of the Chief Secretary. New South Wales State Fisheries,

Sydney Laboratory, Sydney.
Prakash, A., J. C. Medcof & A. D. Tennant. 1973. L'intoxication paralysante par les mollusques dans I'est du Canada. Bulletin Office des Recherches

sur les Pecheries du Canada 177:90 pp.
Medcof J. C. 1974. Some notes on trial fishing, processing and storage of ocean quahogs with appendix on search for ocean quahogs in Port Medway

Harbour, N.S. Fisheries Research Board of Canada, Manuscript Report Series 1322. Biological Station. St. Andrews, N.B., September, 1974.
Medcof J. C. & J. F. Caddy. 1974. Underwater observations on performance of clam dredges of three types. Fisheries Research Board of Canada,

Manuscript Report Series 1313. Biological Station. St. Andrews. N.B.. June. 1974.
Medcof J. C. & W. B. Malcolm. 1974. Making the best use of a natural resource — oyster culture in New South Wales. The Fisherman 40: 1-2:22-23.
Medcof J. C. & M. L. H. Thomas. 1974. Surfacing on ice of frozen-in marine bottom materials. J. Fisheries Res. Board Can. 31:1195-1200.
Medcof J. C. 1975. Living marine animals in a ship's ballast water. Proc. Natl. Shellfish. As.wc. 65:1 1-12.
Medcof J. C. & P. H. Wolf 1975. Pacific oysters [Crassostrea gigasi in New South Wales. Australia. Available from Library, Biological Station, St,

Andrews. N.B.. E5B-2L9.
Medcof J. C. & P. H. Wolf 1975. Spread of Pacific oyster won-ies NSW culturists. Ansl. Fisheries 34:1-7.

Medcof J. C. 1976. Australian oyster and oyster culture: a partial bibliography. New South Wales State Fisheries, Technical Report 1, January. 1976.
Medcof J. C. 1979. Iron-manganese concretions from New Brunswick lakes. J. New Brunswick Museum 4:125-131.
Medcof J. C. 1979. Lake Utopia Concretions "Still a Mystery". The St. Croix Courier. 1 August 1979.

Joiirmil uf Shellfish Research. Vol. 19, No. 1,7-12. 2000.

IN MEMORIAM

RUTH DIXON TURNER

1914-2000

Ruth Turner was bom in Melrose, Massachusetts December 7. 1914. She attended Bridge water State College, MA and graduated with
a B.S. in 1936. She became a school teacher, teaching in both Bondville, Vermont and North Reading. Massachusetts before accepting
the appointment of Assistant Director of Education at the New England Museum of Natural History (now the Boston Museum of
Science). Ruth subsequently became Assistant Curator of Birds at the Museum, before moving to Vassar College as an Instructor in the
Biology Department. During this same period she completed a M.A. at Cornell. By this time Ruth was an accomplished ornithologist,
an interest that she maintained throughout her life. Indeed, it was her interest in birds that first lead her to the Museum of Comparative
Zoology at Harvard. While serving as a volunteer in the Ornithology Department she met William J. Clench, Curator of MoUusks. Clench
introduced Ruth to Dr. William Clapp, a pioneer in the study of marine wood borers, and in 1944 she moved to the William F. Clapp
Laboratories in Duxbury. Massachusetts. It was here that her career as a malacologist became firmly established. She returned to Harvard
University two years later to work with Clench.

Harvard remained her scientific home and a source of great pride to her throughout her career. One of her early field trips with Clench
was to Cuba in 1949 to examine local terrestrial and marine mollusks. She received her Ph.D. from Radcliffe College. Harvard University
in 1954. Her dissertation on the Teredinidae remains a standard work to this day. From 1954 through 1975 she served as Research
Associate in the Department of Mollusks at the Museum of Comparative Zoology, Alexander Agassiz Fellow in Zoology and Ocean-
ography, and Lecturer in Biology at Harvard. In 1976 she became Professor of Biology, Curator in Malacology at the Museum of
Comparative Zoology, and joint editor of the scientific journal Johnsonia. Ruth received honorary D.Sc. degrees from New England
College and Plymouth State College of the University of New Hampshire, and held honorary appointments at the Academy of Natural
Sciences in Philadelphia, the Woods Hole Oceanographic Institution, the Gray Museum at the Marine Biological Laboratory at Woods
Hole, Leigh University. CSIRO and the University of New South Wales in Australia, the University of Puerto Rico, and as an FAO
Fellow in India. Ruth was honored by the Woods Hole Oceanographic Institution as a "Women Pioneer in Oceanography."" On a lighter
but no less important note. Ruth was named "'Diver of the Year"" by the Boston Sea Rovers, an educational society of which she was
a very proud member. Ruth served terms as President of both the American Malacological Union and the Boston Malacological Club.
She was a Honorary Life Member of the National Shellfisheries Association.

Ruth was a pioneer in the field of marine biodeterioration research, and enjoyed a long term relationship with the Office of Naval
Research. This, combined with her work in invertebrate and larval ecology, took her to many corners of the globe including France,
Belgium. Netherlands, England, Germany, Denmark. Puerto Rico, India, Pakistan, many locations in Australasia. South America, and
the former Soviet Union. Although a leading researcher, she enjoyed teaching at all levels from special courses for public school teachers,
to undergraduate and graduate teaching, to her gentle persuasion of fellow researchers to look at a problem or a data set in another light.
Ruth"s work also took her on many oceanographic cruises and to the depths of the ocean. On August 13, 1971, she became the first
woman to dive in the deep submergence research vehicle ALVIN. This was the first of many dives and a deep sea career that included
long term biodeterioration and species diversity work in the deep ocean (it was Ruth who explained why so little wood remained on the
Titanic when it was found deep in the North Atlantic Ocean), and participation in multi-investigator cruises to the Galapagos Rift system.

8 In Memoriam: Ruth Dixon Turner

Despite a career filled with discovery, innovation, and firsts, the most memorable component of Ruth"s character that remains with
the majority of people who met her is her warmth and friendliness, and her desire to show the excitement of science to all. Her love of
science was effusive. She had a unique ability to share her science with audiences of all ages and skill levels. She was equally a superb
teacher, illustrator, and practical scientist from fine work with the electron microscope to dissections of material from the field. Ruth
taught and mentored many scientists at many levels, unselfishly giving of her time and energy to advance their careers. I consider myself
fortunate to have enjoyed such direction from Ruth. Ruth shared much of her science through her publications, over 100 in all. Ruth
worked actively until well after her 80th birthday. She left unfinished two major works, a monograph on her studies of deep sea borers
and a comprehensive illustrated catalog of the pholads. Her colleagues have committed to finish these.

Throughout her career Ruth made unique contributions in malacology and deep sea biology. She was an internationally respected
educator and researcher, an ambassador for marine and biodeterioration studies, and an outstanding role model for women in science.
Ruth is survived by her sisters Winifred Garrity and Lina MacNeil. Ruth was predeceased by her brothers Henry and Arthur, and her
sisters Jessie. Mary, and Frances. Ruth never married, but had a large extended family of colleagues and students. She will be sadly
missed,

Roger Mann
Professor of Marine Science-
School of Marine Science
Virginia Institute of Marine Science
College of William and Mary
Gloucester Point. VA 23062

PUBLICATIONS

1942. Editor. Biillelin of New Bird Life. vol. 6(8-1 2):56-104.

1943. Birding the first year of the war. Bull. Mass. Audubon Society 28(2):33-42.

1944. Vassar birds. Vassar Alumnae Magazine 33(4):15-17

1946. The genus Bankia in the Western Atlantic. Johnsonia 2(l9):l-28. 16 pis. (with W. J. Clench).

1946. John Gould Anthony, with a Bibliography and Catalogue of his Species. Occ. Papers on MoUusks 1(8):81-108, 15 pi.

1947-1948. Republication; Henry Krebs — 1864 The West Indian marine shells. Rev. Soc. Malacologica Carlos de la Torre (Habana) 5:23—10; 59-80;
91-116 and 6;1 1-43; 45-48. (with W. J. Clench and C. G. Aguayo).

1947. Review; Fauna of New England. List of Mollusca, by C. W. Johnson, 1915. Johnsonia 2(231:92.

1947. Review: A List of the Mollusca of the Atlantic Coast from Labrador to Texas, by Johnson, C. W. 1934. Johnsonia 2(23):92.

1947. Procedimientos para recolectar bromas y otros moluscos perforentes marinos. Rev. Soc. Malacologica Carlos de la Torre (Habana) 5(2):43— 14.

1947. Collecting shipworms. Limnological Soc. America, spec. publ. no. 19:1-8, text figs.

1948. A new Thais from Angola and notes on Thais haemastoma Linne. American Mus. Novilates, no. 1374:1-14. 1 pi.
1948. The genus Truncatella in the western Atlantic. Johnsonia 2:149-164, pis. 65-73 (with W. J. Clench).

1948. William Henry Fluck. 1870-1948. Nautilus 62:69-70.

1948. A catalogue of the family Truncatellidae with notes and descriptions of new species. Occ. Papers on MoUusks 1:157-212, pis. 22-24. (with W. J.
Clench ).

1948. The family Tonnidae in the western Atlantic. Johnsonia 2(26):165-192. 1 1 pis.

1949. Sea shells |determination of all shells figured). Life Magazine 27(7):72-75. (with W. J. Clench).

1949. Review: A manual of the Recent and fossil marine pelecypod moUusks of the Hawaiian Islands, by W. H. Dall, P. Barlsch and H. A. Rehder, 1938.

Occ. Papers on MoUusks l(14):231.
1949. Review; Reef and Shore Fauna of Hawaii, by H. H. Edmondson 1933. Occ. Papers on MoUusks 1( 14):231-232.

1949. Review: A collection of Japanese shells with illustrations in natural color, by S. Hirase, 1934. Occ. Papers on MoUusks 1(I4):232.

1950. The western Atlantic marine mollusks described by C. B. Adams. Occ. Papers on MoUusks l( 151:233-403. pis. 29—49. (with W. J. Clench).
1950. The voyage of the Tomas BaiTera. Johnsonia 2(28):220.

1950. The genera Sihenorylis. Cirsolrenui. Acirsa. Opalia. and ,'\inaea in the western Atlantic. Johnsonia 2:221-248. pis. 96-107. (with W. J. Clench).
1950. Review: Sullivan, M. C. 1942. Bivalve Larvae of Malpeque Bay. Prince Edward Id.. Bulletin 77. Fisheries Research Board of Canada, pp. 1-36,
22 pis. Johnsonia 2, p. 248.

1950. Edward Chitty, with a bibliography and a catalogue of his species of Jamaican land mollusks. Oic. Papers Mus. Inst. Jamaica, no. 1:1-12. 1 pi.
(with W.J. Clench).

1951. The genus Epitonium in the western Atlantic. Part I: subgenera Epiioniuni s.s.. Cycloscala. G\roscala. Johnsonia 2:249-288, 23 pis. (with W.J.
Clench).

1951. Review: The Shell Collectors Handbook, by A. H. Verrill. Natural History 60(5):199.
1951. Review: The Sea Shore, by C. M. Yonge 1949. Occ. Papers on Mollu.sks 1( 16):4I0-41 1.

1951. Review: Natural History ot Marine Animals, by G. E. and Nettie MacGinilie 1949. Occ. Papers on Mollusks 1( 16):4I 1— tl 2.

1952. Some problems in the Pholadidae. Hidl. Am, Malacological Union .-Xnn. Kepi, for 1951:9-10.
1952. Me.sunella. a new genus in the Camaenidae. Nautilus 66:32 (with W. J. Clench).

1952. La Rocolte des Tarets. Catalogues VIII, Xylophages et Petricoles Quest Africains. Institut Francais d Alriquc Noirre. pp. 130-134. figs. 156-158.
[translation of paper published in Special Publication #19 of the Limnological Society of America].

1952. The genera Epitcmium (Pan II (subgenera /l.syjcri.Ko/d and Boreoscala]). Dcpressiscala. Cylindriscala. Nystiella and Soluliscala in the western
Atlantic. Johnsonia 2:289-3.56, pis. 131-177 (with W.J. Clench).

1953. New England malacologisls. Am Malacological I'nitm Ann. Rcpt. for 1952:4-6.

1953. The Genera Epitonium. Opalia. and Cylindriscala in the Western Atlantic. Johnsimia 2:361-363, pi. 180,

In Memoriam: Ruth Dixon Turner 9

I9?3. Monographs of the Marine Mollusks of the Western Atlantic. Jolinsonia 2( l9-32):357-359.

1953. [Supplement to| The Genus Bankia in the Western Atlantic. Johiisonia 2(34):357-359. (with D. J. Brown).

1953. Recent works on the marine inollusks of Argentina. Johnsonia 2:380.

1954. The family Pholadidae in the western Atlantic and the eastern Pacific, Part 1: Pholadinae. Johnsonia 3:1-63. pis. 1-34.
1954. Supplement to John Gould Anthony (Occ. Pap. no. 8). Occ. Papers on Mollusks 1(18):442.

1954. Supplement to the Catalogue of the Family Truncatellidae (Occ. Pap. no. 13). Occ. Papers on Mollusks 1(18):445. (with W. J. Clench).

1954. Supplement to Western Atlantic Marine Mollusks Described by C. B. Adams. (Occ. Pap. no. 15). Occ. Papers on Mollusks 1( 18):447. (with W. J.

Clench).
1954. Review: Ensaio de Catalogo dos Moluscos do Brasil. by Frederico Lange de Morretes 1949. Occ. Papers on Mollusks 1(18):449.

1954. Review: Catalogo de la Malacofauna .Antarctica Argentina by A. R. Carcelles. Johnsonia 3:64.

1955. The family Pholadidae in the western Atlantic and the eastern Pacific. Part II: Martesiinae, Juannetiinae and Xylophaginae. Johnsonia 3:65-100,
pis. 35-93.

1955. The North American genus Lioplax in the family Viviparidae. Occ. Papers on Mollusks 2:1-20. pis. 1^ (with W. J. Clench).

1955. Scaphopods of the Atlantis dredgings in the western Atlantic with a catalogue of the scaphopod types in the Museum of Comparative Zoology.

Deep Sea Research, suppl. to vol. 3. pp. 309-320.
1955. The Genus Melongena (abstract). A.M.U. — 20th Ann. Meeting p. 10.
1955. Collecting shipwomis. [in] How to collect shells, pp. 32-35 (American Malacological Union).

1955. The work of Charles B. Adams in the West Indies and Panama. Am. Malacological Union Ann. Rept. for 1955 pp. 7-8 (abstract).

1956. The family Melonginidae in the western Atlantic. Johnsonia 3:161-188. pis. 94—109 (with W.J. Clench).
1956. Melongena corona Gmelin. an excellent marine laboratory mollusk. Turto.x News 34:106-108. pis. 1-2.
1956. Notes on Xylophaga washingtona Bartsch and on the genus. Nautilus 70:10-12.

1956. Additions to the Western .Atlantic Marine Mollusks described by C. B. Adams. Occ. Papers on Mollusks 2:134-136. 1 pi.

1956. Additions to the Pholadidae— Part II. Johnsonia 3(35):188.

1956. The eastern Pacific mollusks described by C. B. Adams. Occ. Papers on Mollusks 2:21-133. pis. 5-21.

1956. Freshwater mollusks of Alabama. Georgia and Florida from the Escambia to the Suwannee River. Florida State Mus. Bull. 1:97-239, 9 pis. (with
W. J. Clench).

1957. Charles Johnson Maynard and his work in malacology. Occ. Papers on Mollusks 2:137-152. 1 pi.

1957. Molluscan wood borers, [in] Symposium on wood for marine use and its protection from marine organisms. American Soc. Testing Materials. Spec.
Tech. Publ. no. 200:10-13.

1957. The family Cymatiidae in the western Atlantic. Johnsonia 3:189-244. pi. 1 10-135 (with W. J. Clench).

1958. The genus Hemitrochus in Puerto Rico. Occ. Papers on Mollusks 2:153-180, pis. 23-30.

1958. The family Pinnidae in the western Atlantic. Johnsonia 3:283-326. pis. 149-171 (with J. Rosewater).
1958. The works of Georgius Everhardus Rumphius. Johnsonia 3:326-327.

1958. Review: Voyage Aux lies de Teneriffe. La Trinite Saint-Thomas. Saint Croix et Porto Rico by Andre Pierre LeDru. Occ. Papers on Mollusks
2(22):179-180.

1958. Review: The Museum Boltenianum or the Bolten Catalogue. Johnsonia 3:283-284.

1959. Notes on the genus Taheilia {Truncatellidae) in New Guinea with the description of a new species. Occ. Papers on Mollusks 2:181-188. pis. 31-32.
1959. The genera Hemiioma and Diodora in the western Atlantic. Johnsonia 3:334-344. pis. 176-179.

1959. Henry A. Pilsbry. Johnsonia 3: introduction ii-iv. 2 pis.

1959. Notes on the feeding oi Melongena corona. Nautilus 73:1 1-13.

1959. Melongena egg cases. Nautilus 73:77.

1959. The status of systematic work in the Teredinidae. Symposium on marine boring and fouling organisms. Univ. Washington Press, pp. 124-136.

1959. Two new genera of land mollusks (Papuininae) from the Central Highlands of New Guinea. J Malacological Soc. Australia no. 3:4-9. pi. 1. text
figs. 1-3 (with W.J. Clench).

1960. Some techniques for anatomical work. Ann. Rept. Am. Malacological Union for 1959:6-8.
1960. Land shells of Navassa Island. West Indies. Mus. Comp. Zool. Bull. 122:233-244. 7 pis.
I960. Mounting minute radulae. Nautilus 73:135-137.

I960. A new Meliobba from Schrader Range. New Guinea. J. Malacological Soc. Australia no. 4:30-31. 1 pi. (with W. J. Clench).

1960. The occurrence of a nematode parasite in the genus Stylodon. J. Malacological Soc. Australia no. 4:56-59, text fig. 1, pi. 7 (with M. A. Pini).

1960. The genus Calliostoma in the western Atlantic. Johnsonia 4:1-880, pis. 1-56. I text fig. (with W.J. Clench).

1960. Teredo s en de mens. Correspondentiblad van Nederlandse Malacologische Vereniging. no. 91:924—925. [Translated into Dutch by C.O.V.
Regteren. Altena].

1961. Heli-x pomatia Linne. colonized at Plymouth. Mass. Nautilus 74:122.

1961. Natural history museums of Europe. Am. Malacological Union Rept. for 1960:13-14.

1961. Report on the American Malacological Union meeting at McGill University. Am. Malacological Union Rept. for 1960:28-32.

1961. Review: Traite de Zoologie. Vol. 5 fascicule 2. Embranchement des Mollusques pp. 1625-2164. Occ. Papers on Mollusks 2:260.

1961. Pleurotomariidae in Bermuda waters. Nautilus 74:162-163.

1961. Remarks on Nettastomella and Jouannetia. Am. Malacological Union Rept. for 1961:17-18.

1961. The genus Lignopholas Turner (Mollusca: Pholadidae). Mitl. Zool. Mus. Berlin 37:287-295.

1962. Nettastomella japonica Yokoyama in North America and notes on the Pholadidae. Occ. Papers on Mollusks 2:289-308. 7 pis.

1962. New names introduced by H. A. Pilsbry in the Mollusca and Crustacea. Acad. Nat. Sci. Philadelphia, spec. publ. no. 4:1-218 (with W. J. Clench).
1962. Books help beachcombers play the shell game. Natural Histoiy 71(7):4-7.
1962. The genus Lithophaga in the western Atlantic. Johnsonia 4:81-1 16. 19 pis. (with K. J. Boss).

1962. Review: British Prosobranch Molluscs, their functional anatomy and ecology, by V. Fretter and A. Graham. Johnsonia 4:1 16.
1962. James H. Orton. his contributions to the Held of fossil and Recent mollusks. Rev. Mus. Argentina Cienc. Nat. Bernardino Rivadavia. Buenos Aires.
8:89-99.

10 In Memoriam: Ruth Dixon Turner

1963. Monographs of the genera Pupiisnla. FoicarUa. and Meliobha (Papuininae: Camaenidae). J. Matucological Soc. Auslmliu no. 6;3-33 (with W. J.
Clenchj.

1963. Nest building in the bivalve moilusk genera. Musculiis and Limu. The Veliger 6:55-59 (with A. S. Merrill).

1964. The subfamilies Volutinae. Zidoninae, Odontocymbiolinae and Calliotectinae in the western Atlantic. Johiisonia 4:129-180. 30 pis. (with W. J.
Clench).

1964. Snail. Encyclopedia Britannica p. 848 A-848 H. 1 1 figs.
1964. Review: Fauna und Flora der Adria. by R. Riedl. Johiisonia 4:180.

1964. Monographs of the genera Megalacron and Rhylidoconcha (Papuininae: Camaenidae). / Mulacologlcul Soc. Australia no. 8:36-71 (with W. J.
Clench).

1964. Anatomical relationships in the Teredinidae. Ann. Rept. American Malacological Union for 1964:16-17.

1965. Mussel, [in] Encyclopedia Britannica. pp. 1096-1098. 2 figs.; 1964. ibid.. Snail, pp. 848A-848H. II figs. (Other articles in the Encyclopaedia
Britannica include: Moilusk. Periwinkle. Cockle. Quahog. Piddock, Teredo, Whelk, Scallop, and Chiton).

1965. Introduction. Occ, Papers on MoUusks 2:l-xvi.

1965. Joseph C. Bequaert. Occ. Papers on MoHusks 2:i-ix. 3 pis.

1966. Monograph of the genus Rhynchotrochus (Papuininae. Camaenidae). J. Malacol. Soc. Australia, no. 9:59-95. text figs. 1-6, pis. 15-22 (with W. J.
Clench).

1966. A survey and illustrated catalogue of the Teredinidae. Spec. puhl. Museum of Comparative Zoology. 265 pp. 64 pis.. 25 text figs.

1966. Some results of deep water testing. Ann. Rept. Am. Malacological Union for 1965. pp. 9-1 1.

1966. Report to the government of India on systematic and biological research on marine wood-boring Mollusca. FAO Report TA 2155. pp. 1-30.

1966. Implications of recent research in the Teredinidae. Beihefte zu Material und Organismen. Berlin. Heft 1. pp. 437—446.

1966. Marine borer research in cooperation with the Office of Naval Research. Report of First Inter-American Naval Research Congress.

1967. A new species of Lyria (Volutidae) from Hispaniola. Nautilus 80:83-84, figs. 2-3.
1967. Teredo. Encyclopedia Britannica pp. 861-862.

1967. A new species of fossil Chlamys from Wright Valley. McMurdo Sound. Antarctica. New Zealand J. Geology Geophysics 10:446-455. figs. 1-5.

1968. The Xylophagainae and the Teredinidae — a study in contrasts. Ann. Rept. Am. Malacological Union for 1967. pp. 46-^8.

1968. Monograph of the genus Letitia (Papuininae: Camaenidae). / Malacological Soc. Australia, no. 1 1:32-49. pis. 3-7. text figs. 1-2 (with W. J.
Clench).

1969. Biological studies in marine wood borers. Arm. Rept. Am. Malacological Union for 1968. pp. 14-16. (with A. C. Johnson).
1969. Review: The shell, five hundred million years of inspired design, by H. & M. Stix and R. T. Abbott. Natural History 78:60-62.

1969. Pholadacea. [in] R. Moore (ed.). Treatise on Invertebrate Paleontology. (N) Mollusca 6(2 of 3):702-742. figs. 162-214.

1970. Some problems and techniques in rearing bivalve larvae. Ann. Rept. Am. Malacological Union for 1969. pp. 9-12. I pi. (with A. C. Johnson).
1970. Richard Winslow Foster. Johnsonia 4:ii-v. 2 figs.

1970. The family Volutidae in the western Atlantic. Johnsonia 4(48):369-372. pis. 172-174. (with W.J. Clench).

1971. Some anatomical and life history studies of wood boring bivalve systematics. Ann. Rept. Am. Malacological Union for 1970. pp. 65-66 (with John
Culliney).

1971. Identification of marine wood boring mollusks of the world. |in) Marine Borers. Fungi and Fouling Organisms of Wood. Chapter I. pp. 18-64.

Published by the OECD.
1971. Biology of the marine wood boring mollusks of the world. Ibid.. Chapter 13. pp. 259-301. (with A. C. Johnson).
1971. Review: Beneath Australian seas, by Walter Deas and Clarrie Lavvler. .Australian Newsletter N. S. no. 2. p. 9.

1971. Australian shipworms. Australian Natural History, Sydney. I7(4):139-145. 4 pis.

1972. Land and freshwater snails of Savo Island, Solomons, with anatomical descriptions (Mollusca, Gastropoda). Steenstrupia (Zool. Mus. Univ.
Copenhagen). 2( l5):207-232. pis. I-I3 (with W.J. Clench).

1972. Results of an international cooperative research program on the biodeterioration of timber submerged ni the sea. Material und Organismen

7(2):93-l 18 (with E. B. G. Jones. H. Kuhne and P. C. Trussell).
1972. A new genus and species of deep water wood-boring bivalve (Mollusca. Pholadidae. Xylophagainae). Basleria 36:97-104. figs. 1-12.
1972. Teredicola typicus C. B. Wilson. 1942 (Copepoda, Cyclopoida) from shipworms in Australia. New Zealand, and Japan. .Australian ./. Marine and

Freshwater Res. 23( l):63-72. figs. 1-16 (with A. G. Humes).
1972. Line photo micrography: A tool in biological studies. Bulletin of the American Malacological Union, p. 30.
1972. Cinemicrographic studies of crawling behavior in larval and juvenile bivalves, (with J. L. Culliney).

1972. Xyloredo. new leredinid-like abyssal wood-borers (Mollusca. Pholadidae. Xylophagainae). Breviora. MCZ. no. 397: 1-19. pis. 1-6.

1973. Wood-boring bivalves, opportunistic .species in the deep sea. Science 180:1377-1379. 2 figs.. I table.

1973. Deep water wood-boring mollusks. Proc. Third International Congress on Marine Corrosion and Fouling. Nat. Bur. Standards. Gaithersburg.
Maryland, pp. 8.36-841.

1973. The biologists view of the Tereduiidae and then" control (with a documentary film on the life history of the Teredinidae). Proc. Third Inlernational
Congress on Marine Corrosion and F<iuling. Nat. Bur. Standards. Gaithersburg. Maryland, pp. 83-87 (with J. L. Culliney).

1974. In the path of a warm saline eftluent. Am. Malacological Union Bull, for 1973. pp. 36—44. figs. 1-3.

1974. A new blind F'hysa from Wyoming with notes on its adaptation to the ca\e environment. Nautilus SS(3):S()-S5. 19 figs, (with W.J. Clench).

1974. New approaches and techniques for studying bivalve larvae |in| W. I.. Smith & M. H. Chanley (eds.). Culture of Marine Invertebrate Animals, pp.
2.57-271, 2 figs. Plenum Publishing Corp.. N.Y. (with J. L. Culliney and P.J. Boyle).

1975. Review: The Shell Makers, Introducing Mollusks. by Alan Solem. J. Fisheries Res. Board Canada 32( 5 ):7 19-720.

1 975. Studies of bivalve larvae using the scanning electron microscope and critical point drying. Bull. Am. Malacological t 'nion lor 1 974. pp. 59-65 (with
P. J. Boyle).

1976. Larval development of the wood boring piddock Martesia striata (Linnaeus) (Mollusca: Pholadidae). 7. E.xper. Marine Biol, and Ecology 22:55-68.
text figs. 1-4 (with P. J. Boyle).

1976. Fixation and prcser\'ation of marine /ooplankton. |inl II. F. Slcedman (ed.). SCOR/UNESCO Handbook Zooplankton Fixation and Preservation.
Chap. 8 section on Mollusca. Part 1 1, pp. 290-304. Lhie.sco Press. Paris.

In Memoriam: Ri'th Dixon Turner 1 1

1976. Search for a weak link. Proc. Workshop on Biodeterioration of Tropical Woods. (D. Bultman, editor). Naval Res. Lab. Washington. D.C.. pp.

31-tO.
1976. Some factors involved in the settlement and metamorphosis of marine bivalve larvae, [in] Sharpley & Kaplan (eds.i. Proc. 3rd. International

Biodegradation Symposium, pp. 409—416.
1976. Larval development of the deep-water wood boring bivalve Xylophaga atlaniica Richards (Mollusca. Bivalvia. Pholadidae). Ophelia 15(2):149-161

(with J. L. Culliney).
1976. Marine biodeteriogenic organisms. I. Lignicolous fungi and bacteria and the wood boring Mollusca and Crustacea. Intern. Biodeierior. Bull.

12(4):120-134 (with G. Jones. S. E. Funado and H. Kuhne).
1976. Reproductive pattern in an abyssal snail. Anwr. Zool. 16(2):269 (with M. A. Rex and C. A. Van Ummensen).

1976. Bivalve larvae, their behavior, dispersal and identification. Proc. U.S. — U.S.S.R. Workshop in Biological productivity and biochemistry of the
worlds oceans, pp. 23-25 [in] J. Costlow (ed.). Ecology of Fouling Communities.

1977. Control of marine borer attack on wood. U.S. Patent 4.012.529 (with J. D. Bultman & L. Jurd).

1977. Genetic similarities of wood-boring bivalves (Pholadidae and Teredinidae) based on comparisons of allozymes. Biol. Bull. 153(2):420 (with T. J.
Cole).

1977. Development, metamorphosis and natural history of the nudibranch Doriclclla ohsciira Verrill (Corambidae: Opisthobranchia). J. Exp. Mar. Biol.
Ecol. 27:171-185 (with F. E. Perron).

1978. Contribution of field and life history studies to an understanding of some cases of opportunism. [in| U.S.S.R. — U.S.A. Symposium on the Program
Biological Productivity and Biochemistry of the Words Oceans, pp. 241-2-14.

1978. Wood, mollusks. and deep-sea food chains. Bull. Am. Malacological Union for 1977, pp. 13-19, figs. 1-3.
1978. Genetic relations of deep-sea wood-borers. Bull. Am. Malacological Union for 1977, pp. 19-25 (with T. Cole).

1978. The feeding behaviour and diet of Calliostoma occidentale. a coelenterate-associated prosobranch gastropod. J. Moll. Stud. -14:100-103 (with F.
Perron).

1979. Mollusks as prey of ariid catfish in the Fly River. New Guinea. Bull. Am. Malacological Union for 1978, pp. 33—10. pis, 1-6 (with T. R. Roberts).
1979. New techniques for preparing shells of bivalve larvae for examination vvith the scanning electron microscope. Bull. ,Am. Malacological Union for

1978. pp. 17-24. pis. 1-3 (with C. B. Calloway).
1979. The role of phytoplankton in the diets of adult and larval shipworms. Lyrodus pedicellatus (Bivalvia: Teredinidae). Estuaries 2(1 ):58-60 (with J. A.

Pechenik and F. A. Perron).
1979. Bankia neztalia n. sp. (Bivalvia: Teredinidae) from Australia-New Zealand, and its relationships. J. Royal. Soc. New Zealand 9(4):465— 173 (with

J. L. McKoy).
1979. High Larval Dispersal Capability of a Deep-sea Hydrothermal Vent Bivalve from the Galapagos Rift. .American Society of Zoologists Meeting

[abstracts!. Dec. 27-30, 1979.
1979. Reproductive pattern in the abyssal snail. Benthonella tenella (Jeffreys), [in] S. Stancyk (ed. I. Reproductive Ecology of Marine Invertebrates. Belle

W. Baruch Library in Marine Science, publ. no. 9. pp. 173-188 (with M. A. Rex and C. A. Van Ummerson).
1979. Biology, life history and relationships of Zachsia zenkeailschi. XIV Pacific Science Congress. Khabarovsk. Abstracts. Committee F. Sec. 1 la, pp.

139-141 (with Y. M. Yakovlev).

1979. Galapagos 79: Initial findings of a deep-sea biological quest. Oceanus 22(2): 1-10 (with F. Grassle and members of the cruise).

1980. Macrobiodegradation of plastics. Proc. 4th International Biodeterioration Symposium, Berlin-Dalhem, pp. 1 17-122 (with G. J. L. Griffin).
1980. Range extension of teredinids (shipworms) and polychaetes in the vicinity of a temperate-zone nuclear generating station. Marine Biology 5S:55-(A

(with K. E. Hoagland).
1980. The giant white clam from the Galapagos Rift. Calyptogena magnifica n. sp. (Bivalvia; Vesicomyidae). Malacologia 20(1 ): 161-194 (with K. J.

Boss).
1980. Larval dispersal of a deep-sea hydrothermal vent bivalve from the Galapagos Rift. Marine Biology 57:127-133 (with R. A. Lutz. D. Jablonski. and

D. C. Rhoads).
1980. Evolution and adaptive radiation of shipworms. Haliotis 10(2):68 (with K. E. Hoagland).

1980. Effects of closed-culture competitive interactions on growth of Teredo navalis. Biological Bulletin I59(2):465 (with G. A. Tracy and C. J. Bergl.

1981. Wood Islands and Thermal Vents as centers of diverse communities in the deep-sea. Biologia Morya. no. I. pp. .VIO [in Russian, translation by
Plenum Publishing Co.].

1981. Physiological aspects of wood consumption, growth, and reproduction in the shipworm, Lyrodus pedicellatus Quatrefages (Bivalvia: Teredinidae).

/. Exp. Mar. Biol, and Ecol. 52:63-76 (with S. Gallager and C. Berg).
1981. Preliminary observations of bacteria and shipworms (Bivalvia: Teredinidae). Biol. Bull. 161:332 (with A. Wright. C. Cavanaugh, R. Mann).
1981. Evolution and adaptive radiation of shipworms. Malacologia 21(1-2):1 1 1-148 (with K. E. Hoagland).

1981. Life cycle of Zachsia zenkewitschi. bivalve mollusk with dwarf males, [in] Sixth All-Union Conference on Embryology Abstracts of paper [in
Russian]. Nauka. Moscow p. 207.

1982. Feeding types in vent macro-organisms. Abstracts of Papers of the 148th Natl Meeting AAAS. 1982:34.

1983. The ecology and reproduction of Zachsia zenkewitschi, a teredinid with dwarf males. Proc. XIV Pacific Science Congress, Khabarovsk.
USSR, August 1979. Section Marine Biology 2, Genetics and Reproduction of Marine Organisms, pp. 215-219, figs. 1-5 (with Y. Yakovlev). [in
Russian].

1983. Documentation and implications of rapid successive brooding in the shipworm. Lyrodus floridanus (Mollusca: Bivalvia). Proc. XIV Pacific Science
Congress. Khabarovsk, USSR, August 1979. Section Marine Biology 2. Genetics and Reproduction of Marine Organisms, pp. 172-177, figs, 1-2
(with C. B. Calloway). ]in Russian].

1983. Dwarf males in the Teredinidae (Bivalvia: Pholadacea). Science 219:1077-1078 (with Y. Yakovlev).

1983. A Cellulolytic nitrogen-fixing bacterium cultured from the gland of Deshayes in shipworms (Bivalvia: Teredinidae). Science 221:1401-1043 (with
J. Waterbury and C. B. Calloway).

1983. Some aspects of the life history of a bivalve mollusc. Zachsia zenkewitschi. Biologiya Morya 9(5):27-34 (with Y. Yakov lev and E. M. Karaseva).
[in Russian!.

Documentation and implications of rapid successive gametogenic cycles and broods in the shipworm. Lyrodus floridanus (Bansch) (Bivalvia: Tere-
dinidae). J. Shellfish Res. 3(l):65-69 (with C. B. Calloway). [Sept. 1984].

12 In Memoriam: Ruth Dixon Turner

1984. An iiverview of research on marine borers: past progress and future directions, [in] J. D. Costlow and R. C. Tipper (eds.). Marine biodeterioration:

an interdisciplinary study, pp. 3-16. Naval Institute Press. Annapolis. Maryland.
1984. Some aspects of the life history of Zachsia zenkewiischi (Teredinidae. Bivalvia). The Soviet Journal of Marine Biology 9(5):257-264. Plenum

Publishing Corp.. N.Y.. Translation from the Russian-Biologiya Morya 1983. (with Y. M. Yakovlev with E. M. Karaseua).
1984. Growth and distribution of moUusks at deep-sea vents and seeps. Oceaims 27(3):55-62 (with R. A. Lutzl.
1984. Larval development and dispersal at deep-sea hydrothernial vents. Science 226:1451-1454 (with R. A. Lutz and D. Jablonski).

1984. Larval ecology of mollusks at deep-sea hydrothermal vents. Bull. Am. Malacological Union. Annual Meeting, Norfolk, Virginia, July 1984 (with
Phillippe Bouchet and Richard A. Lutz).

1985. Notes on mollusks of deep-sea vents and reducing sediments. American Malacological Bulletin. Special Edition No. 1: 23-34. In Perspectives in
Malacology: A Symposium to Honor — Dr. Melbourne and R. Carriker.

1985. Modes of larval development at deep-sea hydrothermal vents, [in] M. L. Jones (ed. ). Hydrothermal vents of the eastern Pacific: an overview. Bull.

Biol. Soc. Washington, no. 6. pp. 167-184. figs. 1-28. (with R. A. Lutz and D. Jablonski).
1985. Squat lobsters. Munidopsis, associated with mesh enclosed wood panels submerged in the deep-sea. American Zoologist 25(4):141A [abstract].

(with A. B. Williams).
1985. Hydrothermal vents, sulfide seeps and mollusks. Am. Malacological Bulletin 3(1):96 (abstract for 1984 meeting).
1985. Description of a hydrocarbon seep community on the Louisiana slope. Am. Zoologist 25(4): lOA [abstract], (with C. J. Denoux, M. C. Kennicutt,

R. R. Bidigare, J. M. Brooks, R. R. Fay, M. L. Jones).

1985. William J. Clench. October 24. 1 897-February 1984. Malacological Rev. 18:123-124.

1986. Larval ecology of mollusks at deep-.sea hydrothermal vents. Am. Malacological Bulletin 4( 1 ):49-54 (with R. A. Lutz. P. Bouchet. D. Jablonski.
and A. Waren).

1986. The language of benthic marme invertebrate development patterns: problems and needs, [in) M.-F. Thompson. R. Sarojini and R. Nagabhushanam
[eds.). Biology of benthic marine organisms: Techniques and methods as applied to the Indian Ocean. Bombay: Oxford and IBH Publishing Co. pp.
227-235, figs. 1-10 (with J. A. Pechenik and C. B. Calloway).

1986. Squat lob,sters (Galatheidae: Munidopsis) associated with mesh-enclosed wood panels submerged in the deep sea. J. Crustacean Biology 6(3):
617-624 (with A. B. Williams).

1986. The biology of molluscan hard-substrate borers. International Conference on Marine Sciences of the Arabian Sea. March 2S-Apri! 2. 1986, Karachi.
Pakistan. Abstracts p. 35.

1987. Seasonal recruitment of marine invertebrates to hard substrates on Georges Bank and the eastern continental shelf of the United States. Nautilus
101(1 ):19-24 (with C.J. Berg. B. Butman and J. A. Early).

1987. Species pairs in the Teredinidae. International research group on wood preservation. Document No: IRG/WP/4142: 1-2 (with C. B. Calloway).
1987. Species pairs in the Teredinidae. American Malacological Union Annual Meeting July 19-23 Key West, Florida. Program p. 44 (Abstract), (with
C. B. Calloway).

1987. Introduction to Symposium on Deep-Sea Hydrothermal Vents and Cold- Water Seeps. 153rd National Meeting of the American Academy for the
Advancement of Science, Chicago, 14-18 February, Abstracts of Papers p. 21.

1988. Biodeterioration — Multidisciplinary, collaborative research, [in) M-F. Thompson, R. Sarojini and R. Nagabhushanam (eds.) Marine Biodeterio-
ration— Advanced Techniques Applicable to the Indian Ocean. Bombay. India Oxford and IBH Publishing Co. PVT. LTD. pp. 3-12.

1988. Biodeterioration — Multidisciplinary, collaborative research, [in) M-F. Thompson. R. Sarojini and R. Nagabhushanam (eds.) Marine Biodeterio-
ration— Brooding in the Teredinidae (Mollusca: Bivalvial. Bombay. India Oxford and IBH Publishing Co. PVT. LTD. pp. 215-226 (with C. B.
Calloway).

1988. Recruitment of marine invertebrates to hard substrates at deep-sea hydrothermal vents on the East Pacific Rise and Galapagos spreading center.
Deep-Sea Research 35( 10/1 1 ):183-V1849 (with C. L. vanDover. and C. J. Berg).

1988. Wood, phytoplankton. dissolved organic material and nitrogen in teredinid nutrition (Mollusca: Bivalvia: Teredinidae). [in] M-F. Thompson and
N. Tirmizi (eds.) Marine Science of the Arabian Sea. Proceedings of the International Conference — Washington, D.C.; Institute of Biological
Sciences, pp. 585-606.

1988. Cellulolytic nitrogen-fixing bacteria in the Teredinidae (Mollusca: Bivalvia). ]iii| Biodeterioration 7; pp. 743-748. (Dr. Houghton, R. N. Smith and
H. O. W. Eggins. Editors).

1989. The Pholadacea [in] Fauna of Australia. P. Beesley Ed.

1989. The Genera Martcsia and Ligiiopliolas in the Indo-Pacific. (Mollusca: Bivalvia: Pholadidae) Ophelia 3(.)(3):I55-156. (with L. N. Santhakumaran).

1990. Xylophile ostracoila in the deep-sea. Proc. Aberwystwyth conference on ostracods (with P. L. Steineck. R. F. Maddocks. G. Coles and R. Whatley).
[in) Ostracoda and Global Events, pp. 307-319 (R. Whatley and C. Maybury. Editors).

1990. Species Richness and Diversity of Algal-A.ssocialed Micromolluscan communities from Sao Miguel. Acores. Acoreana.

1990. Supplement: pp. 39-58 (with R. C. Bullock, and R. A. Fralick).

1990. Bivalves of hydrothermal vents and reducing sediments. Fourth International Congress of Systematic and E\ciluiionary Biology. University of

Maryland: College Park, Maryland. July. 1990. (Abstract) (with E. A. Cobabe).
1992. Characterization and site description of .Solemya horealis. (Bivalvia; Solemyidae). another bivalve, bacteria symbiosis. Marine Biology 1 12:601-

613. (with N. M. Conway, B. L, Howes, J. E. Capu/zo, and C. M. Cavanaugh).
1992. Types and Prevention of Biodeterioration in the Deep Sea. Indo-United Slates Meeting on Recent Developments in Bioloiilmg Control. Bangalore.

(Abstracts Plenary Session; C).
1992. Deep Sea Wood Borers and Ancient Wrecks. American Malacological Union Bulletin. Annual Meeting August 2-7. Sarasota. Florida (Abstract

p. 38).

Jaiimal of Shellfish Rc.winh. Vol. 19, No. 1. 13-14. 2000.

Kenneth Kendall Chew
Honored Life Member

Dr. K. (Ken) K. Chew, is a recognised authority in the field of molluscan biology who has contributed extensively to invertebrate
research and the continuing development of the shellfish industry on the west coast of North America. His contributions in the field of
molluscan aquaculture are recognised world wide and his advice and consultation are frequently sought by industries in many countries.

Ken was born in Red Bluff. California in 1933 and received his elementary and secondary education there. While growing up, he
worked in the family restaurant where he learned the fine art of Chinese cuisine. Those of us who have tasted Ken's cooking can attest
to the fact that he mastered this fine art.

He obtained his B.A. from Chico State College in 1955 and then decided to attend the School of Fisheries of the University of
Washington and become a fisheries biologist. Ken received a fellowship to begin graduate work on trout and salmon but Dr. Van Cleve
sent him to the Washington State Department of Fisheries Shellfish Laboratory at Brinnon for the summer. He became involved in
shellfish work at the lab and decided that his future lay with invertebrates, mainly molluscs, rather than fish and he entered the world
of molluscan biology. He obtained his M.S. degree in 1958, studying the food preference of the Japanese oyster drill, and his PhD in
1962. The title of his PhD thesis was, "The growth of a population of Pacific oysters, Crassostrea gigas, when transplanted to three
different areas in the state of Washington." His supervisor was Al Sparks, a former president of the National Shellfisheries Association.

After recei\ing his PhD. Ken joined the staff of the School of Fisheries at the University of Washington and has remained there since.
He has held several positions at the School and has taught a variety of invertebrate courses. At present he is a Professor in the School
of Fisheries. In 1989 he became the Director of the Western Regional Aquaculture Center (WRAC) which is one of five aquaculture
centers designated by the U.S. Department of Agriculture to foster development of aquaculture in the United States. He resigned that
position in 1996 to be the interim director of the School of Fisheries. He was then appointed Associate Dean, College of Ocean and
Fisheries Sciences at the University of Washington in 1998, the position he holds presently. In addition to other duties. Ken is now busily
engaged assisting with expansion of the College and seeking support for the College from industry.

Ken's research interests cover a wide spectrum that include shellfish biology and aquaculture, paralytic shellfish poisoning, and
problems related to baseline ecological studies involving benthic intertidal and subtidal invertebrate communities. He has published over
100 papers on a wide range of shellfish subjects in scientific Journals. Technical Reports. Conference Proceedings, chapters of books
and in columns of trade publications.

13

14 Honored Life Member: Kenneth Kendall Chew

Teaching and maintaining a close rapport witln students has been an important part of Ken's life and he has inspired many students
to continue with studies in molluscan biology. During his career, about 100 students obtained graduate degrees under his supervision.
In recognition of his outstanding teaching ability he received the 1993 Distinguished Undergraduate Teaching Award from the College
of Oceans and Fisheries Sciences at the University of Washington.

Throughout his professional career. Ken maintained a close working relationship with the molluscan shellfish industry, particularly
the industry in the Pacific Northwest. Much of his research and that of his graduate students focussed on finding solutions to problems
to aid development of the industry. The present healthy state of the shellfish industry in the Pacific Northwest is due in a large measure
to the efforts of Ken Chew and his co-workers. His talents and devotion to the shellfish industry were recognised when he was made
Director of WRAC. In this position he devoted considerable time and energy testifying before Congressional Committees in Washington,
DC on issues related to development of aquaculture.

Ken's influence in the shellfish industry has not been confined to the Pacific Northwest. He has provided advice and consultation to
many countries throughout the world including; Australia, Canada, China, Chile, Japan, Thailand, Taiwan, Hong Kong and the
Philippines. He has lectured on molluscan biology and culture in many countries. He was an invited guest lecturer in China and in 1987
was appointed for life as a visiting professor at Shandong College of Oceanography.

Ken has been actively involved with the National Shellfisheries Association since he first joined in 1958. He served on the Board,
was Vice President from 1970-71, President from 1971-72 and served as an Associate Editor for the Journal of Shellfish Research from
1989-92. He has organized annual meetings held in Seattle. Another important function Ken has undertaken is to arrange Chinese
Dinners at annual meetings. Many of us have enjoyed the fine cuisine and companionship that these evenings have afforded.

In addition to his involvement with the parent National Shellfisheries Association. Ken has played a major role to preserve and foster
the as.sociation between the Pacific Coast Oyster Growers Association and the West Coast Section of the National Shellfisheries
Association. From 1975-1990 Ken was the main reason this association survived and he devoted considerable time and energy into
preserving this close association between industry and the scientific community of NSA. It is now a large and dynamic association and
serves as an excellent forum for people from industry, government, and academia to come together and discuss shellfish work and
problems. Many students have presented their first paper at these meetings. The present healthy state of this association is a monument
to Ken's organisational skills and abilities.

As a result of his work and association with NSA, Ken was awarded the first David H. Wallace Award given by the Association in
1982 for his dedicated service in promoting research, understanding and co-operation among shellfisheries scientists, culturists, man-
agers, producers and regulators. In further recognition of his contribution to NSA, he was elected to Honored Life Member in 1989.

Ken has been active in other organizations as well. He was a member of the Board of the World Aquaculture Society from 1973-76,
President in 1977 and an associate editor of the Proceedings of the Society from 1985-89. He was editor for the North American Oyster
Workshop that appeared as a special publication of the World Aquaculture Society in 1983. He received an Honorary Life Member award
from the World Aquaculture Society in 1995. At present he is a columnist for Aquaculture Magazine.

Ken is an avid sportsman and, when time pemiits, relishes hunting and fishing. He is an excellent taxidermist and has mounted several
species of birds he collected. He is a keen hand ball player and more than one meeting has been delayed so he could complete a game
of hand ball.

Along with all his shellfish activities. Ken has found time to be an exemplary family man. He and his wife. Maegan. have raised four
children and now have three grandchildren. They now have time to relax and enjoy their family and the view of Puget Sound from their
wonderful house in Seattle. The shellfish world and NSA owe much to Ken Chew for his past contributions and his friends and
co-workers know his influence will continue to be felt for many future years.

Neil Bourne

Department of Fisheries and Oceans

Pacific Biological Station

Nanaimo, British Columbia V9R 5K6

Canada

Journal oj Shuiljhh Research. Vol. 19, No. 1, 15-16. 2000.

.t >^

Victor Samuel Stuart Kennedy
Honored Life Member

Dr. "Vic" Kennedy, a long time NSA member. Vice President (1988-1989). and President (1990-1991). was born in Ireland in 1942.
Although long established in the U.S., he still maintains a Canadian, United Kingdom citizenship. His early education was at Sir George
Williams University in Montreal. Canada, where he received his B.Sc. in zoology (1962). He continued graduate education at Memorial
University at St. John's. Newfoundland, with a M.S. in fisheries biology ( 1964). Vic then entered the University of Rhode Island for his
Ph.D. where Dr. Paul Saila was his major professor.

I first remember Vic by a phone call in 1967. whereby he introduced himself and said Dr. Saila suggested he call me because of our
Chesapeake work in thermal ecology. He expressed an interest in doing the same with a shellfish species at our Chesapeake Biological
Laboratory's field station at Hallowing Point about 20 miles up estuary from CBL. I invited him down and vividly remember our lunch
at old famous Shorter' s Restaurant in Benedict on the Patuxent Estuary. He impressed me with his quick mind, familiarity with the
literature, and obvious intense interest in aquatic and shellfish ecology. I hired him on the spot and offered a pre-doc position. I called
Dr. Saila, (an old friend that first befriended me as a graduate student at an American Fisheries Society meeting) afterward. I thought
a graduate student accepted by Saul was surely good enough for me too.

Vic completed his Ph.D. on the role of temperature on the soft shell clam. Mya arcnaria. in 1970 and has maintained a peripatetic
professional career. After completing his Ph.D.. he was visiting Assistant Professor at Chapman College in California where among other
activities he spent two semesters traveling the world and teaching on board their "World Campus Afloat" vessel. He came back to
Maryland in 1972-73 to continue thermal research, then left in 1973 for a Post-Doc fellowship at the University of Canterbury in New
Zealand where he taught and completed research on mussels. In 1974. a Post-Doc was accepted at Newfoundland Biological Station in
St. John's where he investigated the role of arsenic in a marine food web and on benthic soft bottom communities.

In 1976 he returned to the University of Maryland System's Horn Point Laboratory as an Assistant Professor and continued his
teaching efforts as well as benthic ecology research. Again he left the Maryland area in 1983. and spent a sabbatical year as a W. F. Jones
Fellow at St. Francis Xavier University in Nova Scotia where he continued his benthic research and taught a marine ecology course. In
1984 he returned to the Horn Point Laboratory, was promoted to Full Professor in 1986 and named Assistant Director in 1989. Again
he left Maryland on a sabbatical and served as visiting Professor on board the .S"5 Universe Campus. University of PittsburgI ship, for
the Semester at Sea Program. Vic has remained at Horn Point since! Throughout all this substantial traveling (40 countries), teaching

15

16 Honored Life Member: Victor Samuel Stuart Kennedy

and current administrative duties. Vic hias continued a vigorous research! program dealing witli shiellfish reproduction and larval behavior,
as well as crustacean and fish foraging behavior as reflected in the selected publications listing. His activities have covered both the
littoral and sublittoral benthic habitats and communities. His long term interest in thermal ecology has now evolved into the global
climate change arena.

Vic has over 45 journal refereed publications, over 10 chapters in books and conference proceedings and 5 written or edited books.
He enjoys a special reputation for his publication efforts dealing with morphology, biology, ecology, and management history of the
eastern oyster. Crassoslrea virgiiiica. A most important current activity is completing editorship of a 13 chapter book on blue crabs,
which he has been working on with Dr. L. Eugene Cronin. (See Vic's In Memoriam to Dr. Cronin in J. Shellfish Res. 18(1): 1-3. 1999).
Another substantial service he has provided to the research and management communities are publications of 5 extensive bibliographies
that have covered the world's literature in their respected areas.

Concerning professional societies. Vic has assumed numerous responsibilities over the years, including President of the Atlantic
Estuarine Research Society, with the aforementioned National Shellfish Association and a governing board member of the Estuarine
Research Federation. He has had editorial responsibilities for the Transactions of the American Fisheries Society, American Malaco-
logicai Bulletin, and Estuaries, among others. Vic's service also includes numerous requests for research propo,sal reviews by NSF, Sea
Grant. Hudson River Foundation, and the Smithsonian Institution. Beyond his usual numerous editing services, he regularly undertakes
requests for reviews on books dealing with aquatic and coastal habitats and processes. This extensive editing experience he has translated
to a very popular graduate course entitled "Scientific Writing and Communication" in which his last class had 38 students, an almost
unheard of number in a graduate course.

In addition to the W. F. Jones Fellow honor he also won as NSA Thurlow C. Nelson award in his junior investigator days (1968).
was noted for outstanding service by AFS. and given an Honored Life Member Award by NSA in 1995.

Vic shows no sign of slackening in his science enthusiasm and his very active and diversified professional involvements. Indeed, with
his two children off in Canada, one in the creative arts and the other with her family working with the native Inuits on Baffin Island,
I suspect he may even pick up the pace if his wife Debbie will allow!

Joseph A. Mihursky

Professor

Chesapeake Biological Laboratory

Journal oj Shellfish Reseuich. Vol. 19, No. 1. 1 7- IS, 2000.

Sammy M. Ray
Honored Life Member

The scientific contributions of Dr. Sammy Ray to oyster disease research are widely acclaimed, due in no small part to the diagnostic
method he developed to detect the disease agent Dennocystidiwn mariiutm. Dr. Ray was one of a handful of investigators in the early
1950's to explore this new oyster disease found in the Gulf of Mexico. Now the disease agent is called Perkinsiis inarimis and molecular
techniques can be used to specifically diagnose the protozoan pathogen. Nonetheless, the highly reliable diagnostic technique developed
by Dr. Ray is still the most widely used in oyster disease studies.

Dr. Ray was bom in Mulberry KS, attended Mississippi Delta Junior College. Louisiana State University, and received his M.A.
(Biology. 1952) and Ph.D. (Biology. 1954) degrees at Rice University in Texas. His postgraduate career began with the U.S. Fish and
Wildlife Service as a Fishery Research Biologist and he joined the Texas A&M staff in 1957 at the Research Foundation Laboratory in
Grande Isle, LA. He became an Associate Professor (1963) in Oceanography and Wildlife and Fisheries Science and was named Director
of the Marine Laboratory at Galveston. As he reached Full Professor (1972), Dr. Ray was named Head of the Department of Marine
Sciences. Since then he has held positions as Dean of the Moody College of Marine Technology and interim President of Texas A&M
University at Galveston. Dr. Ray officially retired in 1990, but remains active as an ad\isor and coordinator of student programs and
several community outreach programs.

Several academic honors have been awarded to Dr. Ray, including a Faculty Distinguished Achievement Award in Research at Texas
A&M University at Galveston (TAMUG). the William Paul Ricker Award for Distinguished Faculty-Staff Achievement (TAMUG), a
Distinguished Alumnus Award from the Mississippi Delta College, and a Piper Professor Award. He was awarded a lifetime honorary
membership in the National Shellfisheries Association at the 1990 meeting in Maine.

Dr. Ray has been a reliable source of scientific information and advice for the State of Texas for many decades. He remains actively
engaged in the interpretation of scientific knowledge for competent management decisions related to oyster and shrimp fisheries in the
Gulf of Mexico. He has, over the last 10 years, participated in both the Joint Interim Committee on the Texas Shrimp and Oyster Industry
and the Gulf of Mexico Fishery Management Council. Dr. Ray is a past chair of the Scientific and Technical advisory Committee for
the Galveston Bay National Estuary Program and is a member of the Board of Trustees of the Galveston Bay Foundation.

17

18

Honored Life Member: Sammy M. Ray

Perhaps the most rewarding achievement of this exceptional career is the initiation of Sea Camp, "a hands-on marine adventure" for
summer students aged 10-16. currently sponsored by TAMUG and the Texas Sea Grant College Program. Students attending the 5-day
camps are given the opportunity to explore the Galveston Island area in research vessels, visit laboratory facilities and use scientific
equipment to study marine organisms. Dr. Ray served as Director of the Sea Camp until 1993 and. in a similar capacity, is the Director
of the Community & Youth Program for TAMUG. Dr. Ray and his wife Charlotte, an accomplished pianist now playing organ for the
St. Luke's Episcopal Church, have four children and reside in Galveston.

William Fisher

EPA Laboratory

GB/ERL

Sabine Island

Gulf Breeze. FL 32561

Journal of Slu'llfish Research. Vol. l^. No. I. l9-:2. 2000.

HABITAT AND REPRODUCTIVE BIOLOGY OF ANGELWINGS,
PHOLAS ORIENTAL! S (GMELIN)

LIBERATO V. LAURETA AND EVELYN T. MARASIGAN

Institute of Aqiiacultiire

College of Fisheries

University of the Phillipines in the Visayas

Miagao, lloilo, Philippines 5023

ABSTRACT The anaelwina. Pholas orientaUs (Gmelin) is indigenous to the coastal waters of the Provinces of Negros Occidental.
Caniz and Iloilo in Central Philippines. Thev burrow into either muddy sand substratum in the littoral zone or compact bluish-gray
muddv sand in the sub-littoral zone. They burrow to a depth of over 0.3 m and once extracted can never return. Specimens studied were
invanablv dioecious without apparent sexual dimorphism. Sexual mawrity is reached at a shell length of 59 mm and 64 mm for males
and females respectivelv. Each sexuallv mature individual possesses a gonad that is imbedded in the ventral side of the viscera. Both
male and female gonads are arborescent in form and have the same coloration. Samples collected from Barotac Nuevo, Iloilo showed
that the peak of spawning occurred from June through October and gametogenesis started in October.

KEY WORDS: Pholas orientaUs. angelwing. reproductive cycle, gonad, spawning

INTRODUCTION

The angelwing. Pholas orientaUs Gmelin, is one of the species
of the family Pholadidae found in the Philippines. The other spe-
cies are: Barneci dilatata. B. manillensis. and Martesia striata.
Pholas orientaUs is edible and is marketed either fresh or dried in
Hongkong (haw chung). Malaysia (sipiit selat batu). Thailand (hoy
pirn), and Philippines (diwal) (Ablan 1938; Davidson 1976; Saraya
1982; Young and Sema 1982; Tokrisana et. al. 1985; Amomjaru-
chit 19881. It has a sweet, juicy and tender taste, making it one of
the most highly sought bivalves in Central Philippines. However,
indiscriminate harvesting has resulted in the depletion of most of
the natural beds.

To date, the study of Ablan (1938) contains the only available
information on the ecology and utilization of this species. To re-
habilitate the depleted P. orientaUs beds, detailed ecological and
biological information is required. According to Rosell ( 1979). any
attempt to manage the resource in the absence of baseline infor-
mation is an exercise in futility. Thus this study was conducted to
describe habitat and reproductive biology of the species.

MATERIALS AND METHODS

Habitat Adaptation

The study sites were Barotac Nuevo, Iloilo (122°47'N and
10°55'E) along Guimaras Strait and Roxas City. Capiz ( 122°45'N
and 1 l''37'E) adjoining Pilar Bay. both in Central Philippines (Fig.
I). Ecological data from five random stations in each area were
monitored during the study period. The grain size characteristics of
the bottom sediments were determined after the procedure de-
scribed by Buchanan (1971). Water temperature was measured
using a calibrated laboratory thermometer and salinity was moni-
tored using a refractometer. The pH of the water was determined
using a pH meter. Monitoring of the environmental parameters was
conducted from May 1994 to August 1995 at the Barotac Nuevo
site and August 1996 to July 1997 in the Roxas City area.

Determination of Reproductive Biology

The specimens (n = 6-20) used for the study on reproductive
biology were collected every month (May 1994 to August 1995)

from the waters of the Barotac Nuevo site. Specimens were
brought to the laboratory, where the size lengths were measured
using a caliper, then shucked, and the gonads dissected. A portion
of the gonad was examined with a Nikon Optiphot microscope to
determine the sexes. The stages of maturity and gametogenic
cycles were determined from histological preparations. Permanent
mounts of the gonads were prepared following the modified Bell
and Lightner (1989) method. The description of the gonadal stages
were made following developmental stages for other clams (Jones
1981; Nash et al. 1986; Hesselman et al. 1989; Shafee and Daoudi
1991; Ponurovsky and Yakovlev 1992).

RESULTS

Habitat Adaptation

The characteristics of the two natural beds of Pholas orientalis
in Central Philippines are shown in Table 1. P. orientalis from
Barotac Nuevo were found to burrow in compact muddy sand
(particles < 0.25 mm) covered with a thin layer of silt in littoral
areas. No specimens were found in the sandy substratum of the
littoral zone and on the deeper water. Few mangrove trees were
found in the area, and seagrasses and macrobenthic algae were not
observed. At Roxas City area, the angelwings occurred in the
sublittoral areas to a depth of 8 m during the highest high tide, the
bed being bluish gray compact muddy sand (coarse silt). No P.
orientalis were found in the sandy mud bottom of the littoral zone.
The natural bed was wholly devoid of mangrove trees and any
rooted plants. In both locations, the angelwings burrowed in the
substrata to a depth of about 0.3 m. On some occasions, burrows
were found almost adjoining and may have met and crossed one
another.

The trends of physico-chemical parameters (temperature, salin-
ity and pH) in the two study sites during the period of observation
are shown in Figure 2. The ambient water temperature in Barotac
Nuevo ranged from 28 °C to 30 X. and did not fluctuate widely.
The lowest recorded temperature readings were in the months ot
December through February. At the Roxas City site, wider fluc-
tuations in water temperature were observed (24 °C to 3 1 °C) with
December to February being the coldest months. At both sites, the
salinity readings were between the range of 30-35 ppt. A pH range

19

20

Laureta and Marasigan

Figure 1. The natural beds { — ) of P. orientalis in Central Philippines.
(•) Barotac Nuevo study site, and (*1 Roxas City study site.

from 7.8 to 8.2 was recorded throughout the study period at both
study areas.

Reproduction

Sex

Out of total 147 sexually matured specimens that were used in
this study, no hermaphroditic individuals were observed. Angel-
wings were dioecious without apparent external dimorphism. Once
sexual maturity was attained, the single gonad was fused or im-
bedded on the ventral side of the visceral mass, extending from the
anterior to the posterior part. Ripe male and female gonads had a
creamy coloration, and were arborescent in form (Fig. 3A).
whereas spent gonads were yellowish and flaccid (Fig. 3B). The
epithelial walls of the viscera also reflected an almost creamy
coloration, causing difficulty in sex differentiation and deterniina-

TABLE 1.

Ecological information on the two natural beds of Pholas orientalis
in Central Philippines.

Maximum
Water
Habilal Depth

Study Site Type (ml

Substrate Type Vegetation

Barotac Nuevo intcrtldal

>1

Roxas City

sublilloriil

muddy sand
(panicles
<0.25 mm)

compaci bluish
gray muddy
sand (particles
<1.00 mm)

mansirove

40

35-

30-

2S-

20

15

10

5

0

Barotac Nuevo

Roxas City

-Temp

-Sal

-pH

AflAftftAflflfla'^ft

MAMJ J ASONDJ FWIAMJ J AASONDJ FMAMJ J
1994 1995 1996 1997

Sampling Period

Figure 2. Some physico-chemical characteristics of water in the two
natural beds of P. orientalis in Central Philippines.

tion of the size of the gonad and gonadal index. Of the same 147
total gonads that were dissected. 78 (53%) were males, and 69
(47%) females.

Sexual Maturity

The specimens examined in this study ranged from 50-156 mm
shell length. Most were found to be sexually mature. The mini-
mum shell lenath of clams containina maturing gametes was 59

Figure .<. /'. orienlatis with (A) ripe gonad, and (B) spent gonad.

Habitat and Reproductive Biology of Angelwings

21

mm and 64 mm for males and females, respectively. The ages of
the angelv^ings, howeser were not determined.

Gonadal Phase and Spawning

The gonadal state in both sexes was divided uito five phases:
early active, late active, ripe, partially spent and spent. The per-
centage occurrence of gonadal stages of male and female P. ori-
ermilis from Barotac Nuevo is shown in Fig. 4A and B. respec-
tively.

Early active stage. Females follicles were empty and lined with
small developing oocytes and oogonia. In males, few and loosely
an'anged spermatozoa were found in the center of the lumen of the
follicle. These conditions occuiTed during the months of October
to January. During this period, 14% of the male and 33% of the
female angelwings population were in the early active phase.

Late active phase. In females, increased numbers of enlarging
oocytes were freed in the lumen of the follicles. Oocytes were
irregular in shape and had a wide range of sizes. In males, sper-
matocytes predominated the basal membrane of the follicle and
numerous spermatids were found at the center of the follicle lu-
men. For both male and female gonads, about 17 to 60% were in
the late active phase during the period December to May.

Ripe pliase. In the female gonad rounded and ripe oocytes (with
nucleus and nucleolus) were free in the lutnen. In males, the gonad
was predominated by mature spermatozoa in the lumen of the
follicle; the acidophilic sperm tails formed lines radiating from the
center of the follicle lumen. Specimens with ripe gonads were

collected during the months of December to July. The percentage
of ripe females ranged from 14.3 to 66.7, whereas males with ripe
gonads ranged between 11.1 and 66.7.

Partially spent. Male gonads had spermatozoa missing in the
central lumen of the follicle. Female gonads contained fewer ripe
oocytes and appeared flaccid. Both types of gonad occurred in the
months of May to October with percentage occurrence at 14.3 to
57.1.

Spent. Empty shrunken follicles were characteristic of spent
gonads. This gonadal phase was observed from the months of June
to October. By October, most of the angelwings had spawned.

DISCUSSION

P. orientalis is a commercially important yet poorly understood
bivalve species found in Central Philippines. An early survey of
Ablan (1938) showed that angelwings are indigenous to the coastal
waters of Hinigaran, Pontevedra. Valladolid, and San Enrique in
Negros Occidental, Philippines. A more recent survey indicated
the presence of this species in the coastal waters of Barotac Nuevo
toward San Dionisio in the Province of Iloilo and in Ivisan, Sapian,
Panay, Pilar, Pontevedra and Roxas City all in the Province of
Capiz (Fortes, unpublished). Apart from these areas no other site
has been identified for the collection of the angelwings in the
country. All the locations were within 3 to 100 miles at each other.

In this study, angelwings were found in either compact muddy
sand or bluish gray muddy sand (with coarse silt) of the littoral or
sublittoral zones. Ablan (1938) found the angelwings in a muddy

MAMJJASONDJFMAMJJA
1994 1995

Sampling Period

B 100

g 80-1

o 60 ^

c

0)

g- 40-1

0)

i 20-

0

IJii

MAMJJASONDJFMAMJJA

D Spent

D Partially Spent

■ Ripe

m Late Active

a Early Active

D Spent

D Rartially Spent

■ Ripe

II Late Active

H Early Active

1994 1995

Sampling Period

Figure 4. Reproductive cycle of P. orientalis In Barotac Nuevo, Central Philippines. Relative frequency of gonadal stages of (A) male, and (B)
female between March 1994 to August 1995.

22

Laureta and Marasigan

coastal land of Negros Occidental. A related species. Cyrtopleura
costata has been observed to inhabit the sandy mud substratum in
shallow waters from southern Massachusetts. USA. to Brazil
(Turner 1954: Abbott 1974; Rios 1973). No clear explanation
could be offered for the limited distribution of angelwings in the
Philippines, and their contrasting ecological habitats (i.e.. type of
bottom sediments, water depth).

The pholads are capable of burrowing to a depth over 0.3 m
(Ablan 1938; Allan 1959; this study). They live in the burrows for
life (Allan 1959). and once extracted from their lodge they are
unable to return. The burrowing ability is necessary to protect
themselves from predators and the adverse effects of the physical
environment as their shells are fragile. For C. costata. they begin
burrowing after larval settlement, and recorded effective burrow-
ing size was at a mean shell length of 1 1.7 mm (Gustafson et. al.
1991). Larger individuals (> 15 mm) of the same species were
unable to rebury and had to be manually placed beneath the sedi-
ments during field-planting. However, effective burrowing size for
the P. orientalis is not known yet.

Angelwings seem to have an extended annual breeding cycle.
where initiation of gametogenesis begins almost after spawning. It
was observed that sizes of specimens had no effect on the timing
of gametogenesis. Small or large specimens, as long as they are
sexually mature exhibited almost simultaneous gametogenesis.
Gametogenesis was observed in the months of October to January.
The month of October was period when most of the clams were

partially spent or spent. The peak of spawning occurred in the
months of June and October, at onset of the rainy season in the
Philippines. Chanley and Andrews (1971) reported the spawning
period from May through September for C. costata from Virginia.
USA. whereas specimens from subtropical Florida were ripe in the
summer months of June through August. The cyclical reproductive
pattern observed in P. orientalis. however, cannot be definitely and
clearly related to temporal changes in temperature and salinity.
The lack of effect of temperature on the reproductive cycle was
similarly observed on venerid clams like Megapitaria auranliaca,
M. squalida. and Dosinia ponderosa from Bahia Zihuatanejo.
Mexico (Baqueiro and Stuardo 1977 cited by Garcia-Dominguez
et al. 1998). and the giant reef clam Periglypta multicoslata in Isia
Espiritu Santo. Baja California Sur. Mexico (Garcia-Dominguez et
al. 1998).

ACKNOWLEDGMENTS

We wish to thank the Fisheries Sector Program of the Depart-
ment of Agriculture and the Sangguniang Panglungsod and the
Mayor of the City Government of Roxas City. Philippines for
funding this work. Special thanks are due also to the lA staff
particularly to Ms. Janet O. Fernandez. Ms. Jane Apines and Ms.
Shirley Miagao; and the fisheries staff of LGU-Roxas City, par-
ticularly Mrs. Belinda Garido. for their technical assistance. We
sincerely thank Dr. Amulfo Marasigan for improving the manu-
script.

LITERATURE CITED

Abbott. R. T. 1974. American Seashells. 2"" ed. Van Nostrand Reinhold,
New York. 663pp.

Ablan, G. L. 1938. The diwal fishery of Occidental Negros. Philipp. ./. Sci.
66(3):379-385.

Allan. J. I9.';9. Australian Shells. The Griftln Press. Adelaide, pp. 354-356.

Amornjaruchit. S. 19S8. Economically important molluscan shellfish of
Thailand. In: McCoy, E. W. and T. Chongpeepien. (Eds.). Bivalve
Mollusc Culture Research in Thailand. ICLARM Tech. Rep. 19. De-
partment of Fisheries. Bangkok, Thailand: International Center for Liv-
ing Aquatic Resources Management, Manila, Philippines: and Deut-
sche Gesellschaft fur Technische Zusammenarbeil (GTZ) GmbH, Es-
chborn. Federal Republic of Germany, pp. 1-8.

Baquierio. E. and J. Stuardo. 1977. Observaciones sobre la biologia, eco-
logia y Explotacion de Megiipitaria aiiruiilimu (Sow 1835), M.
sciuulidci (Sow 1835) Dosiniu ponderosa (Gray 1838) (Bivalvia: Ven-
eridae) de la Bahia de Zihulanejo e Isia Ixtapa, Gro. Mexico. An.
Centro Cienc. Del Mar y Limnol. Univ. Nat. Auton. Mexio 4:161-208.

Bell. T. A. and D. V. Lightner. 1989. A handbook of normal penaeid
shrimp histology. World Aquaculture Society, pp. 58-63.

Buchanan. J. B. 1971. Measurements of the physical and chemical envi-
ronment, pp. 30-58. In: Holme. N. and A. D. Mclnlyre (eds.). Methods
tor the Sludy of Marine Benthos. IBP Handbook 16. Blackwell Scien-
tific Publication.

Chanley, P. E. and J. D. Andrews. 1971. Aids for identification of bivalve
larvae of Virginia. Malacolngia 1 1:45-1 19.

Davidson. A. 1976. .Seafood of South-Easl Asia. Federal Publications.
Singapore. 366 pp.

Garcia-Doniingue/.. F., B. P. Cehallos-Vasquez. M. Villalejo-Fuerle and
M. Arellano-Mailine/. 1998. Reproductive cycle ofthe giant reefclam
Periftlypui iimllicnMiiUi (Sowerby 1835) (Pelecypoda: Veneridael al
Isia Espiritu Santo. Baja California Sur. Mexico. ./. Shclllixh Res. 17(4):
1009-1013.

Gustafson. R. G., R. L. Crewell, T. R. Jacobsen and I). L. Vaughan. 1991.
Larval biology and mariculiure of the angel wing clam. Cyriopleiini
coslalii. Ai/uucullure 95:257-279.

Hesselman, D. M.. B.J. Barber and N.J. Blake. 1989. The reproduclive

cycle of adult hard clams, Mercenaria spp. in the Indian River lagoon.
Florida. J. Slwllfish Res. 8( 1 ):43-I9.

Jones, D. S. 1981. Reproductive cycles of the Atlantic surf clam. Spisiila
solidissiiiHi. and the ocean quahog Arcliva islnndiea off New Jersey. ./.
ShellJ'Lsh Res. l(l):23-32.

Nash, W. J., R. G. Pearson and S. P. Westniore. 1986. A histological study
of reproduction in the giant clam, Tridacna .^igas in the North-Central
Great Barrier Reef In: Giant Clams in Asia and the Pacific. Coplana.
J. W. and J. S. Lucas (eds.) pp. 86-96.

Ponurovsky. S. K. and Y. M. Yakovlev. 1992. The reproductive biology of
the Japanese littleneck. Tapes philippinanim (A. Adams and Reeve
1850) (Bivalvia: Venerididae). J. Shellfish Res. 1 1(2):265-277.

Rios. E. C. 1975. Brazilian Marine Mollusks Iconography. Fundacao Uni-
versidade de Rio Grande Centro de Ciencias do mar Museo
Oceonografico. Rio Grande, Brazil, 331pp.

Rosell. N. C. 1979. A sludy on the biology and ecology of PUwwia pla-
centa Linne. Natural and Applied Science Bulletin. 3 1 {3^1:203-25 1.

Saraya, A. 1982. Thailand. In: Davy. F. B. and M. Graham (eds.). Bivalve
Culture in Asia and the Pacific, Proc. Workshop held in Singapore.
16-19 February 1982. Int. Dev. Res. Center, Ollawa, Ontario. Canada,
pp. 73-78.

Shafee, M. S. and M. Daoudi. 1991. Gametogenesis and spawning in the
carpel shell clam. Riidilapes deriissalus (L.) (Mollusca: Bivalvia). from
the Atlanlic coast of Morocco. .'Kqiutculttire and fisheries Managenieul
22:20.3-216.

Tokhsana, R., S. Tugsinavisuili, M. Muangkoe and S. Kao-iun. 1985.
Marketing system of shellfish products. Asian Fish. Soc. Res. Network
Thailand and Dept. Agric. Econ., Kasctsarl University. Bangkok.
264pp. (in Thaii.Turner, R. D. I9.'i4. The family Pholadidae in the
western Allanlic and ihe easlern Pacific. Part I. Pholadidae. Johnsonia
3:1-63.

'loung. A. and E. .Serna. 1982. Philippines. In: Davy, F. B. and M. Graham,
(eds.). Bivalve Culture in Asia and the Pacific: Proc. Workshop held in
Singapore. 16-19 Fcbniary 1982. Int. Dev. Res. Center, Ottawa. On-
tario. Canada, pp. 55-68.

Joimuil of Shellfish Reseanli. Vol. \9. No. 1. 23-28, 2000.

INFLUENCE OF DIET ON SURVIVAL, GROWTH, AND PHYSIOLOGICAL CONDITION OF
FINGERNAIL CLAMS MUSCULIUM TRANSVERSUM

TERESA J. NAIMO,' W. GREGORY COPE," EMY M. MONROE,'
JERRY L. FARRIS,^ AND CRISTIN D. MILAM'

^U.S. Geological Siin-ey. Upper Midwest Environmental Sciences Center,

2630 Fanta Reed Road,

La Crosse, Wisconsin 54603
'North Carolina State Uriiversity,

Department of Toxicology, Box 7633,

Raleigh, North Carolina 27695
^Arkansas State University,

Department of Biology,

P.O. Box 599,

State Universir\; Arkansas 72467

ABSTRACT The effects of diet and lahoratory holding time on survival, growth, and physiological condition of fingernail clams
Musculiwn transversum were evaluated in a 1 12-day study. The diets included a commercial oyster diet, a suspension of commercial
rabbit pellets, a suspension of fine, organic-rich sediment, and a complete sediment renewal every 14 days. Sediment and clams were
obtained from a relatively uncontaminated site in the Upper Mississippi River. The e.\perimental design consisted of 18 370-mL
beakers per diet, each containing 5 cm of surficial sediment and 15 clams. Survival of clams was measured daily in each unit. Three
units from each diet were randomly removed on days 7, 14, 21. 28, 56, and 112. and clams were measured for shell length. Glycogen
and cellulase activity were measured in composite samples (5 clams per sample) at each of the six time intervals. Cellulase activity
did not vary among diets or with time. Survival, growth, and glycogen varied significantly among diets, and glycogen concentrations
varied with time, regardless of diet. Clams exposed to the two sediment diets were 2.4 times more likely to survive than clams exposed
to the commercial diets. Survival of clams in all diets exceeded 80% through day 2 1 . Although clams maintained an acceptable survival
rate for 21 days, their physiological condition was compromised much earlier, given that glycogen reserves were reduced by 14-54%
after only 7 days. Thus, laboratory tests with fingernail clams should include physiological measures, in addition to survival, to ensure
that clams are in suitable condition before and during testing.

KEY WORDS: Diet. Muscidium transversum. survival, growth, biomarker

INTRODUCTION

Fingernail clams are an important component in the benthic
invertebrate community of many large rivers and. in the Upper
Mississippi River, have undergone periodic, pronounced declines
in abundance in recent decades (Wilson et al. 1995). For example,
densities in Pool 19 (near Keokuk, lA) averaged 32,000/nr in
1985 and progressively declined to 0 in 1990, and river- wide re-
covery has been slow. Toxicity of bulk sediment or pore water has
been suggested as a factor contributing to the decline in fingernail
clams in the river (Wilson et al. 1995). In particular, concentrations
of un-ionized ammonia in sediment pore water from the Upper
Mississippi River often exceed concentrations demonstrated to in-
hibit growth of tlngemail clams in laboratory studies (Frazieret al.
1996). To assess these and other potential causes of the decline in
abundance requires that clams be collected from the field, held in
the laboratory, and tested through controlled experimentation.
However, information on the relative condition of clams during
long-term holding and its effect on the outcome of laboratory tests
is lacking (Naimo et al. 2000).

The physiological condition of an organism is dependent upon
its nutritional status (Lanno et al. 1989, Foster etal. 1993). Yet. the
importance of nutrition as a factor modifying physiological con-
dition has been largely overlooked. Data on how the condition of
an organism responds to its nutritional status are critical for un-
derstanding the importance of diet as a variable in designing ex-
perimental studies with benthic organisms.

Recently, physiological indicators of condition such as glyco-
gen concentration and cellulase activity have been used to assess
the relative health of bivalve mollusks (Hemelraad et al. 1990.
Haag et al. 1993, Farris et al. 1994, Naimo et al. 1998). Glycogen
is the most readily available storage form of glucose in many
animals, including freshwater mussels. As such, glycogen concen-
trations have been used successfully as an indicator of physiologi-
cal condition in unionid mussels after exposure to contaminants
(Hemelraad et al. 1990) and after infestation by zebra mussels
(Haag et al. 1993). Similarly, cellulase activity is an indirect mea-
sure of feeding because it measures the rate of breakdown of
complex sugars into simple molecules (Farris et al. 1988). Exten-
sive use of cellulase activity in monitoring programs for molluscs
has shown that responses at the biochemical level can be measured
where pollutants or stress first exert their effect (Beeby 1993.
Milam and Farris 1998). In these studies, the predictive capability
of the enzyme assay has been compared with extensive testing of
more traditional biological endpoints in toxicity assessments. Con-
trolled laboratory and field exposures have provided evidence that
reductions in enzyme activity are related to the eventual survival of
the animal and to more subtle changes that occur in filtration,
growth, and bioaccumulation rates (Farris et al. 1994, Milam and
Farris 1998).

We examined survival, growth, and physiological condition in
clams provided different food sources in a 112-day laboratory
study. Our specific objective was to evaluate the effect of diet on
the survival, growth, and physiological condition of fingernail

23

24

Naimo et al.

clams MuscLilium transversum (Say 1829). Furthermore, because
we were interested in the transferability of these data to standard-
ized tests with benthic invertebrates, we examined differences in
survival, growth, and physiological condition between clams fed
two commercially available diets (easily reproducible, but a non-
indigenous diet) and two diets containing sediment (not as repro-
ducible, but more indigenous).

MATERIALS AND METHODS

Experimental Design

We obtained about 600 fingernail clams with a Ponar dredge
from Pool 13 of the Upper Mississippi River for use in the labo-
ratory test. During collection, clams were placed in ice chests
containing sediment and water from the river. The water in the ice
chests was aerated and its dissolved oxygen content was measured
at .30-min intervals to maintain concentrations above 60% of satu-
ration. To obtain an estimate of the physiological condition of
clams at this point in time, we obtained an additional 15 clams,
placed them on dry ice in the field, and stored them at -84 °C in
the laboratory before analysis of glycogen concentration and cel-
lulase activity.

The uppermost 5 cm of sediment from a single sampling site in
Pool 7 of the Upper Mississippi River (Lake Onalaska, river mile
704.5) that contained an abundant fingernail clam population was
obtained with a van Veen dredge. Sediment was placed into 4-L
glass jars, held on ice. transported to the laboratory, and stored in
a refrigerator for no more than 5 days before the start of the test.
Three subsamples of homogenized sediment (each 20-25 g wet
weight) were analyzed to describe textural composition (Guy
1969. Plumb 1981) and volatile matter content (American Public
Health Association et al. 1992). Sediments averaged (mean ± 1
standard error [SE]) 4 ± 0.2% sand. 54 ± 2.4% silt. 42 ± 1.8% clay.
and 7.8 ± 0.9% volatile matter.

The experimental unit was a .^70-mL beaker. All experimental
units were placed into one of two 900-L water baths (3 m length
X 0.8 m width x 0.4 m height). Each water bath was partitioned
lengthwise with Plexiglas to provide four compartments, one for
each diet. Eighteen experimental units were randomly allocated
into each compartment. A temperature of 17 ± 2 °C was main-
tained with submersible quartz healers. About 24 h before the
addition of clams. 1 84-1 88 g of surficial sediment (about 4-5 cm)
and 200 niL of well water from the Upper Midwest Environmental
Sciences Center were added to each experimental unit. On day 0.
we randomly allocated 15 clams, each measuring 4-6 mm in shell
length, into each experimental unit.

We measured the temperature. pH. and dissolved oxygen of the
overlying water every Monday. Wednesday, and Friday in five
randomly selected experimental units in each diet. Because finger-
nail clams are particularly sensitive to un-ioniz,ed ammonia
(Hickey and Vickers 1994). we measured concentrations of total
and un-ionized ammonia in three randomly selected experimental
units every 14 days (Fra/ier et al. 1996). On days 7. 14. 2 1 . 28. 56.
and 112. clams from three randomly selected experimental units
from each diet were sieved from test sediments, counted, recorded
as dead or alive, measured for shell length to the nearest 0.1 mm,
and stored at -84°C for later analysis of glycogen concentrations
and ccllulase activity. Glycogen concentrations (Naimo et al.
IWS) and ccllulase activity (Farris et al. 1988) were measured on
composite samples containing five individuals from each experi-
mental unit. Glycogen concentrations were reported as mg/g wet

weight, and cellulase activity was expressed as a product (exocel-
lulase activity times endocellulase activity in [units/g dry
weight]"). One unit of the enzyme is defined as the amount of
enzyme required to liberate 1 mg of reducing sugar equivalent to
that of glucose per hour with carboxymethylcellulose as a sub-
strate.

Diet and Ration

Clams were fed one of four diets daily; two were commercially
available diets, and two were formulated with sediments from the
Upper Mississippi River (sediment diets). The commercial diets
included an oyster diet, which was a mixture of two marine dia-
toms (50% Thatassiosira pseudoana and 50% Skeletoneina sp.)
fed at a rate of about 7(jLL/clam/day (8-10 x 10'^ cells/mL; Pacific
Oyster Diet B. Coast Seafood Company. Quilcene. WA). The sec;
ond commercial diet was a suspension of Kaytee * rabbit feed, with
pellets made largely from alfalfa, fed at a rate of 2.5 mg/clam/day.
The two sediment diets contained organic-rich sediments from
relatively uncontaminated areas in the river and were the same
sediment used as the substrate in all experimental units. One was
a suspension of fine sediment fed at a rate of 2.5 mg/clam/day. and
the other was a complete sediment renewal every 14 days.

The oyster diet, rabbit pellet diet, and suspended sediment diet
were prepared about 2 days before the start of the experiment. The
oyster diet comes in liquid form and was kept refrigerated. The
rabbit pellet and the suspended sediment diets were prepared by
blending 38 g of rabbit pellets or sediment with 400 mL of well
water in a commercial blender for 5 min. The contents of the
blender were transferred into a l.OOO-niL volumetric tlask and
filled to the meniscus with well water. This process was repeated
until we obtained 32 140-mL bottles of each diet. Once a week,
one bottle of food for each diet was removed from a -20°C freezer
and placed into a refrigerator; the quantity of food in each bottle
was sufficient to feed all clams receiving those diets for 1 wk.
Clams in the sediment-renewal diet were sieved from test sedi-
ments every 14 days, and another aliquot of sediment was replaced
into each experimental unit. Sediments for this diet were the same
sediments that were obtained at the start of the test, stored in a
refrigerator until needed.

Statistical Analyses

Survival of clams was assessed by daily counts of dead shells
on the sediment surface. In addition, at the six time intervals in
which clams from three beakers were removed for physiological
measurements, we also made direct mortality estimates; these data
allowed us to check the accuracy of the daily mortality counts.
Because these two estimates agreed more than 90% of the time,
analyses of survival rate were performed on daily survival counts.
We used the Cox proportional hazards model to determine whether
survival rates of clams varied among diets (Cox 1972). To test for
differences in survival between the commercial and sediment diets.
we used the Wald lest of equality (Parniar and Machin 1945).

We analyzed growth, glycogen concentrations, and cellulase
activity with analysis of covariance (ANCOVA). with time in the
laboratory as the covariale. Because most clams did not survive
after day 56. statistical analyses were only conducted until day 56.
Orlliogonal contrasts were used to compare differences in growth
and physiological condition between the corumerclal and sediment
diets when the ANCOVA was significant. We did not record the
shell leuL'th i.-\\' each clam on dav 0; instead, we ensured that all

Influence of Diet on Musculium

25

clams ranged from 4 to 6 mm in length. Because shell length did
not differ among diets at day 1 (P = 0.21). subsequent analyses
were performed on shell length measures from day 7 through day
56. A type I error a of 0.03 was used to reject all null hypotheses.

RESULTS

The quality of the overlying test water was similar among diets
(Fig. 1 ). For example, grand means (averaged over all diets and
time periods) ranged from 15.4°C to 15.7°C for temperature, 8.2 to
8.3 for pH, and 9.7 to 9.8 mg/L for dissolved oxygen. Concentra-
tions of total (range, 0.03-0.13 mg/L) and un-ionized (0.002-0.008
mg/L) ammonia were well below concentrations that adversely
affect fingernail clams in laboratory exposures (Sparks and
Sandusky 1981).

The survival rate of fingernail clams varied significantly among
diets (P = 0.0001). Survival rates were lowest in clams fed the
oyster diet, whereas survival was highest in clams receiving the
sediment-renewal treatment (Fig. 2). For example, survival aver-
aged 44% in the oyster diet, 66% in the rabbit-pellet diet, 73% in
the suspended-sediment diet, and 84% in the sediment-renewal
diet at day 56. By day 112, only 6% of the clams in the sediment-
renewal treatment were alive, and none survived in the other three
dietary treatments.

Survival was significantly greater in clams provided the sedi-
ment diets, relative to the commercial diets (P = 0.0001 ). After 56
days in the laboratory, for example, survival of clams fed the
sediment diets averaged 79%, whereas survival averaged 55% in

100 ■

18 1
17 ■

M

Temperature

16 ■

15 -

%

^

d

^

^

^

^

^

X

o

Z 9

— o— oyster diet
— •— rabbit pellets
— o— suspended sediment
— • — sediment renewal

Dissolved oxygen

20 40 60

Day of experiment

80

Figure 1. Mean temperature, pH, and dissolved oxygen in overlying
test water from five randomly selected experimental units containing
flngernail clams Musculium transversum fed one of four diets daily for
112 days.

>

c
u

Day of experiment

Figure 2. Survival of fingernail clams Musculium transversum fed one
of four diets in a 112-day laboratory test.

clams fed the commercial diets. However, there was little differ-
ence in survival of clams among diets early in the test: survival of
clams in all diets exceeded 80% through 21 days of exposure. A
unique feature of the proportional hazards model is the ability to
calculate a risk ratio, or the estimated hazard of surviving in one
diet versus another. For example, clams provided the oyster diet
were 1.9 times more likely to die than clams fed rabbit pellets
(Table 1). Additionally, clams fed the oyster diet were almost 5
times more likely to die than clams in the sediment-renewal treat-
ment. Furthermore, clams fed the commercial diets were 2.4 times
more likely to die than clams fed the two sediment diets.

The shell length of fingernail clams also varied significantly
among diets (P = 0.02). Clams receiving the sediment-renewal
treatment were significantly larger than clams in the other three
dietary treatments. For example, clams in the sediment-renewal

TABLE 1.

Estimated probability values, risk ratios, and upper and lower 95%

confidence limits from the survival rate analysis in fingernail clams

fed four different diets in a 112-day laboratory experiment.

Lower 95%

Upper 95%

P

Risk

Confidence

Confidence

Contrast

Value

Ratio

Limit

Limit

Oyster diet.

suspended sediment

0.0001

2.6

1.9

3.5

Rabbit pellets.

suspended sediment

0. 1 1 24

1.3

0.9

1.9

Sediment renewal.

suspended sediment

0.0120

0.5

0.3

0.9

Oyster diet, rabbit

pellets

0.0001

1.9

1.9

2.0

Oyster diet, sediment

renewal

0.0001

4.8

4.0

5.8

Rabbit pellets.

sediment renewal

0.0002

2.4

2.2

2.8

Commercial diets.

sediment diets

0.0001

2.4

1.9

3.2

The risk ratio is the estimated hazard of surviving in one diet versus
another diet; for example, clams fed the oyster diet were 2.6 times more
likely to die than clams fed the suspended-sediment diet.

26

Naimo et al.

treatment averaged 4.8 mm in length over the 56-day duration,
whereas clams in the other three dietary treatments ranged from
4.3 to 4.4 mm. Furthermore, the size of clams did not differ be-
tween clams provided the commercial and sediment diets (P =
0.50), nor did shell length vary with time in the laboratory (P ~
0.23; Fig. 3a). At day 7, clams ranged in length from 4.2 to 4.8 mm
and at day 56, they ranged in length from 4.5 to 4.8 mm.

Glycogen concentrations in clams varied significantly among
diets (/* = 0.049: Fig. 3b). In particular, glycogen concentrations
differed between the commercial and sediment diets (P = 0.02).
For example, mean glycogen concentration was 3.5 mg/g in clams
fed the oyster diet and 4.1 mg/g in clams fed the rabbit pellets. In
contrast, glycogen concentrations averaged 2.8 mg/g in the sus-
pended-sediment diet and 3.0 mg/g in the sediment-renewal treat-
ment. However, glycogen concentrations declined significantly
with time in the laboratory, regardless of diet {P = 0.0001). For
example, glycogen concentrations in clams in the sediment-
renewal treatment averaged 4.6 mg/g at day 7 and had declined to
only 2.2 mg/g by day 56. Moreover, because there was no
diet*time interaction (P = 0.49), the response of glycogen with
time was similar among diets. For reference, glycogen concentra-
tions averaged 5.4 ± 0.5 (SE) mg/g in clams when they were
removed from the Mississippi River.

t

•a

00 *

8 I

b

b^/^

— Q-- oyster diet
— •— rabbit pellets
— o— ■ suspended sediment
^-^ — •— sediment renewal

y/^

F^

r^^

— -~^ ^^^~~~~~~---^

Y

1^^-===^==^^

O IT

p u

o a.

16 '

c

12 •

f^,^--^^!

8 ■

J

\J

1 \1T

4 '

\

/^ \ir'>»

___^ I

f

^.^'^

Day of experiment

Figure 3. Mean (a) srowlh, (b) glycogen concenl rations, and (c) eei-
lulase activity in Ungernail clani.s Miisciiliiim Iransvcrsiiin fed one of
four diets in a 1 l2-da\ lahoratorv lest. (Ilycogen (mg/g «et "eight) and
cellulase activity (junits/g dry « eight j') were measured on a composite
of five clams from each of lliree experimental units sampled on days 7,
14, 21, 2S, and 56. Data point al day 0 is the mean (±1 .SE) glycogen and
cellulase in clams at the time they were collected from the Upper
Mississippi River.

In contrast, cellulase activity did not vary among diets (P =
0.12) nor with time held in the laboratory (P = 0.32; Fig. 3c).
Cellulase activity, averaged over the 56-day exposure, ranged from
0.8 to 5.3 (units/g dry weight") in the oyster diet, 0.8 to 4.8 in the
rabbit pellets, 1.1 to 14.7 in the suspended sediment, and 0.6 to
19.8 in the sediment renewal. Likewise, cellulase activity remained
similar throughout exposure (averaged over all diets) and ranged
from 1.6 to 10.5 at day 7 and from 1.8 to 14.3 at day 56. The lack
of significant diet or time effects was presumably due to the large
variance in cellulase activity among replicates. The coefficient of
variation (CV) usually averaged well over 50%, likely obscuring
any diet or time effects. For reference, cellulase activity averaged
7.3 ± 1.6 (SE) in clams when collected from the Mississippi River.

DISCUSSION

Survival of fingernail clams was greater in treatments contain-
ing sediment from the Upper Mississippi River than in treatments
with commercial diets. A similar observation was made by
Gatenby et al. (1996) with juvenile Villosa iris. In a 45-day labo-
ratory experiment, juvenile mussels reared on sediment and algae
had significantly higher survival {6T7r) than juveniles reared with-
out sediment and fed only algae (227^). Although several investi-
gators have observed higher survival rates in molluscs in experi-
ments with sediment, relative to no sediment (Gatenby et al. 1996,
Naimo et al. 2000, present study), the mechanism(s) contributing
to this are largely unknown. It has been hypothesized that the
addition of a food source, along with fine sediments and their
associated resident bacteria, may enhance digestion in molluscs
(Crosby et al. 1990, Naimo et al. 2000). However, the addition of
bacteria common to riverine systems did not improve survival or
enhance growth in laboratory studies with juvenile Villosa iris
(Gatenby et al. 1996). Naimo et al. (2000) hypothesized that physi-
cal contact with sediment may enhance the survival of fingernail
clams relative to exposures without direct sediment contact. They
observed that Miisciiliitm transversiiiii were twice as likely to sur-
vive when provided with direct sediment contact, suggesting that
clams received nutritional benefit from sediment contact by feed-
ing directly on indigenous, sediment-associated food sources.

Although survival of fingernail clams differed substantially
among diets after 1 12 days, survival exceeded SO'/r through day 21
in all diets. In standardized toxicity tests with bcnthic inverte-
brates, 21-28 days is a standard test duration (American Society
for Testing and Materials 1992), and tests are generally considered
unacceptable if survival of control animals is less than 80%. Thus,
in short-term standardized tests with fingernail clams, excessive
mortality in control organisms would not invalidate test results.

Growth of fingernail clams in the laboratory was minimal over
the 56-day duration. Clams in the sediment renewal treatment
seemed to maintain their size, whereas shell growth in clams in the
other diets was variable. Differences in shell growth in the sedi-
ment-renewal treatment, relative to the other diets, may be related
to the volume of available food (i.e., sediment). Clams in the
sediment renewal treatment received about 736 g of sediment over
56 days, whereas clams in the suspended-sediment and labbit-
pcllel treatments received only 2.1 g of food over this duration.
Although food quality as well as quantity are important, the mag-
nitude of the difference in quantity may have contributed to dil-
Icrcnces in growth among diets. In addition, the magnitude of shell
growth observed in our study (0.1-0.6 mm over 56 days) was
sufficicntiv small such that variation in measureinent of shell

Influence of Diet on Muscuuum

27

length could be a major source of variation and uncertainty in tinis
analysis. Thus, future studies should measure individually marked
organisms and should use techniques appropriate for detecting
small changes in size. The lack of shell growth in this experiment
was not unexpected. For example. Gale (1977) observed that
Sphaeriiim tmnsversiiiii maintained in the laboratory in chambers
containing silt from the Mississippi River grew slowly, with a
mean length increase of 1.3 mm after 33 days.

Glycogen concentrations have been used extensively in bi-
valves as an indicator of physiological health (Haag et al. 1993,
Naimo et al. 1998); however, it is unclear how much glycogen is
required for maintenance, growth, and reproduction. In the present
experiment, we documented significant differences in glycogen
concentrations among diets, particularly between the commercial
diets and the sediment diets. However, the pattern in glycogen
concentrations was such that glycogen was elevated in clams fed
the commercial diets, relative to the sediment diets, in contrast to
the patterns in survival. Two alternate hypotheses for the reduction
in glycogen in the sediment diets include ( 1 ) clams were getting
enough nourishment from the sediment for maintenance metabo-
lism but were unable to store glycogen and (2) clams were not
getting enough nourishment from the sediment and were catabo-
lizing carbohydrate stores. Whichever the case, glycogen concen-
trations declined with time in all dietary treatments, suggesting that
health was declining over this time period. Glycogen concentra-
tions declined by 14-54% by day 7 and 50-70% by day 56. rela-
tive to concentrations in clams when they were taken from the
river.

Some researchers have suggested that the benefit of addition of
sediment to juvenile bivalve cultures is to provide resident bacteria

to enhance enzymatic activity (Crosby et al. 1990). However, we
did not observe any enhancement in cellulase activity between
clams maintained in sediment and clams fed commercial diets.
Cellulase activity in clams was highly variable (mean CV = 67%),
making detection of dietary effects at an acceptable statistical level
difficult. To our knowledge, measurement of cellulase activity has
not been previously performed on fingernail clams; thus, further
refinement of methods could reduce variation associated with this
measure.

In conclusion, we observed significant differences in survival,
shell growth, and glycogen concentrations of fingernail clams fed
different diets, implying that some diets were better than others.
However, the general negative slope of most response variables
(survival, shell growth, and glycogen) suggests that clams were
declining in health with time in the laboratory, regardless of diet.
Therefore, a better diet is needed to maintain clams in a healthy
state in the laboratory. Although clams maintained an acceptable
survival rate for 21 days in the laboratory, their physiological
condition was compromised much earlier. Thus, valid short-term
toxicity tests with fingernail clams can be conducted in the labo-
ratory, but their ability to predict toxicity to field populations is
uncertain. Therefore, laboratory tests with clams should include a
physiological measure, such as glycogen, in addition to survival to
ensure that clams are in suitable condition before and during test-
ing in laboratory studies.

ACKNOWLEDGMENTS

Technical assistance in the field and laboratory was provided
by Michelle Bartsch and Peter Rust. Steve Gutreuter provided
statistical guidance.

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Joiinuil of Shellfish Research. Vol. 19. No. 1, 29-34. 2000.

LOSS OF GENETIC VARIATION IN A STRONGLY ISOLATED AZOREAN POPULATION OF

THE EDIBLE CLAM, TAPES DECUSSATUS

KURT JORDAENS,'* HANS DE WOLF,' TANIA WILLEMS,'

STEFAN VAN DONGEN,^ CARLOS BRITO,'

ANTONIO M. FRIAS MARTINS,' AND THIERRY BACKELJAU' ^

' Department of Biology

University of Antwerp (RUCA)

Groenenborgerlaan 171

B-2020 Antwerp. Belgium
'Department of Biology

University of Antwerp (UIA)

Universiteitsplein I

B-2610 Wilrijk. Belgium
' Department of Biology

University of the Azores

Rua da Mae de Dens 58

Apartado 1422

P-9502 Ponta Delgada

Azores, Portugal
^Royal Belgian Institute of Natural Sciences

Vautierstraat 29

B-1000 Brussels, Belgium

ABSTRACT We used allozyme electrophoresis to compare the genetic variation of an introduced and strongly isolated population
of the edible clam Tapes decussariis in the Azores (Lagoa de Santo Cristo. Sao Jorge) with populations from the main range of the
species (Ria and Thau). Observed and expected heterozygosity values, number of polymorphic loci, and mean number of alleles per
locus in the main-range populations fall within the limits reported for T. decussanis and other Venerid clams. In contrast to previous
studies on Venerid clams, we observed no heterozygote deficiencies. In the introduced Lagoa population, we observed a strong
reduction of allelic diversity and expected heterozygosities and an effective population size of only 5.30. The Lagoa population is only
slightly differentiated from populations from the species' main range and may thus be of low "biological value." Exploitation of T.
decussaius could therefore be allowed to continue but must follow strict collection guidelines, especially given that only 15% of the
area is suitable for exploitation. Otherwise, a unique component of the Azorean fauna that also serves as a fishery resource may be lost.

KEY WORDS: Azores, allozymes, founder effect. Tapes decussaius. population genetics, conservation

INTRODUCTION structure before adequate measures can be taken. In this study, we

estimated the effective population size and investigated whether

Small or isolated populations can contribute substantially to genetic variation is reduced in an introduced, isolated population

biodiversity, and the conservation of such populations must be an of the commercial edible clam Tapes decussaius (Linnaeus. 1 758).

important part of any effective Biodiversity Action Plan (Usher The main range of T. deai.ssaws extends from Great Britain in

1997). The genetic effects in small populations are manifold (Har- 'he north to Senegal in the south, along the Iberian peninsula, and

ris 1984. Usher 1987). Small effective population sizes (A',) often '"'0 the Mediterranean to the east (Tebble 1966). Outside its main

show a loss ofgenetic variability (i.e.. founder effects, bottlenecks) ^nge. the species has been introduced in the Lagoa de Santo

caused by genetic drift (Lacy 1987). Apart from losing (rare) al- Cristo. a small and isolated lagoon situated at the north coast of the

leles, small populations often lose common alleles by chance (Nei '^'and of Sao Jorge in the Azores, approximately 1,400 km from

et al. 1975. Simberloff 1988) and may show elevated inbreeding, 'he African/European coasts, where it was discovered for the first

which mav impair reproductive fitness. In addition, the loss of "me in 1967 (Morton 1967). This lagoon has a total area of 0.86

genetic variability may limit the ability of a population to adapt to km- (length, 500 m; width, 250 m: and maximum depth, 6 m) and

changing environments (Frankel and Soule 1981, Thorpe et al. harbors a unique fauna (Morton 1967. Santos 1985. Santos and

1995). Over the long term, these effects may enhance the risk of Martins 1986, Morton and Tristao da Cunha 1993, Morton et al.

extinction (Soule 1987). Effective conservation or management '^98). The lagoon was classified as a Natural Partial Reserve in

plans require a thorough knowledge of the genetic population 1984 on the basis of its unique origin, geology, and the presence of

the edible clam T. decussanis. In 1989. it was also declared a

Special Ecological Area, to safeguard the unique breeding popu-

*Corresponding author. lation of T. decussatus and to maintain the ecological equilibrium

29

30

JORDAENS ET AL.

of the area. Although there is no written record. T. deciissatus was
probably introduced in the lagoon by humans, especially since the
species occurs nowhere else in the Azores (Morton 1967, Morton
and Tristao da Cunha 1993). Moreover, the planktonic stage of the
larvae lasts approximately 10 days, during which larvae are trans-
ported by sea currents over a distance of 10-100 km (Borsa et al.
1991). Larval transport from the main range to the Azores by sea
currents seems therefore unlikely.

At this moment, T. detussauts is the main commercially ex-
ploited species of the lagoon (Fonseca et al. 1995). Santos and
Martins (1987), Santos et al. (1989), and Gon(;alves and Martins
(1991) showed that the population of T. deciissatus in the Lagoa de
Santo Cristo was declining through overexploitation, especially in
the intertidal parts of the lagoon, where clam collection is easy.
The intense fishery resulted in smaller individuals in the intertidal
area. These potential detrimental impacts on the clams and other
species of the lagoon have obliged the Azorean government to
establish a management program for the Lagoa de Santo Cristo.
Therefore, the clam fishery at the lagoon is nowadays closed dur-
ing a period that largely coincides with the breeding season of the
species (May 15 to August 15: Santos and Martins 1987. 1991).
The present research was performed to provide genetic data that
may be relevant for further substantial management of the clam
population.

MATERIALS AND METHODS

Four samples of T. decussatus were collected from three sites:
Lagoa de Santo Cristo (SC: July 1992 and June 1993). Etang de
Thau (Thau: French Mediterranean coast: August 1993). and Playa
do Testal (Ria: Ria de Muros y Noya. Galicia, Spain: December
1993). Specimens were immediately frozen in liquid nitrogen for
transport to the laboratory, where they were stored at -80 °C.

Forty specimens of each sample were surveyed for allozyme
variation with vertical polyacrylamide gel electrophoresis (PAGE).
Individual tissue homogenates were prepared by dissecting speci-
mens in ice-cold distilled water and removing the digestive gland,
the gills, the foot muscle, and the adductor muscles. Each of the
tissues was separately weighted and homogenized in a 20% (w/v)
aqueous sucrose solution (5 |j.L sucrose solution per mg tissue).
Crude homogenates were centrifuged for 45 min at ±27,000 g at
5 °C to obtain clear supernatants for electrophoresis.

PAGE was performed as described by Backeljau (1987. 1989).
Two electrophoretic buffer systems were used: ( I ) Tris/glycine pH
9.0 in the tray and Tris/HCl pH 9.0 in the gels and (2) Tris/citric

acid pH 8.0 in both the tray and the gels. Enzyme staining recipes
were adapted from Harris and Hopkinson (1976).

Twenty-six enzyme systems were screened in the four tissues
(see Backeljau et al. 1994). Seven of these enzymes yielded inter-
pretable genetic polymorphisms and were retained for further
analysis (Table 1 ).

Alleles were designated alphabetically according to decreasing
electrophoretic mobilities (A = most anodal = fastest-migrating
allele). Previously typed specimens were included with each run to
compare different gels. The BIOSYS-1 version 1.7 package
(Swofford and Selander 1981) was used for estimating allele fre-
quencies, mean numbers of alleles per locus (MNA). observed
heterozygosities (W^,. direct count) and Nei's ( 1978) unbiased ex-
pected heterozygosities {Hj. Numbers of polymorphic loci (P)
were simply counted. Weir and Cockerham's (1984) fixation ia-
dices (F,J were estimated with GENEPOP version 3.0 (Raymond
and Rousset 1995). and genotype frequencies were evaluated for
departures from Hardy-Weinberg (HW) equilibrium expectations
with the probability test implemented by the same program. The
significance of F,^ values was tested with FSTAT version 1.2
(Goudet 1995). Linkage disequilibria (LD) between loci were
tested with the exact probability test in GENEPOP version 3.0.
Whenever needed, testing procedures were corrected for multiple
testing with the sequential Bonferroni method (Rice 1989). Nei's
(1978) unbiased genetic distance between populations was calcu-
lated with BlOSYS-1 version 1.7.

The effective population size (A'^) of the population from the
Lagoa was estimated in two different ways. One method estimates
A'^ from the changes in expected heterozygosity. In a population of
size A'^.. the initial heterozygosity (//„) will decrease to W, after /
generations. The relationship between W,, and W, is given by the
equation «, = //„( 1 - 1/2A'^)' (Crow and Kimura 1970). A second
method (i.e.. the temporal method) estimates A'^. from temporal
changes of gene frequencies as described by Waples (1989) and
Hedgecock et al. ( 1992). Although a few T. decussatus individuals
may spawn in their first year (Vilela 1950). the vast majority of
individuals reach their sexual maturity at the beginning of their
second year (Gallois 1977). Therefore, we used a generation time
of 1 y for T. decussatus. An assumption of both methods is that the
allozyme polymorphisms studied are selectively neutral. To test
this, we performed the Ewens-Watterson test using the algorithm
given in Manly (1985) and implemented by the program
POPGENE version 1.31 (updated version of POPGENE version
1.2 of Yeh and Boyle |1997|).

Because many bivalves show a positive correlation between

TABLE L

Enzymes studied, E.C. numbers, en/.vmc codes, the tissue from which the enz>me was extracted, and the buffer system (TC
acid; T(; = Tris/glycine) used to examine senetic variation in four T. deciissalus populations.

Tris/citric

Enzyme

EC Number

Code

Tissue

BulTer

M;ilatc dchydri'gcriasc
D-Odopinc clehydrcigcnase
IsDcitrale dehydrogenase (NADP*)
Phosphogluconale dehydrogenase
.l-Hydro.xyhulyralc dehydrogenase
Leucylalanine peptidase
PhosphogJuconiuUise

1. 1. 1. .^7

1.1.1,42

1.1.1.44

i.l.l..^()

.V4.LVI1

.'i.4.2.2

MJh
Opdh

Iclhp
I'ildl,
lllolh
I'cp

.Adductor muscle
Adductor muscle
Digestive gland
Digestive gland
Digestive gland
Gills
Adductor muscle

TC
TC
TC
TC
TG
TG
TG

Genetic Variation in Azorean Tapes decussatus

31

shell size and individual heterozygosity (e.g.. Zouros and Foltz
1984), we checked for such a relationship to avoid the possibility
that discrepancies in //„ values would merely reflect size differ-
ences between populations. Therefore. Pearson's product-moment
correlation was calculated between shell length and numbers of
heterozygous loci, as outlined by Diehl and Koehn (1985) and
Fevolden (1992).

RESULTS

Pep revealed two independent banding zones, the cathodal of
which was clearly polymorphic in the Thau and Ria populations.
but monomorphic in the Lagoa population. Yet, because the bands
in this zone were often confused, they were not used for genotypic
analysis. The six remaining enzymes yielded information for seven
putative loci (Table 1 ). the population genetic data of which are
provided in Tables 2 and 3. Out of 18 HW tests, only 2 were
significant (Pgm in Thau and /rf/;/) in Ria; Table 2), but this was no
longer so after sequential Bonferroni correction. Not surprisingly,
F,^ values taken over all loci in all populations were not signifi-
cantly different from 0 (0.193 < P < 0.27). However, compared
with the Lagoa population, the Thau and Ria populations had
higher heterozygosity levels and nearly twice as many polymor-
phic loci and mean numbers of alleles per locus (Table 2). Only
two of the 31 LD tests were significant (data not shown), but both
cases were no longer significant after sequential Bonferroni cor-
rection. Nei's (1978) unbiased genetic distance between the
samples ranged from 0.036 (between two samples from the
Azores) to 0.23 (between Thau and two samples from the Azores)
(Table 3).

The estimate of N^ with the temporal method was infinity. This
result is probably an artifact caused by the small number of loci
analyzed in = 3) (Table 2). It simply indicates that the change in
allozyme frequencies observed between the 2 years was not large
enough to be distinguished from sampling error. The estimate of
N^ obtained from the reduction of heterozygosity was 5.30. The
test for neutrality gave nonsignificant results.

We found no significant correlation between individual het-
erozygosity and shell length (Thau, r = 0.173, P = 0.733; Ria, /■
= 0.36, P = 0.556; and Lagoa (pooled samples), /• = 0.48, P =
0.409).

DISCUSSION

Observed and expected heterozygosity values, number of poly-
morphic loci, and mean number of alleles per locus in the Ria and
Thau populations fall within the limits reported for T. decussatus
and the palourde Rudimpes pinlippinarum (Table 4). As in many
other bivalve species, heterozygote deficiencies have often been
reported in T. decussatus and R. philippinarum (see references in
Table 4), but at present the causes of this remain unclear (Zouros
et al. 1988). Yet, in our study, we observed no heterozygote de-
ficiencies. Nevertheless, our population genetic data of the Thau
population are very similar to the results obtained by Jame et al.
(1988), Borsa and Thiriot-Quievreux (1990), and Borsa et al.
(1994) for the same population and for the nearby population of
Etang du Prevost (Worms and Pasteur 1982). Moreover, genetic
distances between our populations are similar to those reported by
Jame et al. ( 1988) (compare our Table 3 with their Table 4).

However, in the Lagoa population of T. decussatus in the
Azores, we observed a strong reduction of allelic diversity and
expected heterozygosities, but not heterozygote deficiencies, com-
pared with main-range populations. Substantial losses of genetic
diversity have also been observed in bivalves for which hatchery
stocks have been established from only a few individuals (e.g., the
oysters Crassostrea gigas [Gosling 1982, Hedgecock and Sly
1990] and C. virginica [Vrijenhoek et al. 1990, Gaffney et al.
1992]). This may have important implications when management
and exploitation practices are developed. Many hatchery stocks or
introduced populations have a low N^ value despite densities that
can be very high (e.g., Saavedra 1997 and references therein). In
the Lagoa, population densities of T. decussatus may reach 400
individuals/m" (Gonijalves and Martins 1991). Yet we estimated
an effective population size of only 5.30 individuals. Founder ef-
fects, genetic drift, intentional selection, and inadvertent selection
during culture are likely to reduce the genetic diversity of the
Lagoa population further. The introduction of a small number of
individuals a few decades ago probably resulted in the loss of
genetic variation via founder effects. The strong isolation of this
population probably does not allow transport of larvae from nearby
populations (see Introduction), and genetic drift and inbreeding
may further reduce genetic variability. These effects are probably
reinforced by human activities such as selection during harvesting
(e.g., the collection of only large adults). Indeed, the exploitation
of T. decussatus in the Lagoa follows a classic "fishery" picture
with old (i.e., large) shells lacking among empty shells in the
lagoon because they were collected for consumption when alive
(Morton and Tristao da Cunha 1993). It is unclear whether such
selective harvesting affects the genetic structure of the population,
because there was no association between individual heterozygos-
ity and size. Yet this topic deserves further study, as Borsa et al.
(1994) and Passamonti et al. (1997) found a high level of intra-
population structuring, probably related to year-cohort heteroge-
neities, that perhaps indicate short-term selection or genetic drift
(Borsa et al. 1994). Thus, harvesting a single age cohort (i.e.,
oldest and largest individuals) could affect the genetic population
structure.

In none of the populations did we observe a significant corre-
lation between shell size and individual heterozygosity. Some
other studies also failed to show a relationship between individual
heterozygosity and morphological traits such as size and growth
(Adamkewicz et al. 1984, Volckaert and Zouros 1989, Gaffney
1990. Slattery et al. 1991), but others report negative (Wilkins
1978) or positive (Garton et al. 1984, Koehn and Gaffney 1984,
Zouros and Foltz 1984, Gaffney 1990) associations, although as-
sociations may differ among populations (Gaffney 1990).

A positive relation between heterozygosity, body size, and sur-
vival was found in a population of T. decussatus that survived
natural anoxic stress (Borsa et al. 1992). However, in other popu-
lations of the same species, Jame et al. ( 1988) observed no asso-
ciation between asymmetry of left and right valves (as a measure
of fitness, i.e.. the more asymmetric the less fit) and heterozygos-
ity, and an increased variance for morphological traits in the
classes with low heterozygosity. This also appears to be the case
for some of the R. philippinariini populations in the Po river lagoon
in Italy (Fava et al. 1994). In that study, individual heterozygosity
and phenotypic variability appeared to be negatively correlated,
but the relationship was heterogeneous between populations (Fava
et al. 1994).

32

JORDAENS ET AL.

TABLE 2.

Allozyme variation in four populations of T. decussatus (for full
population names we refer to the text).

Thau (n = 40) Ria (n = 40) SC92 (« = 40) SC93 (n = 40)

TABLE 2.
Continued

Mdh

A

0.837

B

0.163

H=

0.272

Ho

0.325

F.S

-0.182

p

' exact

0.564

Opdh

A

0.625

B

0.213

C

0.162

H.

0.538

Ho

0.575

fis

-0.057

p

' exacl

0.500

Idhp

A

0.113

B

0.887

/^e

0.200

»o

0.125

F\.

0.385

"exacl

0.057

Pgdh

A

0.138

B

0.200

C

0.349

D

0.175

E

0.138

H.

0.769

Ho

0.700

fis

0.102

p

' exact

0.384

Hhdh-I

A

0.250

B

0.724

C

0.013

D

0.013

H.

0.412

Ho

0.400

f,s

0.04 1

"cxiict

0.832

Hbdh-2

A

0.987

B

0.013

//c

0.025

//»

0.025

/^is

-0.013

P

• exact

1.000

Pgm

A

0.400

B

0.537

C

0.063

D

H.

0.547

//.,

0.675

/^,.

-0.222

p

0.011*

1.000

0.538
0.225
0.237
0.604
0.575
0.061
0.801

0.038
0.962
0.072
0.025
0.661
0.038*

0.225

0.613
0.162
0.548
0.525
0.054
0.881

0.225
0.762
0.013
0.013
0.368
0.275
0.264
0.144

1.000

0.586
0.363
0.038
0.013
0.522
0.500
0.055
0.192

1.000

0.488
0.262
0.250
0.631
0.675
-0.057
0.526

1.000

0.462

0.338
0.200
0.632
0.6.50
-0.016
0.973

1.000

1 .000

0.887
0.113

0.200
0.175
0. 1 36
0..396

1.000

0,600
0.212
0.188
0.560
0.575
-0.015
0.458

1.000

0.375

0.400
0.225
0.649
0.650
0.011
0.378

1.000

1.000

0.937
0.063

0. 1! 7

0.125

-0.054

1.000

Thau

Ria

SC92

SC93

(n = 40)

(n = 40)

(H = 40)

(n = 40)

Overall

H,

0.400

0.306

0.212

0.192

(SE)

(0.096)

(0.104)

(0.114)

(0.110)

H.

0.404

0.271

0.214

0.193

(SB)

(0.100)

(0.100)

(0.118)

(0.110)

MNA

3.0

2.4

1.7

1.7

P

in

5/7

3/7

3/7

'^(+Pep)

8/8

6/8

3/8

3/8

//j, expected heterozygosity; W„. observed heterozygosity; f„, fixation
index; P^^^^' exact P-values (*P < 0.05); MNA, mean number of alleles
per locus; P. proportion of polymorphic loci; SE, standard error.

Our allozyme data indicate that the Lagoa population from the
Azores is genetically depauperate and only slightly differentiated
from populations from the main range and may thus be of low
"biological value" (i.e., in terms of biodiversity). Gathering of T.
decussatus could therefore be allowed to continue. Nevertheless,
given the lower genetic diversity of T. decussatus in the Lagoa, the
low effective population size, and the depauperate intertidal region
(Santos et al. 1985. Santos and Martins 1987), exploitation of this
species must follow strict collection guidelines (see also Santos
1989), especially given that only 15% of the area is suitable for
exploitation (Morton and Tristao de Cunha I993J. Otherwise, a
unique component of the Azorean fauna that also serves as a small
fishery resource may be lost. In addition, there is much to compare
between llhtiu de Vila Franca on the island of Sao Miguel in the
Azores and the Lagoa de Santo Cristo. The faunistic and scientific
value of Ilhiju de Vila Franca is strongly reduced because of tour-
ism. Thus, opening up the Lagoa for tourism could be disastrous
for the fauna too. Therefore, in view of the unique origin, geology,
fauna, and flora, the place should be declared a "Site of Special
Scientific Interest" (Morton and Tristao da Cunha 1993).

ACKNOWLEDGMENTS

We are indebted to B. Morton (University of Hong Kong) and
R. Tristao da Cunha (University of the Azores, Portugal) for help-
ing to collect the Azorean T. decussatus. J. Troncoso (University
of Vigo, Spain) provided us with the Ria population. Financial
support was received from the EC program "Biodiversidade no
Arquipelago dos Agores" PRAXIS XXI (EUJNICT) 2/2.1/BlA/
169/94. S. V. D. and H. D. W. are FWO fellows.

TABLE 3.

Nei's (1978) unbiased genetic distance between the four populations
of T. decussatus (for population names we refer to the text).

Thau

Ria

SC92

SC93

Thau
Ria
SC92
SC93

0.152
0.239
0.2.30

0.129
0. 1 29

0.036

Genetic Variation in Azorean Tapes decussatus

33

TABLE 4.
Allozyme variation reported in other studies of T. decussatus and R. pbilippinarum.

Species

H„

//,

MNA

P

Reference

T. clecii.ssaiii.s

0.28

2.75

0.83

Worms and Pasteur (1982)

0.23-0.28

2.18-2.73

0.64-0.73

Jarne et al. (1988)

0.22

0.26

2.33

0.78

Borsa and Thiriot-Quievreux ( 1990)

0.18-0.24

0.23-0.33

1.54-1.99

0.54-0.66

Passamonti et al. (1997)

0.19-0.40

0.19-0.40

1.71-3.00

0.43-1.00

This study (all populations)

R. philipptnanim

0.26

0.26

3.18

0.73

Moraga (1986)

0.16-0.20

0.18-0.22

2.67-3.44

0.22-0.33

Kijimaet al. (1987)

0.17-0.25

0.20-0.27

2.6-3.6

0.43-0.57

Oniwaetal. (1988)

0.33

0.34

2,89

0.89

Borsa and Thiriot-Quievreux (1990)

0.34-0.37

2.80-3.10

0.80-0.93

Fava et al. (1994)

0.19-0.22

0.20-0.27

1.57-1.63

0.54-0.75

Passamonti et al. (1997)

0.27

0.27

3.15-3.35

0.75-0.85

Yokogawa (1998)

H„. observed heterozygosity; H^, expected heterozygosity; MNA. mean number of alleles per locus; P, percentage of polymorphic loci.

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Jdiiiiuil ,>/ Shellfish Research. Vol. 19. No. 1, 35-+I. :0()0.

LIFE HISTORY AND HABITAT OBSERVATIONS OF SOFTSHELL CLAMS MYA ARENARIA IN

NORTHEASTERN NEW JERSEY

CLYDE L. MACKENZIE, JR.,' AND SHAWN M. MCLAUGHLIN^

^National Marine Fisheries Service. National Oceanic and Atmospheric
Administration. James J. Howard Marine Sciences Lalxjratory,
Highlands. NJ 07732

'National Marine Fisheries Service, National Oceanic and Atmospheric
Administration. Cooperative Oxford Laboratory. Oxford, MD 21654

ABSTRACT Population densities, survival, factors associated with mortalities, and growth of softshell clams. Mya arenaria. in two
northeastern New Jersey estuaries were studied from 199.^ through 1997. The study areas were near shore where low-tide water depths
ranged from 15 to 90 cm. Juvenile densities were high only in 199.^. Light sets of juveniles from 1994 to 1997 disappeared by the end
of their first summer. The longest living softshells were the abundant 1993 year class, which survived for 26 mo in the Shrewsbury
River. This contrasts with life spans of 7-12 years for softshells in New England. Mortalities of softshells were correlated with: ( 1 )
predation by the striped killifish. Fundulus inajalis. and mummichog, F. heterocUtus: (2) mats of sea lettuce. Ulva laclusa: and (3) high
temperatures (30-32 °Cl. Softshell sarcoma was also present and may have contributed to mortalities. The effects of the mortality
agents varied among locations and years. The softshells of the Shrewsbury River averaged about 23 mm and 40 mm long at the end
of their first and second growing seasons, respectively.

KEY WORDS: Mya arenaria. settlement densities, survival, mortality factors, growth

INTRODUCTION

The softshell clam, Mya arenaria. ranges along the Atlantic
coast of North America from Labrador (Abbott 1974) to Georgia
(Rasmussen and Heard 1995). with the highest abundances located
from the Bay of Fundy to Chesapeake Bay. The species also occurs
in Europe and has been successfully introduced to the coasts of
western North America (Abbott 1974). Investigators in New En-
gland commented on the wide variations in magnitude of annual
sets and on the subsequent survival of softshells (Belding 19.30,
Turner 1949. Turner 1950. Brousseau 1978b). Softshells can live
as long as 7 y (Brousseau 1978b) to 12 y (Belding 1930), Most
postsetting mortalities of softshells are caused by predation by
shrimp, fish, ducks, brachyuran crabs, xiphosuran crabs, and nati-
cid snails (Belding 1930. Turner 1949. 1950, Foley and Taber
1952. Glude 1955. Smith et al, 1955. Cronin and Hall 1968.
Palmer 1976. Edwards and Huebner 1977. Kelso 1979, Holland et
al, 1980. Commito 1982, Hines et al. 1990. Eggleston et al. 1992.
Rasmussen and Heard 1995). and by breakage and displacement in
storm-exposed areas (Kellogg 1910, Belding 1930. Turner 1950.
MacKenzie and Stehlik 1988). Investigators in Europe also have
reported on the wide annual variability in densities of softshell
juveniles and on their subsequent survival and causes of mortality
(DeVlas 1979. Beukema 1982. Pihl 1982. Moller and Rosenberg
1983, Kube 1996).

Epizootics of softshells reported from New England to Chesa-
peake Bay have been associated with disseminated sarcomas
(Barry and Yevich 1972. Farley 1976. Yevich and Barszcz 1977,
Brown et al. 1977. 1979. Farley et al. 1986. Brousseau 1987.
Barber 1990). The proliferative condition is transmissible, progres-
sive, and usually fatal (Brown 1980, Cooper et al. 1982, Farley et
al. 1986). The etiology of softshell sarcotna is uncertain; environ-
mental factors (Barry and Yevich 1972. Yevich and Barszcz 1977)
and a viral agent (Oprandy and Chang 1981 ) have been suspected.

The Navesink and Shrewsbury Rivers and nearby Raritan Bay
in northeastern New Jersey have produced softshells since prehis-
toric times (MacKenzie 1990, MacKenzie 1992), but in recent
years the stocks have been small, and, consequently, commercial

production usually has been small or nonexistent. Previous studies
of the softshells in this area have described abundances (Dean
1975), longevity (Appeldoorn 1983, Appeldoorn 1995), abun-
dances and effects of stomis (MacKenzie and Stehlik 1988), and
the incidence of sarcoma (Barber 1990). Our study was undertaken
to characterize annual recruitment, survival, factors that cause
mortality, and growth.

Study Areas

The study areas were in the Navesink and Shrewsbury Rivers,
in New Jersey (Fig. 1 ). The primary study site in the Navesink
River was off its southeast shore. The site comprised about 3 acres
of firm muddy-sand bottom and extended from near the shore edge
to about 75 m offshore; water depths were froin 15-90 cm at low
tide. The mean tidal amplitude is about 1.7 in (Jeffries 1962). Mats
of sea letmce, Ulva lactuca, formed in the site, and their aerial dis-
tributions varied widely among years. The study site in the Shrews-
bury River was off its northeast shore at a similar shore position
and water depth, and its bottom sediments were similar. It was
about 1 acre in size. Little sea lettuce grows in that section of the
river. A reason for selecting the two sites was convenient access to
the shore by foot as most all the shoreline areas of the two rivers
are private property. The softshells in the two rivers are subtidal.

The identified predators of softshells in the two rivers were: the
striped killifish, Fundulus majalis; the mummichog, Fundulus het-
erocUtus: and the blue crab. Callinectes sapidus. Schools of
striped killifish and mummichogs were nearly always present in
the study sites, except during the lowest tides, from at least mid-
May into October. The blue crabs were scarce in the rivers from
1993 to 1996, but were more abundant in 1997.

During this study, the salinity at the Navesink River site ranged
from 15 to 25 0/00. and at the Shrewsbury River site from 20 to 25
0/00. Water temperatures were mostly 1 1-12 °C during early May.
18-20 °C during June, and peaked at about 25 °C in late July and
early August, but in mid-afternoon during late July-early August,
1995, water temperatures ranged from 30.0 °-31.8 °C. Tempera-
tures afterward cooled.

35

36

Mackenzie and McLaughlin

Raritan
Bay

a

O
o

CD
03

3

Figure 1. Locations of study and sampling sites in nortlieastern New
Jersey.

The waters of the Navesink and Shrewsbury Rivers interchange
with Raritan Bay. which is contaminated with many types of pol-
lutants (Pearce 1983. Breteler 1984). The pollutants consist of
suspended particulates, oil and grease, many toxic trace metals,
polynuclear aromatic hydrocarbons, polychlorinated biphenyls,
DDT. and dioxins (Stanford and Young 1988, Wolfe et al. 1996).
In 1974. the copper concentration in western Raritan Bay bottom
water was 65 parts per billion (ppb), the highest reported for any
estuary; the copper concentration in the surface water there was 36
ppb. and in mid-Raritan Bay it was 7.9 ppb (Waldhauer et al.
197S). In 1992. the copper concentration in the surface water was
considerably lower: 4.6 ppb in western Raritan Bay, and 4.3 ppb in
mid-Raritan Bay (Anonymous 1992). The buy has extremely high
primary productivity with the annual value in the 1970s at 817 g
C/nr. which was considered among the highest of any estuary
(O'Reilly et al. 1976). In the 196()s. Raritan Bay was classified as
an advanced eutrophic system (Federal Water Pollution Control
Administration 1967), but since the 1970s its water quality has
improved (Brosnan and O'Shea 199.'i). Elevated nitrogenous
wastes nevertheless continue to stimulate the growth of dense phy-
toplankton blooms (Draxler et al. 1984, Brosnan and O'Shea
199.'i); Draxler et al. (1984) had reported Secchi disc readings in
the bay of < 1 .0-2.0 m during most of the spring and summer.
Sleimlc and Caracciolo-Ward (1989) have shown that the den-
sity and diversity of benthic macrofauna in Raritan Bay are rela-
tively low compared with other U.S. east coast estuaries. Similar
determinations of pollution, phytoplankton blooms, and macro-
fauna have not been reported in the Navesink and Shrewsbury
Rivers.

The eutrophicalion of waters probably v\as responsible for pro-
ducing some large mats of sea lettuce observed in the two rivers.
As Hull ( 1987) noted, sea lettuce begins as tiny leaves attached to
shells and other objects in the spring, grows and persists as thick
mats during the sunnner. antl llicn ncarlv disappears in the late fall.

METHODS

Sampling Procedures

Field observations lasted 5 y, 1993 to 1997. Water salinity was
determined quarterly by titration. Surface water temperatures were
measured with a hand-held thermometer daily at 7:30 am at the
Navesink River study site from May into September in 1994, 1993,
and 1996. In 1993, following heavy sets of softshells, densities of
this year class were estimated once a month, except in the coldest
parts of the year, in the Navesink and Shrewsbury River study
sites, by placing a ring that encircled a 0.28 m" area on the sub-
strate and then removing all softshells for counting and measuring.
Three such samples were taken for each determination. From each
monthly sample, a subset of 100 softshells, was measured and
lengths were plotted to determine growth rates. In 1994, samples to'
determine the densities of young-of-the-year (juvenile) softshells
were taken similarly at each site. In 1995, 1996, and 1997, three
0.28-m~ areas or six O.l-m" areas were sampled at each site. Only
two to three samplings were made in each year from 1994 to 1997,
because the low densities of juvenile softshells fell quickly to
nearly zero per sample following the initial samplings in June or
July.

Potential predators of softshells were collected by pulling a
fine-mesh, 15-m seine for about 60 m over an inshore section of
bottom in the study sites in the Navesink and Shrewsbury Rivers.
A single seining was made at each site at half tide during the
outgoing tide in July 1994. Fish and shrimp were collected, but
only the fish were examined. They were placed in a plastic bag,
held on ice in a cooler, and frozen the same day. Later, they were
thawed, and the invertebrates, plants, and other contents in their
stomachs and guts were identified and counted using a dissecting
microscope.

Field Experiment on Fish Gut Evacuation

During August 1996. an experiment was conducted to deter-
mine the evacuation rate of food from the stomachs and guts of the
mummichog, F. hcteroclinis. One hundred mummichogs (mean
length 79.1 mm; range 63-1 10 mm) were seined and divided into
five groups of 20 each. The first group of fish was immediately
iced, then frozen, and later thawed and examined for the quantity
of food in their guts. The other four groups were held in separate
field cages suspended above the bottom for 3, 6, 9, and 24 h at
temperatures of 23.5 °-25.0 °C and then processed similarly to the
first group. A visual estitnate was made of gut fullness.

Diagnosis of Sarcomas

The prevalences of softshell sarcomas were determined using
histological methods (Farley et al. 1986). Samples of 50 softshells,
40-55 mm long, were collected quarterly al four sites, namely, our
two primary study sites in the Navesink and Shrewsbury Rivers, at
Lewis Point (5 km west of our primary study site in the Navesink
River), and in Raritan Bay at the Old Ferry Dock on the west side
of Sandy Hook (Fig. 1 ). The collections eventually ended ni the
Navesink and Shrewsbury Rivers because the softshells had died
or had became too scarce. Following collections, the sollshells
were transported to the Cooperative Oxford Laboratory, Oxford,
MD. Hemolymph was drawn from the adductor muscles into ster-
ile syringes containing ambient sterile seawater. expelled into slide
chambers, and fixed after 30 min in I i;iiJtaraldehvde-4 formalde-

Life History and Habitats of Softshell Clams

37

hyde. The hemolymph preparations were stained with fuelgen pi-
croniethyl and were examined tor sarcomas by light microscopy.

RESULTS

Navesink River

The setting densities of juvenile softshells in our Navesink and
Shrewsbury River study sites were similar to one another each
year. The juveniles were relatively abundant in the two rivers only
in 1993. In the Navesink River, they had set throughout the shal-
lows over a distance of 1 0.5 km off its south and northwest shores.
At the study site, their density at the initial sampling in August
1993 was 1,1 10/0.28 m". Their survival after that was fairly high:
60-69% were alive in late April to late May 1994 (Table 1 ).

In 1993. sea lettuce was relatively sparse in the study site, but
by mid-June to early July 1994, a solid mat of sea lettuce had
formed. The mat was about 25 cm thick and extended from the
shore outward to cover about half of the 3-acre bed. In addition,
some i.solated stationary sea lettuce mats, as small as 2 m across,
formed in areas beyond the main mat. All the observed 1993 year
class of softshells covered by the mats initially extended their
siphons several centimeters out of the sediment, then emerged
from it. laid on its surface beneath the mat. and died. In contrast,
the softshells in unvegetated areas did not extend their siphons,
emerge, and die.

From 1994 through 1997. the sets of juvenile softshells were
light in the river. In 1994, the unvegetated sediments outside any
sea lettuce mats received a set of juveniles; on June 30 of that year,
they had a mean density of 54.7/0.28 nr (three replicates. SE 9),
but by July 8. 1994, their density had fallen to 2.3/0.28 m" (three
replicates, SE 0.7). The 1995 and 1996 sets were much more
sparse than those in 1994 and 1997. On July 28, 1997, the 1997
juveniles had a mean density of 28.8/0.10 m- (six replicates. SE
4.7), but by August 9. 1997, their density had fallen to 3.7/0.10 nr
(six replicates, SE 0.6). Subsequent samplings in August and Sep-
tember each year from 1994 to 1997 found few juveniles in the
site.

On July 7, 1994, when the density of the 1994 year class of
softshells was declining rapidly, a seining was made over the bed
to examine the stomachs and guts of fish. Forty-one of 60 striped
killifish (average length 64 mm, range 46-78 mm) contained an

TABLE 1.

Densities, mean, and standard error (S.E.) of 1993 year class Mya

arenaria at stud> sites in Navesink River and Shrewsbury River.

Densities are expressed as mean per 0.28 m'. S.E. is based on 3

samples on each date.

Navesink River

Shrewsbury River

Date

Mean

S.E.

Date

Mean

S.E.

1 Sep 9.^

1. 110

117

7 Oct 93

849

57

8 Oct 93

1.170

200

1 1 Nov 93

650

62

28 Apr 94

668

37

29 Apr 94

677

45

24 May 94

767

16

28 Jun 94

784

85

29 Jun 94

0

2 Aug 94

586

10

2 Sep 94

520

81

26 Apr 95

573

9

7 Jun 95

456

16

7 Aug 95

0

average of 46 juvenile softshells/fish (range 1-169 softshells). and
one of three mummichogs (average length 97.3 mm. range 84-1 15
mm) contained two juvenile softshells. The softshells ranged from
2-1 I mm long. The remaining striped killifish and mummichogs
had food in their stomachs but no softshells.

Shrewsbury River

In 1993. softshells set densely in the shallows along most of the
north shore of the Shrewsbury River in a band about 7 m wide,
over a distance of about 4.2 km. The density of the 1 993 year class
of softshells at the study site at the initial sampling in October
1993 was 849/0.28 m". After that, their survival was fairly high, as
54-67% were alive in late April-early June 1995 (Table 1). By
August 7, 1 995, about 26 months after setting, this entire year class
of softshells was dead at the site. They died during a period of
unusually high air and water temperatures in late July-early Au-
gust. At 3:00 PM on July 3 1 , the water temperature was 3 1 .8 °C, the
softshells were dying and rotting, and the water over the bed was
a yellow-brown mixture of rotting softshell meats and brown phy-
toplankton. Their mortality apparently was caused by the high
temperatures, because the lethal temperature of adult softshells is
in the temperature range of 30.5 °-32.5 °C (Kennedy and Mihur-
sky 1971).

From 1994 through 1997, juvenile softshells were relatively
scarce throughout the river. At the study site, the small numbers
observed by scraping with a sieve through the surface of sediments
in 10 places in June and July disappeared by August or September
in the years in which they set, similarly as the light sets had
disappeared in the Navesink River.

On July 8, 1994, fish were seined at the study site and their guts
were examined for softshells and other foods. Four striped killifish
(average length 107 mm, range 92-1 13 mm) contained an average
of 26 juvenile softshells/fish (range 21-32 softshells per fish): 123
of 150 mummichogs (average length 69 mm, range 40-93 mm)
had an average of 15.5 juvenile softshells per fish (range 1-53
softshells per fish); and one spot. Leiostomus xanthwus. had 1 15
juvenile softshells. The softshells ranged from 4 to 1 1 mm in
length for all fish. Other items in the guts of striped killifish and
mummichogs in the Navesink and Shrewsbury Rivers were: juve-
nile common Atlantic slippersnails, Crepidula fornicaui: amphi-
pods; isopods; juvenile horseshoe crabs. Limiilus polxphemus
(about 3 mm carapace width); polychaetes; sea lettuce; and detri-
tus.

Food Passage Through Mummichogs

Mummichogs passed food through their stomachs and guts rap-
idly (Fig. 2). In the experiment to estimate the rate, a large decline
(80%) in fullness of their guts was evident after 3 h, and little food
remained after 24 h. The results suggest that the softshells found in
mummichogs that were seined at the sites were eaten within 24 h,
and they imply a high consumption rate.

Histology

In the Navesink River, quarterly samples showed a low sar-
coma prevalence in 1994, but prevalence reached 18%' in Decem-
ber 1995 and decreased slightly to 13% and 14% for the first two
quarters in 1996. while samples from Lewis Point were negative
for sarcomas in 1991 to 1993 (Table 2). In the Shrewsbury River,
quarterly samples of softshells examined for sarcomas were nega-
tive in 1994 and 1995. At the Old Ferry Dock, in collections in

38

Mackenzie and McLaughlin

Fundulus Gut Evacuation Study
(16 Aug 96)

Figure 2. Percentage with food in guts and average fullness of guts of
F. heteroclitus held in field cages at spaced intervals. 0-24 h.

1995. 1996. and 1997. from 10-20% of softshells were infected
with sarcoma on four of seven dates, and from Q^9c were infected
in the remaining three dates.

Growth

The length-frequency curves for the 1993 year class of soft-
shells in the Navesink and Shrewsbury Rivers are presented in
Figure 3. The curves for each time period show a single mode that
broadens somewhat as time passes. In the Navesink River, the
softshells had a mean length of 15.4 mm in September 1993. 22.1

TABLE 2.

Percent prevalences of .softshell sarcomas based on histology
(n = 50).

Lewis

Navesink

Shrewsbury

Old Ferry

Date

Point"

River

River

Dock

7-9-yi

0

9-4-91

0

12-4-91

0

.^-3-92

0

6-.^-92

9-9-92

12-9-92

3-25-93

6-38-93

9-29-93

6-15-94

0

0

9-12-94

4

0

12-6-94

2

0

5-22-95

0

0

6-26-95

2

0

7-25-95

6

0

9-21-95

2

4

12-5-95

18

12

3-27-96

13.3"

10

7-18-96

14.4"=

0

10-3-96

10

2-26-96

20

4-14-97

2

° Location In Navesink River,
"n = 47.
' n = 45.

Shrewsbury River

Navesink River

^^

November '93

April '94

z^..

May '94

j^

.y%V.

Sept '94

.J^.

V

20
10

A

Sept '93

^

10

/\

10

J^

Nov '93

10

^yvvyv

Apr '94

10
5

/v

May '94
^ -

10 13 16 19 22 25 28 31 3J 37

« 43 46 49

Length (mm)

, /I Apr -9!

12 16 20 24 28 32 36 40 44 48 52 56 60 64

Figure. 3. Length-frequency distributions of the 1993 year class Mya
arenaria in the Navesink River, 1993 to 1994, and the Shrewsbury
River, 1993 to 1995.

mm in November 1993, and 31.6 mm in May 1994. In the Shrews-
bury River, their mean lengths were 19.3 mm in October 1993.
22.9 mm in November 1993. 26.6 mm in April 1994. 38.9 mm in
November 1994, 47.6 mm in April 1995, and 48.9 mm in June
1995.

DISCUSSION

In attempting to find reasons for the large annual variability in
setting densities of softshells in Europe. Beukema (1982, 1992).
Jensen and Jensen (1985), and Moller (1986) observed that heavy
sets of softshells and some other bivalves occurred during sum-
mers following cold winters and that light sets followed mild win-
ters. The bivalves were active during the mild winters when little
food was available in the water, and they consequently had ab-
sorbed most of their gonads by the time spawning began in the
spring. Our study was not continued sufficiently long enough to
document such a correlation, but it is likely that dense sets of
softshell juveniles result from certain weather conditions. The
spring and early summer of 1993 when the heavy sets occurred in
the Navesink and Shrewsbury Rivers did feature weather with no
cold easterly winds with rain. During the springs and summers of
1994 to 1997. however, when light sets occurred, several periods
of cold easterly winds and rain, each of 3— t days duration, were
interspersed with periods of v\armcr westerly and southerly winds.
Bclding ( 1930) had noted that the numbers of larvae in the water
declined during periods of cold rains.

Earlier investigators ha\e noted the disappearances of softshell

Life History and Habitats of Softshell Clams

39

juveniles by the end of their first summer in some years (Brous-
seau 1978a. Moller and Rosenberg 1983. Beukema 1979. Pihl
1982). We believe that predation by striped killifish and mummi-
chogs was the principal reason for the sharp declines and disap-
pearances of juxeniles in our study sites during 1994 to 1997. The
observations suggest that any relatively light sets of softshells, as
dense as 500/m" or even higher, could be lost to such predation
every year whenever the fish are abundant in the two rivers. The
fish likely were present and preyed on juvenile softshells in 1993.
but perhaps the juveniles were so abundant that a great many
remained alive by the time they had grown too large for the fish to
prey on them.

Fish also prey on softshells in other regions. Kelso (1979)
described heavy predation of juvenile softshells by mummichogs
in Massachusetts. In our study, the sizes of softshells (2-11 mm
long) taken by the striped killifish and mummichogs were similar
to those that Kelso (1979) reported; probably 1 1 mm is near the
maximum size of a softshell that the fish can devour. More soft-
shells were present in the guts of mummichogs in the Navesink
River (about 46 softshells per fish) than he found in Massachusetts
(6-9 softshells per fish). Perhaps the softshells were more abun-
dant in the Navesink River. Medcof and McPhail (1952) stated that
adult winter flounders. Pleiironecres aineiicainis. about 28 cm
long, consumed whole juvenile softshells and nipped off the si-
phon tips of adult softshells in eastern Canada. In their study, the
softshells with nipped siphons recovered without unusual mortal-
ity. Rasmussen and Heard (1995) stated that Atlantic stingrays.
Dasyatis sabiiui. feed on softshells in Georgia. Pihl (1982) and
Moller and Rosenberg (1983) observed that flounders Platichthys
ftesiis consume large numbers of juvenile softshells. 2-12 mm
long, in Sweden, and DeVlas (1979) observed that flounders P.
flesus and plaice. Pleiironectes platessa. consume juvenile soft-
shells and the siphon tips of older softshells in the Netherlands.
Summer flounders. Paralichthys dentatus. and other fish were
present in the Navesink and Shrewsbury Rivers and might have
preyed on softshells. but they were not observed or collected dur-
ing our visits to the study areas.

Relatively scarce in our study sites from 1993 to 1996, blue
crabs appeared to be a minor predator then, but they were abundant
and may have killed many juvenile softshells in 1997. Since our
observations were limited to periods of low and mid tides and
during daylight, blue crabs and other predators may have entered
the study sites and eaten some juveniles during high tides and at
night during all years. Green crabs, Carcinus maenas. and naticid
snails, both predators of softshells in New England (Belding 1930.
Glude 1955. Smith et al. 1955. Edwards and Huebner 1977. Com-
mito 1982), were not observed in the two rivers during 1993 to
1997 and could not have caused much mortality of the softshells.
Horseshoe crabs, also a softshell predator in New England (Turner
1949. 1950). were scarce and apparently killed few softshells in
the two rivers. The shrimp. Crangon crangon. preys on softshells
as large as 3 mm long in Europe (Moller and Rosenberg 1983).
The seven-spine bay shrimp. Crangon septemspinosus. and the
marsh grass shrimp. Palaemonetes vulgaris, were abundant in our
two study areas but were not examined as predators of small post-
set softshells. and neither were amphipods and isopods.

In eastern North America, greater scaup. Aytliya marila. prey
on a variety of small clams, including softshells. blue mussels.
Mylihis edidis. and snails (Foley and Taber 1952. Cronin and Hull
1968. Barclay pers. commun.. 1998). Black ducks. Anus nihripes.
prey on bivalves, including Macoma balthica. blue mussels, and

marine snails, such as eastern mud snails, llyanassa obseleta
(Palmer 1976). Greater scaup and black ducks were present in the
Navesink and Shrewsbury Rivers, but there were no signs that they
ate softshells in our study areas.

Juvenile softshells also can be killed on exposed shallow habi-
tats during wind storms by having their thin shells ground into
fragments or being washed onto nearby beaches (Kellogg 1910.
Belding 1930, Turner 1950. MacKenzie and Stehlik 1988). This
type of mortality was not observed in our Navesink and Shrews-
bury River study sites, but it was observed in the softshells that had
set along the south shore of Raritan Bay.

Once past their first summer, softshells can survive fairly well
as long as exogenous mortality factors are absent, as shown by
Belding (1930) and Brousseau (1978b) in New England. Kube
(1996) in Europe, and others. In the Navesink River, the 1993 year
class of softshells survived well from September 1993 through
May 1994 until mats of sea lettuce killed them, and in the Shrews-
bury River it survived well from October 1993 through June 1995
when shortly afterward high temperatures apparently killed them.
The age of the Shrewsbury River softshells when they died. 26 mo,
was the maximum that any lived in the two study sites and is far
shorter than softshells lived in New England where their habitat
was undoubtedly much better (Belding 1930, Hanks 1963, Brous-
seau 1978b). Appeldoorn (1995) stated that softshells in the Nave-
sink River could live at least 15 years around the time of his
sampling (1977), but his finding was based on shell markings and
sizes of softshells found during a single collection and might be in
error. Nevertheless, in an earlier paper. Appeldoorn ( 1983). report-
ing on the same 1977 samples, stated that softshells were present
as large as 78 mm long or even larger and were obviously older
than the largest softshells (62 mm) that we found in the Navesink
and Shrewsbury Rivers. The environmental conditions in the two
rivers during 1993-1997. such as extremely high temperatures in
1995. apparently did not allow the softshells to live as long as they
did during the 1970s.

Some earlier workers had shown that algal mats grow over and
kill bivalves, but our study may be the first to document that mats
of U. lactuca kill softshells. Thiel et al. (1998) had similarly found
that overgrowths of the filamentous alga Enteromorpha prolifera
kill softshells in Maine; Breber (1985) found that mats of Ulva
rigida and Cracilaria sp. kill carpet-shell clams. Tapes deciissatits,
in Italy; and Everett (1994) showed that the bent-nose macoma,
Macoma nasiita. was more abundant in areas devoid of Ulva ex-
panse than in areas where it formed mats in California. The same
condition probably develops under U. lactuca mats that Gray
( 1992) described under U. rigida mats in Europe: Anaerobic con-
ditions are reached and sulfide and other toxic compounds are
produced leading to a massive mortality of benthic organisms.

Sarcoma infections occur seasonally (Farley 1976. Farley 1989.
Cooper et al. 1982, Brous.seau 1987, Barber 1990). Perhaps in
collecting the softshells quarterly, we missed detecting some sar-
coma in them. During most collections of adult softshells. a few
recently dead specimens with whole shells were noticed among the
100-200 that were taken. Sarcoma might have been responsible for
some mortality that was not identified to cause, or perhaps the
softshells died from some other cause. We were unable to deter-
mine whether contaminants in the waters and sediments and den.se
phytoplankton blooms affected the longevity of the softshells. Bar-
ber ( 1990) found sarcomas in softshells in the Shrewsbury River in
1986 and 1987 and concluded that annual mortality due to the

40

Mackenzie and McLaughlin

disease was about 3.5% at that time. Our study cannot add much
to his estimate.

The sizes of softshells at certain ages that Appeldoorn ( 1983)
suggested for the Navesink River correspond with our findings in
the Shrewsbury River. For example, at 20 months of age the soft-
shells that Appeldoorn measured were 42.5 mm long and at 28
months they were 47.3 mm long, or similar to the mean lengths of
softshells in the Shrewsbury River at about the same ages in No-
vember 1994 and June 1995. However, the comparisons are too
crude to compare actual growth rates in the 1970s and the 1990s.

The small and sporadic commercial harvests of softshells in
this area likely are due to their low setting densities and poor
survival rates in recent years. The softshells probably would sur-
vive longer if a period of cooler summers and reduced eutrophi-
cation of waters were to follow.

ACKNOWLEDGMENTS

We thank D. Jeffress and F. TrioUo for assistance with the field
work, and J. Buckle. R. Pikanowski. R. N. Reid. and two anony-
mous reviewers for critically reviewing the manuscript.

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Joiirmil of Shellfish Resenrch. Vol. 19. Ni). I, 4.V50. 2000.

QUAHOG PARASITE UNKNOWN (QPX) IN THE NORTHERN QUAHOG MERCENARIA

MERCENARIA (LINNAEUS, 1758) AND M. MERCENARIA VAR. NOTATA FROM ATLANTIC

CANADA, SURVEY RESULTS FROM THREE MARITIME PROVINCES.

GREGORY S. MACCALLUM AND SHARON E. MCGLADDERY

Fisheries and Oceans Canada
Gulf Fisheries Centre
PO Box 5030
Moncton, NB EIC 9B6

ABSTRACT A histology based survey of 3047 quahogs from various sites in three Atlantic Canadian provinces between 1990-98
revealed Quahog Parasite Unknown (QPX) in clams ranging from 18-92 mm in length (> 1 .5 years old). Prevalences ranged from 1.7%
in wild quahogs to SO^r in hatchery broodstock. An additional two year (1996-97) seasonal survey of four sites (St. Andrews and
Shediac Bridge, New Brunswick; Wallace. Nova Scotia; and West River. Prince Edward Island) found QPX in quahogs 43-102 mm
in length (n = 715) at Wallace {7c P = 6J9c) and Si. Andrews iVc P = 209c). Infections were found in spring, summer and fall
samples and no significant difference was found between male and female infected quahogs at either site (1996-97; x". P > 0.10). The
most commonly infected tissues were the gills, mantle and gonads.

KEY WORDS: Quahog Parasite Unknown (QPX). Af mercenaria. M. mercenuria var. nouiia. pathology

INTRODUCTION

Quahog Parasite Unknown (QPX) infects the hard-shell clam
(northern quahog) Mercenaria mercenaria and the selected vari-
ety, M. mercenaria var. notata (Chanley 1961). It has caused cu-
mulative mortalities ranging from 80% in quahogs from New
Brunswick (Drinnan and Henderson 1963), and Cape Cod. Mas-
sachusetts (Smolowitz et al. 1998) to 100% in hatchery broodstock
in Prince Edward Island (Whyte et al. 1994; Bacon et al. 1999). It
also occurs in apparently healthy quahogs from Atlantic Canada
and Virginia (McGladdery et al. 1993: Ragone Calvo et al. 1998).
QPX has also been associated with quahog mortalities from Burton
Bay, Virginia (Ragone Calvo et al. 1997). The Gulf of St.
Lawrence is the northern-most limit of M. mercenaria. thus the
clams may respond differently, both in terms of growth and disease
resistance, from clams in the middle of their geographic distribu-
tion in the U.S. The conditions which trigger pathogenic infections
levels, however, have yet to be determined.

Recent interest in developing the northern quahog for aquacul-
ture in Atlantic Canada revealed a lack of base-line information on
normal parasite and disease profiles for this species. Because cul-
ture involves handling and holding the clams in unnatural condi-
tions, QPX has the potential to become a significant health prob-
lem, especially as hatchery broodstock are developed (Whyte et al,
1994). An accurate understanding of the seasonal and geographic
distribution of QPX in wild and cultured populations throughout
Atlantic Canada was. therefore, required. Throughout the past 10
years, samples of wild and cultured quahogs have been examined
histologically for parasites and pathology, including QPX, as part
of diagnostic services provided by Fisheries and Oceans Canada,
Gulf Fisheries Centre, Moncton, New Brunswick. These data, in
addition to a seasonal histological survey of wild quahogs from
four sites in New Brunswick (NB), Nova Scotia (NS), and Prince
Edward Island (PEI), conducted between 1996 and 1997, were
examined to determine if there were significant population differ-
ences in quahog health profiles. Unlike other bivalve species cul-
tured to date, quahogs in Atlantic Canada have undergone rela-
tively little transfer and population mixing. This was, therefore,

seen as an opportune time to collect base-line health information
for subsequent development of the quahog aquaculture industry.

MATERIALS AND METHODS

Diagnostic Survey 1990-98

A total of 3047 quahogs was examined (Table 1). Quahogs
(wild and notata variety) were collected or shipped live froin aqua-
culture sites and hatcheries in NB, NS and PEI (Table I) to the
Gulf Fisheries Centre, Moncton, within 12-24 h of collection.
Anterior-posterior length (mm) and weight (in shell) were mea-
sured before shucking. A 2-3 mm dorso-ventral cross-section was
removed and fixed in 1% gluteraldehyde/4'7c formaldehyde (How-
ard and Smith 1983) for light microscopy. The tissues for light
microscopy were paraffin embedded, sectioned (6 \x.m ) and
stained using Harris' hematoxylin and eosin.

Tissue sections were examined at 25 and 250 magnification
using a Leitz Dialux 20 compound microscope. Prevalence of QPX
was recorded, along with a qualitative scale for intensity of infec-
tion (light = < 25: organisms, moderate = 25-50 organisms: and
heavy = > 50 organisms) per tissue section. The sex ratio of
mature quahogs. infected with and without QPX, was compared to
a 1 : 1 ratio using a standard Chi Square test (Zar 1984) to determine
if there was any relationship between quahog sex and presence of
QPX.

Seasonal Survey 1996-97

Wild quahogs were collected during the spring (May/June),
summer (August) and fall (October/November) of 1996 and 1997
from: (1) St. Andrews, NB: (2) Wallace, NS: (3) West River, PEI:
and (4) Shediac Bridge, NB (Figure 1 ). Samples of 28-30, total =
715, quahogs were collected and processed as described above,
between May, 1996 and October. 1997 (Table 2). Water tempera-
ture and salinity were taken at the time of collection from all four
sites during both years. In addition, a continuous temperature re-
corder was placed at the Wallace location from May to October
1996 and 1997. The sex ratio of infected quahogs was compared to
a 1:1 ratio using a standard Chi-Square test (Zar 1984).

43

44

MacCallum and McGladdery

TABLE 1.
Collection details and QPX results for 1990-98 survey.

Lengths

***Sex Ratio

examined

Prev.

**Inf.

(Infected

Date

Collection Site

(mm)

No.

(%)

Levels

Quahogsl

1990

Ellerslie, PEI*

>25

5

80.0

H

4U

13-5-91

Bouctouche. NB

55-81

16

0

0

0

22-5-91

Shippagan. NB*

72-90

15

13.3

H

2M

17-6-91

Cocagne. NB

78-85

30

0

0

0

18-7-91

Cocagne. NB

75-89

30

0

0

0

29-7-91

Pictou, NS

83-104

45

0

0

0

6-7-91

Halifax. NS*

>25

10

0

0

0

15-8-91

Shediac Bridge. NB

73-91

30

0

0

0

17-8-91

Cocagne. NB

72-88

30

0

0

0

30-3-92

Shippagan. NB*

25-38

5

0

0

0

1-7-92

Cocagne. NB

71-91

30

0

0

0

26-8-92

Cocagne. NB

70-90

30

0

0

0

15-10-92

Cocagne. NB

45-63

30

0

0

0

7-6-93

Malagash. NS

57-76

30

0

0

0

22-6-93

Cocagne. NB

48-71

30

0

0

0

22-6-93

West River. PEI

54-73

30

0

0

0

13-7-93

Powell cove. NS

32-110

26

7.7

H

IM;1F

14-7-93

Wallace. NS

43-67

39

0

0

0

26-7-93

Brule Harbour. NS

53-180

30

3,3

L

IF

3-8-93

West River. PEI

52-82

30

0

0

0

4-8-93

Wallace. NS

49-62

30

0

0

0

8-8-93

Cocagne, NB

43-61

30

0

0

0

12-10-93

Ellerslie. PEI*

8-15

22

0

0

0

19-10-93

Malagash. NS

52-80

30

0

0

0

26-10-93

West River. PEI

51-96

30

0

0

0

7-6-94

West River. PEI

53-91

30

0

0

0

28-6-94

Cocagne. NB

65-75

30

0

0

0

12-7-94

Malagash. NS

52-63

30

0

0

0

22-8-94

West River. PEI

57-91

30

0

0

0

24-8-94

Cocagne. NB

63-71

30

0

0

0

13-9-94

Malagash. NS

54-84

30

0

0

0

2-11-94

West River. PEI

51-96

30

0

0

0

22-11-94

Malagash. NS

43-83

30

0

0

0

14-6-95

Shippagan. NB*

47-69

120

0

0

0

20-6-95

Shippagan. NB

<2

60

0

0

0

7-10-95

Shippagan. NB

<8

60

0

0

0

22-8-95

Bouctouche. NB

50-79

6

0

0

0

27-10-95

Little Harbour, NS* (m

>25

8

2.5

H

IM

27-10-95

Ellerslie. PEI*

>25

4

0

0

0

14-5-96

Ellerslie. PEI

2-6

60

0

0

0

3-6-96

Ellerslie, PEI*

28-53

25

8.0

L

2M

11-6-96

Shippagan. NB* (n)

30-50

15

47.0

H

2M:4F:U

27-7-96

Ellerslie. NB*

30-40

6

0

0

0

27-7-96

Ellerslie. NB

<8

20

0

0

0

11-8-96

Little Harbour. NS*

72-105

60

0

0

0

29-8-96

Little Harbour. NS*

72-81

1

0

0

0

9-9-96

Orwell. PEI

3-7

\5()

0

0

0

11-4-97

Corkumsls. NS*(n)

3.3-67

26

31.0

H

5M:3F

7-6-97

Ellerslie. PEI

36-71

29

3 1 .0

H

6M:3F

7-6-97

Vernon River. PEI

>25

30

0

0

0

9-6-97

Pugwash. NS

42-86

60

1.7

L

IM

9-6-97

Powell Cove. NS

45-75

60

(1

0

0

9-6-97

Tatamagouche. NS

42-90

60

0

0

0

24-06-97

Shippagan. NB (nl

<I0

48

0

0

0

20-10-97

Shemoguc, NB (n)

7-22

121

0

0

0

20-10-97

Bouctouche, NB (n)

14-21

56

0

0

0

22-10-97

Vernon, R, PEI (n)

>25

30

0

0

0

QPX IN THE Northern Quahog

45

TABLE 1.

Continued.

Lengths

***Sex Ratio

examined

Prev.

**Inf.

(Infected

Date

Collection Site

(mm)

No.

(%)

Levels

Quahogs)

22-10-97

Tatamagouehe. NS (nl

>25

38

(1

(1

0

23-10-97

Bale Ste- Anne. NB (n)

>25

30

0

0

0

11-12-97

Ellerslie. PEl

28-33

30

0

0

0

15-01-98

Ellerslie. PEl* (n)

>25

25

0

0

0

15-01-98

Ellerslie. PEl*

>25

4

0

0

0

4-5-98

Little Harbour. NS*

70-98

60

0

0

0

8-5-98

Shemogue. NB (n)

<10

10

0

0

0

12-5-98

Bouctouche. NB (n)

<10

10

0

0

0

14-5-98

Ellerslie. PEl

<10

40

0

0

0

14-5-98

Ellerslie. PEl

<5

60

0

0

0

24-6-98

Ellerslie. PEl

29-95

60

6.7

H

1M:3F

26-5-98

Shediac Bridge. NB

89-102

6

0

0

0

5-6-98

StCecile. NB(n)

<10

26

0

0

0

15-7-98

St Andrews, NB

44-87

40

10.0

H

3M:1F

30-7-98

Vernon River, PEl

18-25

30

6,7

M

2U

30-7-98

Vernon River. PEl (n)

20-25

30

0

0

0

4-8-98

Wallace. NS

<10

29

0

0

0

4-8-98

Wallace, NS(n)

<I0

31

0

0

0

28-8-98

West River. PEl

80-100

30

3.3

M

IF

22-9-98

Shippagan, NB (n)

16-22

21

0

0

0

23-9-98

St Andrews

40-77

29

6.9

M

2M

6-10-98

StCecile. NB(n)

11-24

40

0

0

0

9-10-98

StCecile. NB(n)

17-27

19

0

0

0

13-10-98

Bouctouche. NB (n)

16-22

9

0

0

0

15-10-98

St Mary's Bay, NS

32-63

60

0

0

0

20-10-98

Shippagan. NB

13-30

60

0

0

0

20-10-98

Shippagan. NB (n)

15-20

23

0

0

0

26-10-98

Vernon River. PEl

19-25

31

42.0

M

4M;9U

26-10-98

Vernon River. PEUn)

19-31

32

0

0

0

27-10-98

Baiede Vin. NB(n)

>25

45

0

0

0

27-10-98

Bale de Vin, NB

9-14

60

0

0

0

27-10-98

Percival River, PEl

43-63

30

3.3

M

IF

2-11-98

Wallace, NS (n)

7-16

27

0

0

0

2-11-98

Wallace. NS(n)

19-31

30

0

0

0

Total

3047

*- hatchery broodstock

(n) - Meicenaiia mercenaria variety notula

**- H-heavy, M-moderate, L-light

*** - M-male. F-female. U-undetermined (restinn/immature)

RESULTS

Diagnostic Survey 1990-98

No gross clinical signs were observed in any of the quahogs
examined for tfie diagnostic survey, including clams with high
intensities of infection detected using histological examination.
QPX was found in M. mercemma and M. m. var. notata from all
three provinces. Prevalences ranged from 1.7% in wild quahogs
from Pugwash, NS, in 1997, to SO'/r in moribund broodstock from
the Ellerslie hatchery, PEl, in 1990 (Table 1). Of 3047 quahogs
examined, 64 showed evidence of QPX infection i% P = 2,2)
(Figure 2). Intensity of infection ranged from light to heavy. The
size range of quahogs infected by QPX ranged from 18.3-92.5 mm
(Table 1), The sex ratio of infected quahogs was 30 male: 1 8 fe-
male: 16 undetermined (resting stage or immature), which was not
significantly different from 1:1 (x". P > O.IOl. The sex ratio of

uninfected clams, however, was significantly different from 1 : 1
(996 male: 862 female: I 125 unidentified (resting stage or imma-
ture): X". P < 0.005).

Of all the infected clams, the most commonly infected tissues
were the gills (34%). mantle (3l7f ) and gonads Ol'^r) (Table 3).
The digestive gland and foot were less commonly infected (12 and
5%, respectively).

Seasonal Suney 1996-97

No gross clinical signs were observed during necropsy of the
quahogs collected for the seasonal survey. Clams from two of the
four sites showed evidence of QPX infections: Wallace (1996
only) and St. Andrews (1996 and 1997) (Table 2), The summer
sample of quahogs from Wallace had a prevalence of 6,7'* QPX
(light intensity). Quahogs from St. Andrews showed prevalences
of QPX ranging from 3.3%^ (spring and fall, 1996), at light inten-
sities, to 209^ (summer 1997) at heavy intensities (Figure 3).

46

MacCallum and McGladdery

Figure I. Map of Atlantic Canada showing sampling sites positive for
QPX from all surveys and diagnostic material examined. The circle
denotes the 1959-63 QPX study of Drinnan and Henderson (1963),
diamonds denote the 1990-98 diagnostic survey, stars denote the 1996-
97 survey and triangles show sites with QPX in hatchery broodstock.
The dashed lines represents the northern-most limit of M. mercenaria.

The mean sample lengths of the quahogs examined (n = 715)
ranged from 60.0 (± 10.7) to 83.5 (± 6.3) mm in 1996 and 61.9 (±
9.9) to 83.2 (± 4,9) mm in 1997. It was difficult to tell whether the
same cohorts were sampled over the two year seasonal survey.
because quahogs grow slower, once mature, in cooler northern
waters than in warmer waters to the south. The highest water
temperatures occurred in August at all sites and temperature ranges
(8-24'C) were relatively consistent between sites for both years
(Table 2). All four sites had moderate to high salinities (20-329?()
which were consistent over the survey period (Table 2). The high-
est prevalence (20'}f ) was found in clams from St. Andrews in the
summer of 1997 (Tabic 2). The second highest prevalence ( 13.3%)
was found in the spring, 1997, sample. QPX was detected in one
sample of clams from Wallace, in the summer of 1996 (6.7%). The
temperature recorder on the Wallace bed recorded air tempera-
tures, at low tide, as low as 0 "C in May. 1996 and as high as 34
"C in August. 1996 and 1997. The sex ratio of QPX-positive
quahogs was 5 male:l female in 1996 and 8 males:4 females in
1997, which was not significantly different from 1 : 1 (x". P > 0. 10.
1996 and P > 0.25, 1997). The sex ratio of uninfected clams was
173 male: 178 female: 1 unidentified (resting s(agc or immature) in
1996. and 187 male: 159 female: 1 unidentified (resting stage or
immature) in 1997. which was not significantly different from 1:1
(X-, P>0.90. I996andx-. P>0.I0, 1997). Of the infected clams,
the most commonly infected tissues were the gonads (28'7r ) and
mantle (22%). although (he digestive gland and foot (17%) and
gills (1 1%) also showed high levels of infection (Table 3).

DISCUSSION

QPX or QPX-like organisms were first found in Atlantic
Canada in the late 1950's/ early 1960's in wild M. mercenaria
from Neguac. NB (Miramichi River estuary) in the Gulf of St
Lawrence (Drinnan and Henderson 1963). Prevalences ranged
from 50% in weak and dead quahogs to 5% in apparently healthy
quahogs (Drinnan and Henderson 1963). Accumulated mortalities
in grow-out tests conducted between 1959 and 1960 ranged from
60-90%' in native quahogs to 20-25% in apparently healthy qua-
hogs transplanted from nearby Miramichi beds (Drinnan and Hen-
derson 1963). QPX was not investigated further until the early
1990's when it was found in moribund quahogs (15-30 mm in
length) being conditioned for spawning at a hatchery in PEI
(Whyte et al. 1994). The connective tissue and muscle were found
to be infected with "an invasive eukaryote organism" identical to
that described by Drinnan and Henderson ( 1 963 ) and was given
the non-taxonomic acronym "QPX" for "Quahog Pararsite Un-
known" (McGladdery et al. 1993; Whyte et al. 1994).

QPX or QPX-like organisms have been found in quahogs from
New Jersey in 1976 (Smolowitz et al. 1998) and more recently in
quahogs from Virginia (Ragone Calvo et al. 1997. Ragone Calvo
et al. 1998) and Massachusetts (Smolowitz ef a/. 1998). During the
summer of 1995. 1.5-2 year old quahogs planted on aquaculture
leases in Cape Cod, experienced mortalities with prevalences rang-
ing from 10% in "non diseased" clams to 90% in di,sea.sed clams
(Smolowitz et al. 1998). Cultured 1-2 year old clams (19-89 inm)
from the eastern shore of Virginia ranged from 8-20% in 1996. to
4-48%' in 1997. with associated mortalities estimated at 10-20% in
the latter (Ragone Calvo et al. 1998).

The Miramichi Estuary of the Gulf of St. Lawrence is the
northern-most geographic limit of M. mercenaria. thus QPX does
not occur in the St. Lawrence River as mentioned in Ford et al.
(1997) and Smolowitz et al. (1998). Prevalences of QPX in M.
mercenaria and M. m. var. notata in the 1990-98 diagnostic survey
ranged from 1.7% in M. mercenaria in Nova Scotia, to 80% in
broodstock being conditioned for spawning at the Ellerslie Shell-
fish Hatchery. PEI (Table 1 ). No mortalities attributed to QPX
have been found in wild quahogs in Atlantic Canada since the
original cases reported by Drinnan and Henderson (1963). how-
ever, open-water mortalities aie known lo have occurred without
being investigated (Drinnan. pei's comm.). The highest prevalences
of QPX recorded in the 1 990-98 diagnostic survey were in both
cultured native and notata variety broodstock from all three Mari-
time provinces (Table 1 ). The 1996-97 survey found 6.7% preva-
lence of QPX in clams from Wallace. N.S. and 3.3-20% QPX in
an isolated native population at St. Andrews. Prevalences in qua-
hogs at both sites were comparable to those found in US wild
clams (8-90%, Smolowitz et al. 1998, Ragone Calvo et al. 1997
and 1998).

The si/e range of infected quahogs in this study I'anged from 1 8
lo 1 10 mm (Tables I and 2). Before 1998, the reported size range
of QPX infected quahogs was > 35mm shell length. Despite their
small size, the 18-25 mm cultured M. mercenaria from Vernon
River. PEL had been in the field for one year and were approxi-
malcly 1.5 years old (Burleigh pers comm.). Ford et al. (1997)
examined tissue .sections of 2203 seed quahogs (< 1-20 mm and no
more than a few months old) from 13 different hatcheries in six
Stales. No evidence of QPX or QPX-like organisms was detected.
QPX was also not detected in 756 hatchery-produced quahogs
after a year of field grow-out (Ford el al. 1997), thus, it was

QPX IN THE Northern Quahog

47

TABLE 2.
Collection details and QPX results for 1996-97 survey

Water

temperature

Lengths

(°C) and salinity

examined

Prev.

**Inf.

***Sex Ratio

Date

Collection Site

{"r,) at collection

(mml

No.

(%)

Levels

(Infected Quahogs)

2-5-96

Wallace. NS

8°

25%<.

63-78

28

0

0

0

21-5-96

St Andrews, NB

10°

26%c

58-83

30

3.3

L

IM

5-6-96

West River. PEI

15°

25%.

69-91

30

0

0

0

11-6-96

Shediac Bridge. NB

15°

26%.

56-89

30

0

0

0

1-8-96

Wallace. NS

24°

30%<.

65-78

30

6.7

L

2M

19-8-96

West River. PEI

22°

26%c

51-86

30

0

0

0

23-8-96

St Andrews. NB

22°

26%o

43-79

30

6.7

M

2M

27-8-96

Shediac Bridge. NB

23°

26%c

57-81

29

0

0

0

1-10-96

Wallace. NS

10°

20%o

64-84

30

0

0

0

17-10-96

West River. PEI

10°

23%o

52-91

30

0

0

0

21-10-96

St Andrews. NB

11°

26%o

43-76

30

3.3

H

IF

25-10-96

Shediac Bridge. NB

9°

30%r

73-95

30

0

0

0

27-5-97

St Andrews. NB

10°

25%r

53-87

30

13.3

L

3M:IF

5-6-97

Shediac Bridge, NB

14°

M%c

38-101

30

0

0

0

5-6-97

Wallace. NS

10°

26%c

51-88

30

0

0

0

9-6-97

West River. NS

16°

26%o

50-95

30

0

0

0

13-8-97

St Andrews. NB

21°

32%,.

46-78

30

20,0

H

4M:2F

18-8-97

Wallace. NS

24°

31%<,

66-93

30

0

0

0

26-8-97

West River. PEI

24°

29%o

50-93

29

0

0

0

29-8-97

Shediac Bridge. NB

21°

31%<,

54-99

29

0

0

0

9-10-97

St Andrews. NB

10°

32%<,

50-79

30

6.7

H

1M:1F

16-10-97

West River. PEI

8°

27%,,

54-102

30

0

0

0

20-10-97

Wallace. NS

11°

32%o

71-93

30

0

0

0

24-10-97

Shediac Bridge, NB

10°

32%.

44-90

30

0

0

0

Total

715

** - H-heavy, M-moderate, L-lighi

*** - M-male. F-female, U-undeterniined (resting/immature)

concluded that hatchery-produced seed are unlikely to be infected
by QPX. Conversely. Whyte et al. (1994) found QPX in infected
hatchery-reared quahogs ranging from 15-30 mm in shell length.
The report did not distinguish the exact size or age of infected
quahogs. and no attempt was made to characterize the relationship
between individual quahog size and presence of QPX (Whyte.
pers, comm,). Due to colder growing conditions in the Gulf of St.

0 +

■ N.R

ap.E.1

■ N.S.

I

m

■nmefYcais)

Figure 2. Historical and geographic summary of QPX in M. merce-
naria and M. mercenaria variety notata from Atlantic Canada.

Lawrence, compared with Massachusetts and Virginia, it is pos-
sible that the < 20mm quahogs examined by Whyte et ai. (1994)
could have been the same age as larger quahogs from further south.
All QPX findings to date in the US have been from quahogs
typically 1 to 2 years-old (Ragone Calvo et al. (1997 and 1998) and
Smolowitz et al. (1998)),

The taxonomic affinity of QPX is currently under investigation
in both Canada and the U.S, (Smolowitz et al. 1998: Maas et al.
1999). Whyte et al, (1994) suggested that the QPX was similar to
the labyrinthulids and thraustochytrids. belonging to the Phylum
Labyrinthomorpha (Pokorny 1985), Members of these groups are
common saprophytes in marine environments (Porter 1990), and
have also been reported to cause disease in a number of molluscs

TABLE 3.
Prevalence of QPX in different tissues of infected quahogs.

Tissues

Gill

Mantle

Gonad

Digestive gland

Foot

1990-98

1996-97

percent of

percent of

nfected clams

infected clams

(n = 64)

(n = 18)

34

11

31

22

31

28

12

17

5

17

48

MacCallum and McGladdery

25

^ 20

«j 15

c

es 10

1

■ St. Andrews
D Wallace

1

1 1

■n

1

■

1

r

iT

1

May-96 Aug-95 OcI-96 Ma.v-97 Aug-97 Oct-97

Time (months)

Figure 3. Prevalence of QPX from the two year repeated survey 1996-
97. Solid black represents St. Andrews (Sam Orr Pond), N.B.) clams;
unfilled box represent Wallace, N.S. clams. Clams from Shediac
Bridge, N.B. and West River, P.E.I, were negative for QPX.

(Polglase 1980: McLean and Porter 1982; Jones and O'Dor 1983;
Bower 1987a). One Labyrinthulid. Liibynnthidoides haliotidis, has
been linked to mortalities of up to 100% of nursery-held juvenile
abalone, Haliotis kamtschatkana. in British Columbia (Bower
1987a). Further investigation found that L. haliotidis is transmitted
directly from abalone to abalone by a flagellated zoospore stage
(Bower 1987b). Motile zoospores have been identified in both
Canadian (Whyte et al. 1994) and U.S. (Kleinschuster et al. 1998)
QPX cultures, therefore, it is likely that QPX is also transmitted
directly. The likelihood of direct transmission would also be ex-
pected to be heightened in holding facilities or nurseries where
clams are held in close proximity to each other in raceways, down-
wellers or upwellers. Further research on QPX transmission to
both M. mercenaria and M. m var. notatci is needed to ftilly un-
derstand the epizootiological potential of this parasite.

Smolowitz et al. (1998) noted thickened, retracted, light tan.
swollen mantle edges in diseased clams from Cape Cod. Occa-
sionally, yellow/tan nodules, 1—4 mm in diameter, were also ob-
.served along the mantle edges or in the mantle areas adjacent to the
anterior adductor muscle. Shell margins were chipped and diseased
quahogs showed variable amounts of sand embedded between the
mantle edge and shell (Smolowitz et al. 1998). Smolowitz el al.
(1998) postulated that shell chipping was a result of quahogs at-
tempting to close their shells on the sand and sediment caught in
the quahog's mucus. Soft tissues and shells were examined for all
clams used in this study, however, no gross pathological changes
have been seen, to date, in infected quahogs from Atlantic Canada,
including heavily infected individuals.

Both sexes of quahog were infected with QPX . Prevalences in
males were significantly higher than in females in the 1990-98
survey, but no significant differences were found between males
and females in the 1996-97 seasonal survey. Uninfected quahogs
examined in both surveys had a sc\ ratio of 1:1. No diffcrejices
between the sex o( infected clams have been reported elsewhere, to
date.

The most commonly infected tissues in infected clams from the
1990-98 diagnostic survey were the gills iM'/i I. mantle (.M '/,) and
gonad (.^K/r). Similar results were found in the 1996-97 seasonal
survey (gonad-28'f and mantle-227f ). Smolowitz et al. (1998)
found that the most commonly infected tissues of infected quahogs
from Cape Cod were the mantle (917,) and gill (6.'^7f). Ragone
Calvo c't ill. (1998) also found the mantle (ft.V;-; ) and gills (.^.S'f i to
be ihe most tVcqucnIly inleclc(.l tissues in infected i.|ualu)gs from

Virginia. The digestive gland (12-17%) and foot (5-17%) were
less commonly infected in both 1990-98 and 1996-97 surveys.
Smolowitz et al. ( 1998) also found the kidney (20-25%), adductor
muscle (0-6%), foot (3-13%), digestive gland (0%), ganglia/
mantle nerves (0%) and palps (0%) to be less heavily infected.
Ragone Calvo et al. (1998) also observed infections in the mus-
culature of the foot, sinuses and connective tissue of the kidney
and connective tissue of the digestive glands (4. 11, and 15%,
respectively). Drinnan and Henderson (1963) found QPX in the
gill, kidney, connective tissue, foot, and heart of infected quahogs
from New Brunswick but did not differentiate between levels of
infection and tissue site. Although not quantified for this study, we
found no evidence of palp, nerve or adductor muscle infections.

There are at least three environmental factors which may
favour the proliferation of QPX in both hatchery and wild clams:
i) stocking density; ii) water temperature; and iii) genetic suscep-
tibility. Stocking density may have played an important part in the
epizootic incident of QPX in wild quahogs from Neguac, N.B.
(Drinnan 1961). The typical or natural stocking density of wild
adult (> 20 mm) quahog populations in Atlantic Canada is ap-
proximately 4-5 clams m"~ (T. Landry. Fisheries and Oceans
Canada, pers. comm.). Historically some quahog farming opera-
tions have planted seed (< 3 mm) at densities ranging from 357-
43,01 1 m"- (Judson et al. 1977: MacPherson et al. 1978: Wither-
spoon 1984) with no outbreaks of QPX reported. To date, only one
report by Kraeuter et al. ( 1998) has examined the effects of plant-
ing density on proliferation of QPX. Juvenile quahogs (< 10 mm)
from New Jersey were planted on intertidal and subtidal sites at
three densities; 215. 430. and 860 clams nr" per plot. The preva-
lence of QPX increased during the four-month experiment, but no
significant effect, due to density or location, was detected (Kraeu-
ter et al. 1998).

Water temperature and/or salinity may also be significant fac-
tors influencing the prevalence of QPX. All four sites in the sea-
sonal survey experienced relatively similar temperature regimes, at
the time of collection, ranging from 8 °C (May, 1996, and October,
1997) to 24 °C (August, 1996-97). Salinities ranged from 25-32%r
between 1996 and 1997. The clam beds at St. Andrews, Shediac
Bridge and West River are all sub-lidal ( 1-3 m depth depending on
tide level), whereas the Wallace site is completely exposed during
each low tide. As a result, seasonal temperatures at the Wallace
site, ranged from 8-28 °C (from May to October, 1996 and 1997).
with air temperatures reaching as high as 34 "C at low tide. QPX
was detected at the St. Andrews location in temperatures ranging
from 10-22 C and salinities ranging between 25-32^^1. Histori-
cally, water temperatures at the St. Andrews site (Sam Orr Pond)
range from -0.1-25 °C (Medcof 1961, S.M.C. Robinson. Fisheries
and Oceans Canada, pers. comm.). The single QPX infection de-
lected at Wallace occurred in August. 1996. when the waler tem-
perature was 24 "C and salinity was 30'/t'(.

Ragone Calvo et al. ( 1998) collected quahogs from 1 S different
sites in Chesapeake Bay and coastal Virginia, where salinities
ranged from 15 to ?i47ti. QPX was only detected in clams from
three coastal lagoons, where salinities ranged from 30 to 34%f
(Ragone Calvo et al. 1998). Theses authors point out that the
absence of QPX from more moderate salinities (l5-25%f) may
have been related to a limitation in QPX's salinity tolerance or
have reflected sampling bias (Ragone Calvo et al. 1998). In sea-
sonal colleclions from one Virginia coastal site, between July.
IWfi and June. 1W7. Raizone Calvo et al. (19981 observed the

QPX IN THE Northern Quahog

49

highest prevalences and most severe infections in November and
May samples. Smolowitz et al. (1998) reported that quahog mor-
talities in Massachusetts, associated with QPX infection, were
highest in August and October. Temperature and salinity are
known to be related to proliferation of other bivalve parasites such
as Perkinstis niariniis. Haplosporidium costale and Haplospo-
ridiiim nelsoni (Bower et al. 1994, Ford et al. 19991, thus it is
possible that QPX proliferation and pathogenicity may also be
influenced by temperature and/or salinity.

Clam harvesting practices may also influence QPX prolifera-
tion. Harvesting of quahogs in Atlantic Canada has, traditionally,
been done by hand (forks, tongs and rakes), although hydraulic
harvesters are also used (Bourne 1989). The population of quahogs
in Neguac, N.B.. were harvested using an escalator harvester when
mortalities started to increase, both in air storage and at Hay Island
holding beds, between 1957 and 1959 (Drinnan 1960). Although
no clear association between harvest methodology and QPX has
been determined, its effect on physiological stress and defense
capability seems worth investigating further.

In conclusion, QPX seems to be ubiquitous in both wild and
cultured quahogs from the Maritime Provinces and is reported for
the first time in quahogs from the Bay of Fundy. In light of past
mortalities associated with this parasite, especially in hatchery
broodstock being conditioned for spawning, QPX may present a
significant challenge to development of quahog aquaculture in our
region. The dynamics of infection and pathogenicity under differ-
ent holding and handling conditions require more investigation to
manage pathogen proliferation. This is especially important as
uninfected populations seem to be few. if any, in Atlantic Canada,
making selection of QPX-free broodstock an impractical solution.

ACKNOWLEDGMENTS

We wish to thank Dr. S.M.C. Robinson, J. Martin, R. Chandler
(Dept. Fisheries and Oceans, Canada), E. Semple (Wallace, N.S.),
J. and R. Caissie (Caissie Cape, N.B.), B. Murley (New Haven,
P.E.I. ), P. Burleigh, N. McNair (P.E.I. Dept. Fisheries and Envi-
ronment) and D. Methe (N.B. Dept. of Fisheries and Aquaculture)
for their assistance with collections. Dr. B.A. MacDonald (Uni-
versity of New Brunswick. Saint John) and R.E. Drinnan (Mus-
quodoboit Harbour, N.S.) kindly reviewed early draft manuscripts.
Mrs. M. Stephenson, Dr. M. Maillet (DFO, Canada) and W. Morris
(U.N. B.S.J) provided valuable technical and statistical support.
This project was funded in part by the New Brunswick Alternate
Shellfish Aquaculture Species Project, part of a Canada/New
Brunswick Cooperation Agreement on Economic Diversification.

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and Propagation of Quahog Parasite Unknown. / Shell. Res. 17:75-78.

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larval and postmetamorphic bay scallops, Argopeclen irradians (Lama-
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from hatchery-spawned seed stock 1975-1978. Prince Edward Island
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& E.C. Bovee (eds). Illustrated Guide to the Protozoa Society of Pro-
tozoologists, Lawrence, KS. pp. 318-321.

Polglase, J.L. 1980. A preliminary report on the Thraustochytrid(s) and
Labyrinthulid(s) associated with a pathological condition in the lesser
octopus (Eledone cirrhosa). Bot. Mar. 23:699-706.

Porter. D. 1980. Phylum Labyrinthomycota. /«: L. Margulis. J.O. Coriiss.
M. Meilkonian & D.J. Chapman (eds). Handbook of Protoctista. (Jones
& BartleU, Boston), pp. 388-398.

50

MacCallum and McGladdery

Ragone Calvo, L.M., J.G. Walker & E.M. Burreson. 1997. Occurrence of
QPX. Quahog Parasite Unknown in Virginia hard quahogs, Mercemiria
mercenaria. J. Shell. Res. 16:335-336

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distribution of Quahog Parasite Unknown, in hard quahogs. Merce-
naria mercenaria. in Virginia. USA. D/.v. Ac/iiar. Org. 33: 209-219.

Smolowitz. R. 1998. QPX. a Protozoan parasite of hard quahogs Proceed-
ings from the 3rd International Symposium on Aquatic Health August
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Smolowitz. R.. D. Leavitt & F. Perkins. 1998. Observations of a Protistan

disease similar to QPX in Mercenaria mercenaria (hard quahogs) from
the coast of Massachusetts. / of Invert. Path. 71:9-25

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Parasite X). a pathogen of northern quahog Mercenaria mercenaria
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Witherspoon. N.B. 1984. An investigation of the aquaculture potential of
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Journul of Shellfish Research. Vol. 19, No. 1. 51-56. 2000.

AGE AND SIZE OF MERCENARIA MERCENARIA IN TWO SISTERS CREEK,

SOUTH CAROLINA

ARNOLD G. EVERSOLE,' NATHALIE DEVILLERS,' and
WILLIAM D. ANDERSON-

^ Department of Aquacidture. Fisheries and Wildlife

Clemson University

Clemson, South Carolina

29634-0362
'South Carolina Department of Natural Resources

Charleston. South Carolina

29422-2559

ABSTRACT Northern quahogs. Merceiiaria mercenaria (L.). were sampled from four sites in Two Sisters Creek, South Carolina.
Shell lengths (SL) were measured and ages estimated from increments in shell sections. Mean SL of individuals collected at the two
sites near the mouth of the creek were significantly larger than those collected in the upper reaches of the tidal creek. The back-
calculated mean SL. however, were similar among sites within most age classes. Mean ages of individuals near the mouth were
significantly older than those from the upper reaches. Differences in the population age structure were also observed among sites.
Several factors are explored to explain the upstream pattern of decreasing SL and ages of quahogs in Two Sisters Creek.

KEY WORDS: Mercenaria. quahogs. clams, age. size, growth

INTRODUCTION

Commercial densities of northern quahogs. Mercenaria merce-
naria (L.), occur in small creeks that dissect extensive tidal
marshes of South Carolina and Georgia (Anderson et al. 1978,
Walker 1987, 1989). Two Sisters Creek, South Carolina, which is
representative of this type of habitat, became part of a State Shell-
fish Ground (SSG-134) in 1986. Reported landings from SSG-134.
which also included Ashepoo River, Rock Creek, Atlantic Intra-
coastal Waterway, and Ashepoo-Coosaw Cut, averaged only 145
bags/year before an exploratory survey of quahog resources in
Two Sisters Creek (S.C. Department of Natural Resources, unpubl.
data). Resource managers perceived that quahog exploitation was
limited before and after Two Sisters Creek became part of SSG-
134. The mean (± SD) shell lengths (anterior posterior axis, SL) of
quahogs collected during an earlier exploratory survey of May 6,
1987 indicated that individuals sampled from two sites nearer the
mouth were larger (88.1 ± 10.37 mm and 87.1 ± 10.30 mm) than
those collected from a mid-way site (69.0 + 14.81 mm) and a site
farther up Two Sisters Creek (61,6 ± 19.26 mm). A similar trend
was observed in Christmas Creek. Cumberland Island. Georgia.
where relatively higher numbers of larger quahogs (i.e., chowders)
were found near the creek's mouth than in the upper reaches of the
tidal creek (Walker 1987). Differences in quahog size among sam-
pling sites could have resulted either from variations in growth rate
or age of the respective populations.

The objectives of this study were to test the null hypotheses that
growth rate and age were similar among quahog populations in-
habiting four different upstream sites in Two Sisters Creek, South
Carolina (Fig. I ). Age estimations, based on annual growth incre-
ments within the shell (e,g„ Arnold et al, 1991, Jones et al. 1990,
Peterson et al. 1985), were used to compare age and SL of differ-
ent-aged quahogs from the four sample sites in Two Sisters Creek.

MATERIALS AND METHODS

Study Sites

Quahogs were sampled from four sites within Two Sisters
Creek, South Carolina, on February 25. 1994. The site closest to

the mouth was designated site 1, and sites 2, 3, and 4 were located
progressively farther up the tidal creek (Fig. 1 ). Midchannel depths
of the four sites at flood tide were 7.60 m, 8.20 m, 4.30 m. and 3.35
m. respectively. Tidal range was about 2 m. Bottom water tem-
peratures and salinities at the time of collection ranged from 12 to
14 °C and 21 to 25 gl"'. An estimate of bottom types indicated that
sites 1, 2, and 3 were a mixture of mud, sand, and shell; whereas,
site 4, although similar, seemed to contain more clay.

Sampling

Quahogs were collected at flood tide from subtidal sites with a
hydraulic escalator harvester configured with a Maryland-type
head. The mesh size of the escalator conveyor would retain qua-
hogs >32 mm SL if not covered by mud or shell. In this event,
smaller quahogs would be harvested. Subtidal bottoms at depths of
2-8 m were sampled across the width of the creek. Sampling at
each site continued until sample sizes were ^ 100 individuals.
Quahogs were returned to Clemson University and frozen until
analysis. Shell length and height (lateral axis. SH) were measured
with calipers to the nearest 0.1 mm. After measuring, individuals
were categorized according to the following commercial size
groups: sublegals. < 44.4 mm SL; littlenecks, 44.4-67.9 mm SL;
cherrystones, 68-78 mm SL; and chowders > 78 mm SL, A sub-
sample of 50 quahogs per site, representative of the distribution at
that site, was used for aging.

Age Determination

Quahogs were shucked, and the better valve was selected for
sectioning. The valve of larger shells was cut from the ventral
margin through the umbo, with a high-speed geological saw
mounted with a diamond blade. Smaller shells were embedded in
resin epoxy to avoid fracture during the sectioning (Kennish et al.
1980). Similarly, embedded shells were cut with a slow-speed saw
mounted with a high-density diamond blade. Valves were polished
with various grit carborundum papers and then etched in \9c hy-
drochloric acid. Age was obtained by counting translucent (dark)
bands on the polished surface of a cut valve. Bands were counted

51

52

EVERSOLE ET AL.

ST. HELENA SOUND
Figure 1. Sampling sites in Two Sisters Creeli, South Carolina.

three times with two bhiid counts by the saine observer. Values
difficult to read were then washed and exposed to acetone before
pressing against an acetate sheet (Kennish et al. 1980, Ropes
1984). The age of these clams was obtained by counting bands
using a microfilm projector. A pattern of alternating translucent
(dark) and opaque (light) increments on sectioned valves of known
aged quahogs cultured in South Carolina waters was used to verify
the formation of annual shell growth increments in the study (De-
villers 1994).

Back-Calculated Shell Length

Shell heights from the umbo to the translucent increment for
each age increment in the sectioned valves of shells were measured
to the nearest 0.1 mm (see Jones et al. 1990). Measurements were
limited to the first 12 increments, because of the difficulty asso-
ciated with correctly measuring small increments thereafter. These
measurements were then converted to SL using the equation (Ever-
sole, unpubl. data):

In SL

.049.^ In SH - 0.0136; r- = 0.997. n

1.171

Statistical Procedures

Analysis of variance (ANOVA) was used to determine signifi-
cant differences in SL between the field sample and subsamples.
Significant differences in age, SL. and back-calculated SL were
also determined by ANOVA. Paired means were compared with
the least significant difference (SAS I98.'i). Alpha level was set at
0.05 for these analyses.

RESULTS

Shell Lengths

Mean SL of the quahogs sampled from sites 1 and 2 were
similar but significantly (P < 0.03) larger than those animals
sampled at sites .3 and 4 (Table 1 ). Individuals from site 3 were
also significantly larger than those quahogs sampled at site 4. The
mean SL and ranges of these quahogs used for age determination
were similar to that observed in the field sample (Table 1 ).

The frequency distributions ot commercial qualiog sizes col-
lected trom the four sites are presented in Figure 2. Chowders
dominated the collections at site I (94.2%). site 2 (96. 2'*). and site
3 (69.8'/f ). Site 4 contained 37.8% littlenecks and similar percent-
age of cherrystones (26..3%) and chowders (27.8%). Only 1.0%.

TABLE L

Mean (± SD) and range (in parenthesis) of the shell lengths (mm) of

Mercenaria mercenaria from four sites in Two Sisters Creek, South

Carolina. Values in a column not sharing the same letter superscript

are significantly different at P < 0.05. There was no significant

difference between field and subsample mean SL.

Field Sample

Subsample

Site

N

Shell Length

104
104
106
151

93.27 ± 11.2-V'
(41.9-110.7)

94.27 ± 1 1 .60-'
(36.0-117.5)

79.05 ± 17.57"
(32.2-101.9)

67.84 ± 14.32'
(33.2-97.6)

N

Shell Length

50

93.08 ±12.68"

(41.9-110.7)

50

94.10+ 11.32"

(37.7-108.8)

50

78.39 ± 17.81"

(32.2-100.*)

50

67.92 ±14.59'

(33.2-93.5)

' Site 1 was closest to the mouth while sites 2, 3 and 4 were progressively
further upstream in Two Sisters Creek.

1.9%, 8.5%, and 7.9% of the quahogs were sublegal size (< 44.4
mm SL) in collections from sites 1. 2. 3, and 4. respectively.

Age

The oldest age of the sampled quahogs was 29 years from site
1. and the youngest was 1 year from sites 2 and 4 (Fig. 3). Sig-
nificant differences (P s 0.05) were detected among the four sites
in the average age of quahogs. Individuals on average (± SD) from
site 2 ( 16.7 ± 4.70 years) were older than animals from site 1 (14.9
± 5.37 years) and in turn, quahogs from site 3 (8.4 ± 3.96 years)
and site 4 (4.7 ± 2.30 years) differed from each other and were
significantly younger than those from sites 1 and 2. The range of
ages in the sample from site I was 28 years with several (n = 10)
unrepresented age classes in the distribution (Fig. 3). The distri-
bution of ages from site 2 was similar (range and number of
missing age classes) but differed from site 1 in having an obvious
dominant age class at 15 years. The range of ages from site 3

100

80

60

?

40

>>

?n

o

c

0

0)

1

c

lOOi

0)

Site 2
(n=104)

Site 3
(n = 106)

80

I6C
40
2C
- 0

(n = 151)

11

■ ■■

y.'

.6*

Figure 2. Relative fre(iuency of commercial sizes of Mercenaria mer-
cenaria from four sampling sites in Two .Sisters Creek, South Carolina.
ComnuTcial sizes were suhlegals (< 44.4 mm SLl, littlenecks (44.-Mi7.9
mm SI.), cherr\ stones (68-78 mm SI.) and chowders (> 78 mm SL).

Age and Size of Quahogs

53

30-

20

Site1

10

.»««. .

ILlUllL^

•-,

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Site 2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Site 4

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Age (years)

Figure 3. Age structure of 50 Mercenaria mercenaria collected from
each of four sites in Two Sisters Creek, South Carolina.

encompassed 18 years with six missing age classes: whereas, those
animals aged from site 4 spanned 10 years with only one missing
age class at 9 years. None of the quahogs sampled from site 4 were
older than 10 years; whereas. 90%. 96% .and 22% of quahogs from
sites 1. 2, and 3 were older than 10 years of age.

Back-Calculated Shell Lengths

The back-calculated mean SL of quahogs from sites 1. 2. and 3
were similar in size from age 1 through 11 years (Table 2). One
significant difference among the four sites occurred from ages 1 to
4 years when quahogs from site 4 were significantly larger (P :£
0.05) than those quahogs from sites 1. 2. and 3. The only other
significant size difference in back-calculated SL occurred at 12
years (Table 2). Quahogs reached reproductive size (35 mm SL,
Eversole in press) between 1 and 2 years and commercial size
(44.4 mm SL) between 2 and 3 years.

DISCUSSION

Comparison of quahog sizes between this and other studies
needs to be done with caution because of different sampling gear
efficiencies and the common problem of the under representation
of small individuals in samples (Fegley. in press). Although
Walker (1987, 1989) used a different collection method, he did
collect a similar range of sizes as those collected in this study and
observed that chowders (> 78 mm SL) were the dominant com-
mercial size class in 43% of the 40 sites sampled in Georgia
waters. He also determined that chowders were more abundant in
areas with little or no harvesting; whereas, littlenecks were more
abundant in heavily fished areas. In a statewide survey of quahog
habitat, littlenecks were found to be the most abundant commercial
size class in South Carolina, which has a viable fishery (Anderson
et al. 1978). In Two Sisters Creek, which has not been extensively
harvested, the dominant commercial size was the chowder in three
of the four sites sampled. Greene and Becker (1978). Malinowski
(1985). Rice et al. (1989). and Walker (1989) have suggested that
the gear used to harvest quahogs is biased towards the larger sizes
resulting in differential removal of larger individuals and a shift in
the population structure toward smaller commercial sizes. Con-
versely, it is anticipated that the larger commercial sizes would

TABLE 2.

Mean (± SD) of the back-calculated shell length (mm) by age (years) of Mercenaria mercenaria from four sites' in Two Sisters Creek, South
Carolina. Values in rows not sharing the same superscript are significantly different at P < 0.05.

Site 1

Site 2

Site 3

Site 4

Age

1

2

3

4

5

6

7

8

9

10

11

12

50
50
49
48
47
46
46
46
46
46
45
40

Mean SD

27.4

± 6.95-'

47.2

± 9.28"

61.8

± 8.69"

72.2

± 8.08"

79.4

± 7.60"

83.8

± 7.29"

88.2

±7.21"

92.0

± 7.24"

95.8

±7.01-'

97.3

± 7.49"

99.9

± 7.74"

101.7

± 8.40"

50
50
49
49
48
48
48
48
48
48
48
48

Mean SD

28.1

± 6.73"

49.6

± 9.09"

62.3

± 7.86"

72.0

± 6.72"

79.0

± 6.23"

84.4

± 6.75"

88.6

± 6.85"

92.3

±7.22''

94.8

±7.16"

97.5

± 7.52"

00.2

± 7.76"

02.5

± 7.90"

50
50
44
41
41
41
39
34
26
14
11
7

Mean SD

27.3

± 9.35"

47.4

± 9.00"

63.4

±9.27"

74.3

± 8.87"

80.4

±9.12"

84.8

± 9.34"

88.6

± 8.83"

91.8

± 9.24"

93.2

± 8.76"

93.0

± 6.79"

95,4

±7.12"

95.6

± 8.45"

50
46
38
32
28
20
12

6

1

1

Mean SD

32.4 ± 6.60"
52.1 ±8.31"
65.9 ± 8.07"

76.1 ±8.54"
81.9 ±8.99"
87.3 ±8.15"

91.2 + 8.72"
95.7 ± 8.23"

101.4
102.3

Site 1 was closest to the mouth while sites 2, 3 and 4 were progressively farther upstream in Two Sisters Creek.

54

EVERSOLE ET AL.

accumulate in areas not heavily harvested. This accumulation may
explain why sites I through 3 in Two Sisters Creek were domi-
nated by chowders (Fig. 2), but it does not adequately explain the
dominance of littlenecks at site 4, which would be the least likely
of the four sites to be commercially harvested or poached because
of its size and location. In addition to the affect of harvesting, size
and age structure of populations are also influenced by growth
rates, recruitment, and mortality (Cerrato 1980). Because quahogs
at site 4 grew at the same rate or faster than the quahogs at the
other three sites, different growth rates can not solely be used as an
alternative hypothesis for explaining differences in sizes among
sampling sites.

Annual cycles of shell growth increment formation have been
observed in shells of quahogs sampled from Rhode Island to
Florida (Arnold et al. 1991. Fritz and Haven 1983, Jones et al.
1989, 1990, Kennish 1978. Peterson et al. 1985) and from South
Carolina (Devillers 1994). Mean age determined from sectioned
shells revealed that animals sampled from the sites nearest the
mouth of Two Sisters Creek were significantly older than indi-
viduals collected from site 4 in the upper reaches of the tidal creek.
Collections from sites 1 and 2 closest to the mouth also contained
the oldest quahogs and the widest spread of ages. Differences in
mean age and age frequency distribution among the sampling sites
could have resulted from sampling error (e.g.. small and under
representative samples); however, on two separate sampling occa-
sions and using the same gear, quahog size (age) decreased from
sites near the mouth to upstream sites in Two Sisters Creek. If
sampling error occurred, it was similar among sites and sampling
occasions.

The absence of quahogs older than 10 years in the collection
from site 4 may have been the consequence of a catastrophic event,
intense predation or the recent successful establishment of the
population. Low salinity periods resulting from hurricanes are re-
ported to cause extensive quahog mortalities (Wells 1961). Al-
though site 4 is more likely to be influenced by a catastrophic
event than the other three sampling sites because of its smaller
size, we have no evidence to indicate such an event occurred in this
section of the coast 10 years ago. Furthermore, it is unlikely that
such a large-scale event would have a stratified effect over such a
restricted area as from site I to site 4.

The subject of settlement and postsettlement roles in defining
macroinvertebrate soft-sediment communities has been exten-
sively reviewed by Butman (1987). Olafsson et al. (1994), and
Snelgrove and Butman (1994). Although these authors discuss
several factors important in defining adult assemblages in soft
sediments, it has not been clearly established whether adull spatial
patterns result from differential larval settlement, differential post-
larval survival, or redistribution (Armonies 1996, Bachelet et al.
1992, Peterson 1986, Wilson 1990).

Existing data indicate that hydrodynamic processes play a ma-
jor role in determining the settlement of bivalves in soft marine
sediments (e.g.. see the review by Butman 1987). Near-bottom
hydrodynamic fortes determine the fate and tlux of bivalve larvae
over a patch of bottom. These forces are particularly important in
the case with M. merceiuuia larvae because of their weak swim-
ming ability (Bachelet et al. 1992). M. nwrcciKiriii exhibited pas-
sive sctlleiiient when exposed to different sedimenl types in still
and tlume-tlow water tests (Butman 1987. Butman et al. 1988).

Field studies evaluating the importance of hydrodynamics to
recruitment are few (e.g., Carriker 1961, Mitchell 1974, Petersen
1986, Pratt 19.53. Wilson 1990). Pratl ( 1953) provided the earliest
suggestion that the distribution of quahogs was similar to the sedi-

ment panicles in Narragansett Bay. Rhode Island, implying hy-
drodynamic processes were important in quahog distribution. He
concluded from measurements of current patterns that early stage
larvae coincided with the dense assemblages of adults and that
hydrographic processes mixed and transported the larvae with time
to potential settlement sites. Carriker (1961) commented that the
mo.st striking feature of the horizontal distribution of larvae in
Little Egg Harbor, New Jersey, was its unevenness and as a con-
sequence, quahogs set in areas that did not have adults. After
studying quahog abundance and distribution in Southampton Wa-
ters. England. Mitchell (1974) came to a similar conclusion that
the distribution of adult quahogs is in part controlled by tidal
transport of the larvae produced by spawning beds. Mitchell
(1974) also hypothesizes that variation in recruitment among years
in different sites in Southampton Waters was related to the suc-
cessful transport and settlement of competent larvae.

Andrews ( 1983) observed that most of the oyster larvae carried
upriver during flood tide were transported down river during ebb
tide with the exception of those few oyster larvae trapped upriver
in oyster beds and small tidal creeks of James River. Virginia.
Andrews (1983) also postulated that upriver entrainment was more
successful in systems with low flushing rates than highly flushed
systems. The four sites in Two Sisters Creek, because of their
channel width and depth, have different flushing rates, with site 4
having the highest projected rate of the sites sampled. Quahog
larvae produced in the main body of Two Sisters Creek probably
could have been entrained in a tidal excursion at site 4. However,
considering the patchy distribution of larval quahogs and the short
window competent larvae have to set at slack tide (Carriker 1961.
Armonies 1996). the probability of setting before being flushed
from the small tidal creek was probably low. If entering and setting
larvae survived predation pressures, perhaps a resident population
of quahogs would have been established and served as a source of
larvae for future recruitment at site 4. Because quahogs have a
tendency to spawn at ebb slack water and be transported upstream
with the subsequent flood tide (Carriker 1961), larvae from an
established population at site 4 would have an increased probabil-
ity of being retained in the lidal creek and recruiting to the popu-
lation.

Another explanation for the different age distributions of qua-
hogs in Two Sisters Creek involves the resuspension and distri-
bution of postlarval individuals. Shifts from the initial distribution
of recently settled Macoma hahhica have been observed in the
Wadden Sea (Armonies and Hellwig-Armonies 1992). Although
postlarval M. meirenarici possess a temporary byssus thread (Car-
riker 1961). it can be released or broken resulting in dislodgment
and resuspension by water flov\ (Butman et al. 1988). Resettlement
of postlarval quahogs at site 4 in Two Sisters Creek would also
require the appropriate hydrodynamic forces for transport and the
subsequent survival of post larvae.

Predation helps shape quahog population structure (Bricelj
1993) by selecting the smaller (younger) moic \ulnerable indi-
viduals in the population (Whetstone and Eversole 1978. 1981 ). Of
the suite of predators consuming quahogs (Gibbons and Blogo-
slawski 1989), crabs are the most important predators in South
Carolina (Whetstone and Eversole 1978). Crab-related mortalities
up lo lOO'/f were observed in juvenile quahogs planted in unpro-
tected sites in Georgia and Florida (Men/el and Sims 1962. God-
win 1968). Quahog survival is improved if small individuals are
provided some protection or if predators are removed (Eldridge et
al. 1979. Peterson 1982). Greene and Becker (1978) observed an
increase in quahog recruitment after a severe winter reduced the

Age and Size of Quahogs

55

number of blue crabs in Great South Bay, New York. Peterson
(1982) demonstrated that the roots and rhizomes of seagrasses
provide protection for infaunal species such as quahogs from some
predators. Both Peterson (1982) and Wilson (1990) concluded that
as much as 507c of the difference in quahog density between
vegetated and unvegetated areas was attributable to enhanced post-
larval survival. There is adequate information to indicate that post-
settlement processes (predation) play an important role in inver-
tebrate populations in soft marine sediments (see Olafsson et al.
1994 review). Although the distribution and abundance of preda-
tors within Two Sisters Creek could have played a role in the age
distribution of quahogs among the four sites, we have no evidence
to indicate predators either eradicated all the quahog sets 1 l-t- years
ago or selectively preyed on the older, larger individuals in site 4.

The maximum ages of quahogs are lower in faster-growing
populations in southern latitudes along the United States coast than
those observed in the slower-growing, more northerly populations
of quahogs (Ansell 1968, Jones et al. 1990). Fewer quahogs would
be expected to attain an older maximum age in site 4 if the faster-
growing individuals at this site died at a younger age than at the
other sites. Differential mortality of the faster-growing quahogs
also helps explain why fewer chowders were observed in site 4
than in the other sites.

Our results illustrate that the differences in quahog sizes among
sampling sites in Two Sisters Creek was attributable to different

age structures at the four sites. Quahogs collected from the upper
reaches of tidal creek (site 4) were younger than those collected
downstream, and none of the quahogs from site 4 was older than
10 years; whereas, the oldest quahogs from sites 1, 2, and 3 were
29. 27, and 19, respectively. Although the first steps in establishing
a population of quahogs involves settlement, the importance of
hydrodynamic processes or predation (mortality) effects on post-
larvae cannot be underestimated. Unfortunately, we have very
little data to support a hypothesis to explain the observed age
distribution in Two Sisters Creek. Future efforts to investigate
quahogs recruitment should include an integrated approach that
simultaneously considers factors such as hydrodynamic processes
and post-settlement survival. Unraveling these causes of recruit-
ment variation will be crucial to understanding the distribution and
abundance of quahogs.

ACKNOWLEDGMENTS

The authors thank Chris Kempton for his help in cutting shells
and preparing illustrations. Special thanks go to Dr. L. W. Grimes
for his help with statistical analysis. Drs. Randy Walker and John
Kraeuter generously provided comments on an earlier draft, which
greatly improved the manuscript. This research was supported by
the S.C. Agriculture Experiment Station, Clemson University, and,
as such, is Technical Contribution No. 4576.

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Annual deposition within shells of the hard clam, Mercenaria merce-
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Shellfish. Assoc. 68:42-48 .

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Jounml of Shellfish Research. Vol. 19. No. 1. 57-62, 2000.

MODELING GEODIJCK, PANOPEA ABRUPTA (CONRAD, 1849) POPULATION

DYNAMICS. I. GROWTH

A. HOFFMANN,' A. BRADBURY," AND C. L. GOODWIN'

Washington Department of Fish & Wikllife

600 Capitol Way North

Olympia. Washington 98501
'Washington Department of Fish & Wildlife

Point Whitney Shellfish Laboratoiy

1000 Point Whitney Road

Brinnon, Washington 98320
' 750 Mountain View Road

Qiiilcene, Washington 98376

ABSTRACT In Washington State, target fishing monahty rates (f ) for the geoducl< clam, Panopea abnipta (Conrad, 1849), are based
on relative changes in biomass and therefore depend on growth patterns. With these policies, higher growth rates lead to larger harvest
quotas so that applying higher rates to areas with slower growth would cause overharvesting. Therefore, in estimating growth patterns,
it is important to recognize the scale to which estimates of growth rates should be applied. In this study, we tested whether growth
parameters differed among regions and among local sites within regions in Washington, and whether they differed enough to compel
managers to create location-specific policies. Von Bertalanffy growth parameters were estimated for 1 1 sites dispersed among four
regions. Among those sites, L, ranged from 13.2 to I7..1 cm. k ranged from 0.1 13 to 0.23.S. and ;„ ranged from -0.029 to 0.806. Of
the three parameters, the growth constant k had far more influence on target fishing mortality rates (F) than either L, or /„. Statistically
significant differences in k were found among all local sites within geographic regions. However, only some of the differences were
of a magnitude to concern management policies. We have proposed a general method for calculating and then tesfing for managerial
significance when a linear relationship exists between k and the fishing mortality rate (F). Our results implied that managerially
significant differences in *: existed among local sites within Washington's geoduck management regions, posing a dilemma for
managers who, by convention, propose a single target fishing mortality rate for each region.

KEY WORDS: Geoduck, growth, hypothesis testing, managerial significance. Panopea abnipta. von Bertalanffy

INTRODUCTION

The Pacific geoduck clam Panopea abnipta is a large hiatellid
bivalve that occurs from Alaska to Baja, CA, and west to southern
Japan (Bernard 1983). Geoducks are one of the largest burrowing
clams in the world, reaching a live whole weight of .3.2.'i kg (Good-
win and Pease 1987). Adults are buried to 1 m in sand and mud
substrates from the lower intertidal to depths of more than 110 m
(Jamison et al. 1984). They dominate the biomass of benthic in-
faunal communities in many parts of Puget Sound, WA, where
they have supported a commercial dive fishery in subtidal waters
since 1970 (Goodwin and Pease 1991). Commercial dive fisheries
also exist in Alaska and British Columbia (Campbell et al. 1998),
and geoducks now provide the most valuable commercial clam
harvest on the Pacific Coast of North America. The average annual
ex-vessel value of Washington's geoduck harvest from 1990 to
1998 was US$14 million. From 1971 through 1997 annual land-
ings have averaged 1 ,540 tons.

The Washington Department of Fish and Wildlife and several
of the Washington tribes manage commercial geoduck harvest on
a regional basis. There are six regions statewide that are based
largely on legally defined tribal fishing boundaries. By coinci-
dence, these boundaries also roughly conform to major oceano-
graphic basins within Puget Sound (Ebbesmeyer et al. 1984). Cur-
rently, four of the regions (Fig. 1 ) are surveyed for biomass. and
geoduck quotas are calculated annually for each of these regions as
the product of biomass and a target fishing mortality rate. The
target fishing mortality rate (F) is based on the output of an age-
based equilibrium yield model (Bradbury and Tagart 20001, which
relies in part on a three-parameter von Bertalanffy growth func-

tion. Past studies on geoduck growth (Goodwin 1976, Breen and
Shields 1983, Anderson 1971 ) have only provided point estimates
for annual growth increments, making it impossible to determine
whether growth rates differed significantly among geographic ar-
eas. In this paper, we first estimated von Bertalanffy growth pa-
rameters for individual geoducks at 1 1 Washington sites and then
conducted hypothesis tests for differences in growth parameters
within and among the management regions.

In conducting a hypothesis test, statistical significance is not
always biologically meaningful. In this study, statistical signifi-
cance refers to whether or not the growth parameters change;
biological significance refers to how much the growth parameters
change. Statistical significance is well defined: however, biologi-
cal significance is not. In this study, determining biological sig-
nificance stemmed from the decision processes that were in place
for managing the geoduck harvest and thus are more appropriately
termed "managerial significance." Managerial significance was
determined by how much the growth parameters must change be-
fore management decisions would be altered, and this degree of
change was factored into the hypothesis-testing procedure. We
concluded that according to the management criteria given, not
only should regional specific growth parameter estimates be used,
but within some regions site-specific estimates should also be
used.

METHODS

Data

Geoducks were collected from 1979 to 1982 at 11 previously
unfished sites in Washington (Fig. 1). The sites were chosen op-

57

58

Hoffmann et al.

Strait

Dallas Bank

Tala Point

Port Gamble control

Port Gamble dredged

Thorndyke Bay

Bangor

Fishermans Point

8 Agate Passage

9 Blake Island

10 Herron Island

11 Hunter Point

Figure T. Sampling sites for geoduck growth. Also shown are bound-
aries for four of Washington's geoduck management regions; the two
regions not shown contained no sampling sites and no surveyed geo-
duck biomass.

portunistically from among those scheduled at the time for pre-
fishing surveys. However, they were spread out over the entire
commercial fishing range of Washington geoducks. Preliminary
dive surveys were conducted at each of the 1 1 study sites to map
their boundaries. The shallow to deep boundaries of a commercial
geoduck tract were set by management to be between 6 and 23 m
mean lower low water. The along-shore boundaries of a commer-
cial tract were subjectively defined on the basis of drops in geo-
duck densities, suitability of substrate, proximity to sewer outfalls
or ferry traffic lanes, etc.

Geoducks were sampled from a series of transects. The
transects were approximately 0.91 m wide, ran perpendicular to
the shore from 6 to 23 m, and were spaced approximately I km
apart (Fig. 2). In some of the larger sites, transect lines were spaced
at systematic intervals wider than 1 km. In each site, the first
transect was located opportunistically along the shoreline at one
end of the mapped bed. Because the divers were unable to see
either the substrate or the geoducks from the survey boat before
selecting the starting point, we made the assumption that the se-
lection represented a random starting point.

For logistical purposes, each transect was divided into 4.i.72-
m-long subsections. Divers used a commercial water jet to dig
geoducks from the approximate center of selected subsections.
They were instructed to dig the first 10 geoducks seen without
regard to size or any other criterion. The subsections selected were
every fourth one, ignoring transect identity, i.e.. as if the transects
were laid end to end. For example, if the first two transects were
each made up of 10 subsections, then the first, fifth, and ninth

-6 m depth

contour

-23 m

depth contour

-

loj

.9 ml at

1 (3) 1 m

Hi (61

|(7)

1

I

1 km

Transect (a)

(8)

to

1 llOi 1 (II)

|<.:.

1 (13)

4 d")

i

Transect (b)

i

1 IIS)

1 (16)

1 (17) 1 (18)

1 (19)

1 (20)

1, (21)

1

Tran.sec( (c)

(22)

1 (2!)

1 wi LOSJ-

, !i '2'*l

1 (27)

1 (28)

1

Transect (d)

Figure 2. A schematic of the sampling design (not to scale or number).
The smaller rectangles represent the hypothetical 46-m-long subjec-
tions (1-28) that make up the hypothetical transects (a-d). Ten geo-
ducks were collected from every fourth subsection (shaded) as if the
transects had been laid end to end.

subsections of the first transect would have been sampled and the
third and seventh subsection of the second transect would have
been sampled, etc. This procedure resulted in samples ranging
from 21 to 258 geoducks, depending on the size of the site
(Table I ).

A total of 1 ,2 16 geoducks were sampled from the 1 1 sites. They
were held in saltwater and returned to the laboratory within a few
days of collection for processing. All geoducks were numbered
and separated by site before processing, and the greatest anterior-
posterior length of the right valve was measured with calipers.
Thirty geoducks per site were subsampled for this growth analysis.
The subsample from each site was drawn randomly from the num-
bered shell samples, ignoring the sub.section identity. Of the 330
sampled, 234 were used in the growth analysis. The 96 geoducks
eliminated from the subsample were discarded either because they
were unreadable or because they were <15 years old.

Annual growth increments were determined using the acetate-
peel method developd by Thompson et al. (1980) and described for
geoducks in Shaul and Goodwin (1982). Growth increments for
ages beyond 25 years were not calculated, because geoducks reach
their asymptotic size between the ages of 15 and 25 years (Shaul
and Goodwin 1982). The yearly rings on individual geoducks were
measured to provide length at age data for individuals. Thus, in
the nh region (/ = 1 . . . 4), the data for the /th individual {/ =
1 . . . n^) consisted of paired observations il^^^, a,^^. j = I, . . . J,^)
where 1,^^ was the length measured for age ringy and fl^^ was the
age assigned to age ring j. The number of individuals varied by
region and the number of age rings varied by individual (because
some geoducks were less than 25 y old). From the paired obser-
vations on each individual, length was regressed on age with a
nonlinear von Bertalanffy function:

/;,

L,.J\

'H '^.1.

where Ki,, ~ MO.ir;;). Thus, each nonlinear regression produced a
set of estimated parameter values {Z.^,^,^,,. f„„| for the /th indi-
vidual (/ = I . . . n^) in the nh region (;• = I . . . 4). The variance
(t;, represented both the measurement error and the uncertainty
caused by the absence of old growth rings. Therefore, the vari-

Geoduck Growth

59

TABLE 1.

Sample size, mean shell length and von Bertalanffv growth parameter estimates (±SE) derived from shell length at age for P. abrupta at 11

sites in Washington.

No.

No.

Mean Shell

Region

Site

Dug

Subsampled

Length (cm)

L^ (cm)

k

'o

South Sound

Hunter Point

71

21

15.2

16.4 (±0.357)

0.2283 (± 0.009)

0.719 (±0.040)

Herron Island

36

23

12.5

13.2 (±0.158)

0.1 544 (±0.006)

0.422 (±0.074)

Central Sound

Agate Passage

208

20

13.6

15.8 (±0.383)

0.1964 (±0.009)

0.183 (±0.066)

Blake Island

19

18

13.0

14.6 (±0.283)

0.1586 (±0.006)

0.806 (±0.071)

Hood Cunal

Bangor

98

25

13.5

14.3 (±0.252)

0.1569 (±0.007)

0.545 (±0.055)

Tala Point

96

24

12.4

13.6 (±0.361)

0.1435 (±0.009)

-0.029 (±0.071)

Port Gamble (dredged)

180

21

13.1

15.2 (±0.283)

0.1810 (±0.007)

0.661 (±0.052)

Port Gamble (control)

80

21

12.7

14.0 (±0.390)

0.1610 (±0.007)

0.599 (± 0.075)

Thorndyke Bay

258

21

12.2

13.0 (±0.201)

0.1421 (±0.005)

0.550 (±0.097)

Fishermans Point

21

19

16.8

17.3 (±0.251)

0.2353 (±0.009)

0.552 (±0.059)

Strait

Dallas Bank

149

21

12.0

13.3 (±0.405)

0.1131 (±0.005)

0.334 (± 0.096)

ances for older geoducks with more growth rings were likely to be
more precise than for younger geoducks. The resulting heterosce-
dasticity for geoducks >15 years of age was thought to be minimal
and was ignored.

Hypothesis Testing

The experimental design was a two-factor analysis of variance
(ANOVA) in which the first factor was region and the second was
sites nested within regions. We first conducted a hypothesis test on
site effects within each region and only conducted a test among
regions if the site effects were nonsignificant. In this case, non-
significance meant not managerially significant. Thus, for all sta-
tistically significant tests. Tukey multiple comparisons (Neter et al.
1985) were used to test for managerial significance. The Tukey
multiple comparisons yielded confidence intervals for the differ-
ences in growth among locations. For any one comparison to be
managerially significant, the difference in growth had to be at least
some constant c. These comparisons were identified by confidence
intervals that excluded the interval {-c. c).

Calculating Managerial Significance (c = 0.027)

Most U.S. and Canadian fisheries, including all of those under
U.S. federal jurisdiction, are managed using biological reference
points (BRPs). BRPs are calculable quantities that describe a popu-
lation's state and are usually used as targets for optimal fishing
(National Research Council 1998). A BRP is most often expressed
as a fishing mortality rate (F); examples include f msy- /^max- ^^'^
fj,9i,. These are the fishing mortality rates that are expected to
achieve, over the long term, maximum sustainable yield, maxi-
mum yield per recruit, and a spawning stock biomass that is xx%
of the unfished level, respectively.

We considered two management criteria in calculating the
threshold of managerial significance for geoduck growth param-
eters: (1) the BRP used by managers in setting the target fishing
mortality rate and (2) the number of significant digits to which this
target fishing mortality rate was calculated. Geoduck managers in
Washington currently use as a BRP the fishing mortality rate cor-
responding to F^tfcf.. a reference point that is widely used for U.S.
West Coast groundfish (Clark 1993). Managers have agreed to
calculate this target fishing mortality rate to three decimal places.
For example, there is a managerially significant difference be-

tween annual fishing mortality rates of 0.027 and 0.028. but not
between 0.027 and 0,0273.

The three von Bertalanffy growth parameters were first evalu-
ated to determine which had the most influence on yield model
predictions. The equilibrium model described in Bradbury and
Tagart (2000) was used to calculate F^^rt, for different values of
{L.^_,kJf,} in the range observed in the data. L^, while it affected
model predictions of absolute yield, did not affect relative
spawner-per-recruit biomass or relative yield per recruit and was
therefore eliminated from further analysis. Figure 3 shows that the
growth parameter k is more infiuential on ^409^ than ?„. Because L-^
had no impact on F^^^^ and t^ had only minimal impact, we con-
ducted univariate hypothesis tests on k.

Changes in k would only affect management decisions if they
caused the model-based fishing mortality rate to change by 0.001
or more. In general, whenever a linear relationship exists between
k and F with slope (3.

0.000

Figure 3. Surface plot of F^a,
and fnS.

080

values as a function of the observed ks

60

Hoffmann ft al.

Ak

= 3 => AA- = — ^

AF,

In this example, a linear regression of the level of change Fj,,,.^ on
k yielded a highly significant slope {P < 0.001) of p = 0.0366.
Using this slope and Washington's management decision to cal-
culate annual fishing mortality rates to three significant digits, c =
0.001/0.0366 = 0.027. Thus, absolute differences in k among sites
(or regions) s0.027 were managerially significant.

RESULTS

Table 1 shows the von Bertalanffy parameter estimates and
their variances for the 1 1 study sites. The resulting growth curves
for the fastest-growing site (Fishermans Point) and the slowest-
growing site (Dallas Bank) are shown in Figure 4; the growth
curves for all other sites lie between these two. Also shown for
comparison is Anderson's (1971) growth curve for geoducks at
Big Beef Creek and Dosewallips beaches in Hood Canal.

Test for Nested Site Effects

The test for site effects within regions was statistically signifi-
cant {Fj22^ = 24.72. P = 0*) for all regions. Further testing for
managerial significance produced mixed results. The Tukey mul-
tiple comparisons between sites within regions showed four com-
parisons in which the differences were managerially significant
(Table 2): between the sites in the South Sound region and among
several sites in the Hood Canal region.

Power of Tukey Multiple Comparisons

Because the null hypothesis of the growth parameters not being
significantly different was not rejected for sites within Hood Canal
and for the two sites in the Central Sound region, we conducted a
power analysis to assess whether or not the nonrejection was
meaningful. The power analysis estimated the probability that any
one of the Tukey multiple comparisons would have excluded the
interval (-0.027, 0.027) if in fact the differences in k among sites
had been at least 0.027. To estimate this probability, we used the
two-sample /-test power analysis option of Power Analysis and
Sample Size (PASS version 6.0, Hintze, 1996). Each of the mul-

10 15 20
Age (yrs)

Figure 4. The von RcrtalanfTy growth curves for geoduck growth ul
the fiistisl gniHth site ( FishiTmans Point! and the slowest growth site
(Dallas Itanki In this stud). .Mso shown is Anderson's (1971) growth
curve for Big Becf/Dosewallips.

tiple comparisons was a /-test that needed a Tukey multiplier. To
adapt the software into giving the appropriate power estimates
(Table 3), we inflated the estimated standard deviation of the com-
parison, (Vm5£ = 0.0309, calculated by the ANOVA) by the ratio
of the Tukey multiplier (3.217) to the analogous Z multiplier
(1.96):

= 3.2l7/1.96VM5e

Power in the Hood Canal Region

0.0507.

In the Hood Canal region, there were 6 sites and 15 compari-
sons, 3 of which were significant. With an average sample size of
22, the estimated probability was 0.4233 (Table 3) for detecting a
0.027 difference in any one of the comparisons. If the actual dif-
ferences in k among sites had been at least 0.027, then one would
expect to detect more than three of them. In fact, the probability of
detecting a difference of 0.027 in at most three comparisons, where
the probability of detection was 0.4233 per comparison, is the
probability that a binomial random variable with N = \5 and P =
0.4233 was less than or equal to 3. This probability was 0.0645.
Given that this probability was very low. there is evidence that
among the sites in Hood Canal, other than Fishermans Point, the
differences in k are not likely to be greater than 0.027 and thus
need not be estimated separately.

Power in the Central Sound Region

In the Central Sound region, there were two sites, and the
comparison was not significant. With a power of 0.4233 of detect-
ing significance in a comparison, the chance of not rejecting the
null hypothesis was 1 - 0.4233 = 0.5767. Because this probability
is high, nonrejection of the null hypothesis was not meaningful:
i.e.. the results are inconclusive.

Because Hood Canal was the only region producing evidence
for common growth rates among sites, we did not pursue a test of
regional differences. Thus, we recommend that with the given
management criteria, separate growth models should be used in the
regions given in Table 4. For the sites within Hood Canal other
than Fishermans Point, the average growth parameter was calcu-
lated as the mean of the average growth parameters in each site
(Table 4).

DISCUSSION

The first result to note is the difference in the growth curves
presented here and that from Anderson ( 197 1 ). We estimated both
a lower rate of growth (k) and a smaller asymptotic size {LJ for
geoducks: however, differences in the target population explain
this discrepancy. Anderson's target population consisted of sub-
tidal and intertidal geoducks between the presumed ages of I and
5 years. Our target population consisted of subtidal geoducks older
than 15 years. Because mean geoduck shell length is inversely
proportional to water depth (Goodwin and Pease 1991). it is ex-
pected that Anderson's sample would ha\e a higher estimate of Z,.,.
Likewise, a higher estimate of A- is expected with a vounger target
population.

Of the three von Bertalanffy giowih paranielers. oiilv one was
determined to be iiinucnlial: (he parameter k. For the criteria given,
managerial significance was calculated to be differences in k of
0.027 or greater among locations. That is, if the growth parameter
differed by more than 0.027 among locations, then location-
specific growth estimates should be used for setting harvest quotas.

Data that were collected in four different regions encompassing

Geoduck Growth

61

TABLE 2.
Confidence intervals for the multiple comparisons of Test 1.

Lower

Upper

Site

Comparison (x,/x,)

A = v, - X,

SD(A)

Bound*

Bound

South Sound

Herron/Huntert

-0.0739

0.0102

-0. 1 1 64

-0.0314

Central Sound

Agate/Blake

0.0379

0.0109

-0.0076

0.0834

Hood Canal

Bangor/Tala

0.0133

0.0088

-0.0151

0.0417

Bangor/Gamdredge

-0.0242

0.0092

-0.0536

0.0053

Bangor/Thomdyke

0.0147

0.0092

-0.0147

0.0442

Bangor/Gamcontrol

-0.0042

0.0092

-0.0336

0.0253

Bangor/Fishermans Pointt

-0.0784

0.0094

-0.1087

-0.0481

Tala/Gamdredge

-0.0375

0.0092

-0.0672

-0.0077

Tala/Thorndvke

0.0014

0.0092

-0.0283

0.03 1 1

Tala/Gamcontrol

-0.0175

0.0092

-0.0472

0.0123

Tala/Fishermans Pointt

-0.0917

0.0095

-0.1223

-0.0612

Gamdredge/Thomdyke

0.0389

0.0095

0.0082

0.0696

Gamdredge/Gamcontrol

0.0200

0.0095

-0.0107

0.0507

Gamdredge/Fishernians

-0.0542

0.0098

-0.0858

-0.0228

Thomdyke/Gamcontrol

-0.0189

0.0095

-0.0496

0.0118

Thomdyke/Fishermanst

-0.0931

0.0098

-0.1247

-0.0616

Gamcontrol/Fi shernianst

-0.0743

0.0098

-0.1058

0.0428

* The confidence intervals were calculated using a Tukey multiplier of 3.2 1 7, i.e.. A
distribution 95th'7r quantile with 1 1 and ^ degrees of freedom.
t Statistically significant data in these rows.

: 3.217* 5D{A). The multiplier corresponded to a studentized range

1 1 different sites were tested for differences among growth pa-
rameters. Statistically significant differences in k were detected
among most of the sites within the three regions Central Sound,
Hood Canal, and South Sound. Further testing showed that in the
South Sound, the sites were also significantly different. In Hood
Canal, only one site was significantly different from the others. In
the Central Sound, the results were inconclusive. Therefore, to
preserve the management sensitivity criterion of 0.001 in the es-
timated Fjijr; levels, we recommend different growth parameter
estimates be used for each site in Straight. Central Sound, and
South Sound and that one common model for the sites in Hood
Canal other than Fishermans Point be used.

We speculate that environmental factors related to tidal flow
may have been a primary cause of the differential growth rates.
Goodwin and Pease ( 1991 ) found that the average shell length of
geoducks in Puget Sound was greatest in sandy substrates and
decreased in both muddier substrates and those composed of pea
gravel. Because size and growth are related, it is reasonable to

conclude that growth is greatest in sites that are subject to inter-
mediate tidal flow (i.e.. those composed primarily of sand) and
decreases in both low-energy (muddy) and high-energy (gravelly)
environments. The substrate was primarily composed of sand at
the three sites in our study with the highest k values (Fishermans
Point. Hunter Point, and Agate Passage). The three sites with the
lowest k values were Dallas Bank, a site composed primarily of
pea gravel, and Tala Point and Thorndyke Bay. both of which are
muddy. Goodwin and Pease (1991) also suggested relationships
betwen geoduck size and environmental factors such as primary
productivity and water temperature. Along with tidal currents,
these factors are likely to vary from site to site, resulting in dif-
ferential growth parameters.

Evidence for site-specific growth differences poses a dilemma
for managers who must recommend a single regional harvest rate.
If growth rates were common ainong sites, a regional estimate
based on any selection of sites would be unbiased. However, we
found that the growth constant can be site specific, requiring ad-

TABLE 3.

Power estimates for a single Tukey multiple comparison for various
sample sizes.*

Sample Size

Power

TABLE 4.
Growth Parameter k estimated by region and site.

20
21
22
23
24
25
30

Region

0.3914

South Sound

0.4075

Central Sound

0.4233

0.4389

Hood Canal

0.4542

0.4693

0.5409

* Power was estimated using the two-sample 7"-test option of PASS version
6 (Hintze 19961 with a standard deviation of 0.0507 and a difference in
means of 0.027.

Strait of Juan de Fuca

Site

Estimated k

Hunter

0.2283

Herron

0.1544

Agate

0.1964

Blake

0.1586

Bangor

0.1569

Tala

0.1569

Gamdredge

0.1569

Thorndyke

0.1569

Gamcontrol

0.1569

Fishermans Point

0.2353

Dallas

0.1131

62

Hoffmann et al.

justments to a sampling plan for estimating an unbiased regional
parameter. Because the sites in this study were not selected at
random, a regional k that is an average of the estimated site As will
be biased. Managers might consider using the lowest estimated
A-value with the expectation that that would be a conservative
approach. Alternatively, another study could be conducted using a
sampling plan designed to yield unbiased regional estimators.

ACKNOWLEDGMENTS

Warren Shaul and Conrad Budd assisted C.L.G. in collecting,
preparing, and analyzing the age-growth data. Michael Ulrich pre-
pared the map. We thank Tom Jagielo for computing assistance in
writing the growth parameter estimation program.

LITERATURE CITED

Anderson, A. M., Jr. 1971. Spawning, growth, and spatial distribution of
the geoduck clam. Panope generosa. Gould, in Hood Canal, Washing-
ton, Ph.D. thesis. University of Washington, Seattle. WA. 133 pp.

Bradbury, A. & J. V. Tagart. 2000. Modeling geoduck Panopea abrupla
(Conrad, 1849) populations dynamics. 11. Natural mortality and equi-
librium yield. / Shellfish Res. 19:63-70.

Breen, P. A. & T. L. Shields. 1983. Age and size structure in five popu-
lations of geoduck clams (Panope generosa) in British Columbia. Ca-
nadian Technical Report. Fish. Aquat. Sci. No. 1169. 62 pp.

Bernard. F. R. 1983. Catalogue of the living Bivalvia of the eastern Pacific
Ocean: Bering Strait to Cape Horn. Can. Spec. Publ. Fish. Aquat. Sci.
No. 61. 102 pp.

Campbell. A., R. M. Harbo & C. M. Hand. 1998. Harvesting and distribu-
tion of Pacific geoduck clams, Panopea abrupla, in British Columbia,
pp. 349-358. In: G. S. Jamieson & A. Campbell (eds.). Proceedings of
the North Pacific Symposium in Invertebrate Stock Assessment and
Management. Can. Spec. Publ. Fish. Aquat. Sci. No. 125.

Clark, W. G. 1993. The effect of recruitment variability on the choice of
target level of spawning biomass per recruit, pp. 233-246. //;.■ G. Kruse.
D. M. Eggers. R.J. Marasco. C. Pautzke. and T.J. Quinn II (eds.).
Proceedings of the International Symposium on Management Strate-
gies for E.xploited Fish Populations. Alaska Sea Grant College Program
Report No. 93-02. University of Ala.ska, Fairbanks.

Ebbesmeyer. C. C. C. A. Coomes, J. M. Cox, J. M. Helseth, L. R.
Hinchey. G. A. Cannon & C. A. Barnes. 1984. Synthesis of current
measurements in Puget Sound. Washington. Vol. 3. Circulation in
Puget Sound: an interpretation based on historical records of currents.
NOAA Technical Memorandum NOS OMS 5. NOAA, Rockville, MD.
73 pp.

Goodwin, C. L. 1976. Observations on spawning and growth of subtidal
geoducks {Panope generosa. Gould). Proc. Natl. Shellfish. Assoc. 65:
49-58.

Goodwin. C. L. & B. C. Pease. 1987. The distribution of geoduck (Panope
uhriipta) size, density, and quality in relation to habitat characteristics
such as geographic area, water depth, sediment type, and associated
flora and fauna in Puget Sound. Washington. Washington Department
of Fisheries Technical Report No. 102. 44 pp.

Goodwin, C. L. & B. C. Pease. 1991. Geoduck (Panope abrupla (Conrad,
1849)) size, density, and quality as related to varous environmental
parameters in Puget Sound. Washington. J. Shellfish Res. 10:65-78.

Hintze, J. L. 1996. Power Analysis and Sample Size (PASS) User's Guide,
Version 6.0. Number Cruncher Statistical Systems, Kayesville, UT.
245 pp.

Jamison. D., R. Heggen & J. Lukes. 1984. Underwater video in a regional
benthos survey, pp. 15-17. In: Proceedings of the Pacific Congress on
Marine Technology. Marine Technology Society. Honolulu.

National Research Council. 1998. Improving Fish Stock Assessments. Na-
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Neter, J., W. Wasserman & M. H. Kutner. 1985. Applied linear statistical
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Shaul, W. & C. L. Goodwin. 1982. Geoduck (Panope generosa: Bivalvia)

age as determined by internal growth lines in the shell. Can. J. Fish.

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Journal of Shellfish Research. Vol. 19, No. 1. 63-70. 2000.

MODELING GEODUCK, PANOPEA ABRUPTA (CONRAD, 1849) POPULATION DYNAMICS. II.
NATURAL MORTALITY AND EQUILIBRIUM YIELD

A. BRADBURY' AND J. V. TAGART^

' Washington Department of Fish and Wildlife

Point Whitney Shellfish Laboratory

1000 Point Whitney Road

Brinnon. Washington 98320
'Washington Department of Fish and Wildlife

600 Capitol Way North

Olympia. Washington 98501

ABSTRACT The natural mortality rate of geoduck clams. Panopea ahrupia (Conrad. 1 849). was estimated from data collected at 14
previously unfished sites in Washington State in order to predict the potential yield of the commercial fishery under various harvest
rate strategies. The instantaneous rate of natural mortality (A/) estimated by the catch curve method for geoducks of ages 28-98 was
0.0226 y"'. Other important life history parameters — growth, schedules of sexual maturity, weight-al-age, and fishery selectivity —
were estimated from the literature and file data. These parameter estimates were used to drive an age-based equilibrium yield model
that predicted yield per recruit (YPR) and spawning biomass per recruit (SPR) over a range of fishing mortality rates. The model
produced values of the instantaneous fishing mortality rate (F) for five commonly used constant harvest rate strategies. The fishing
mortality rate producing maximum YPR (f„a,) ranged from 0.053-0.100 depending on the site growth parameters, but reduced SPR
to 15-21% of the unfished level, f-values for the Fq., strategy ranged from 0.28 to 0.37. reducing SPR to 35-37% of the unfished level.
Three harvest rate strategies that reduce SPR to either 35%, 40%, or 50% of the unfished level were also evaluated, with F-values
ranging from 0.018 to 0.036. The F„j, strategy, currently adopted by Washington managers, was achieved with F = 0.028 (averaged
over all sites), corresponding to an annual harvest rate of 2.7% of the exploitable biomass. The model was most sensitive to estimates
of M. whereas growth, fishery selectivity, and sexual maturity schedules had relatively little effect on yield or SPR. Apparent shifts
in recruitment during the past 30-45 y may have biased the estimate of M. Direct estimates of M and recruitment are therefore a high
research priority if the model outputs are to remain useful.

KEY WORDS: Geoduck. Panopea ahrupia. natural mortality, yield, harvest rate, spawning biomass

INTRODUCTION

The geoduck clam Panopea abrupta (Conrad. 1849) supports
the most economically important clam fishery on the Pacific Coast
of North America (Campbell et al. 1998. Hoffmann et al. 2000).
Since 1967. the Washington Department of Fish and Wildlife has
performed annual dive surveys to estimate the exploitable biomass
of geoducks in Washington. "'Exploitable biomass" here refers
only to geoducks within the legally fishable water depths of 6-23
m. in areas that are not polluted or otherwise unsuitable for com-
mercial fishing and of sufficient size for their siphons to be visible
to divers. Based on market and survey samples in Washington, this
excludes most geoducks <300 g. Of 11.181 geoducks randomly
sampled using commercial methods, only 2'7c were <300 g (Good-
win and Pease 1987). Geoducks usually attain this size in 5-7 y
(Hoffmann etal. 2000).

Exploitable geoduck biomass on a commercial bed is estimated
as the product of the total bed area, the mean weight per geoduck.
and the mean density of geoducks. Mean density is estimated by
counting geoduck siphon "shows'" using a systematic strip transect
technique (Goodwin 1978). Mean weight per geoduck is estimated
from a series of samples dug at systematic intervals along the
transect lines. The sum of the most recent biomass estimates on all
surveyed beds within a management region constitutes the regional
biomass estimate. There are currently six geoduck management
regions in Washington, based largely on legally defined tribal
fishing boundaries. Because only a few beds can be surveyed
intensively in this manner each year, regional biomass estimates
consist of the most recent estimate for each bed. with known
catches subtracted from those beds as they are fished.

To establish annual fishing quotas, managers apply a target

harvest rate to the exploitable biomass estimate in each manage-
ment region. Beginning in 1981. the target harvest rate was fixed
at 2% of the estimated virgin (unfished) biomass on surveyed,
commercially viable beds. This target harvest rate was based on a
Ricker (1975) yield per recruit (YPR) model, but the model out-
puts were never explicitly documented. Furthermore, emphasis in
fisheries management has shifted since that time; harvest strategies
based on YPR analyses (e.g., F^^^ and Fq ,) are now often sup-
planted by strategies that seek instead to preserve the reproductive
potential of the population. These spawning biomass per recruit
(SPR) strategies are increasingly being used in marine fmfisheries
(Clark 1991) and to a lesser extent in shelltTsheries (Quinn and
Szarzi 1993).

In this study, we derive estimates of the natural mortality rate
(M) from geoducks sampled at previously unfished sites in Puget
Sound and the Strait of Juan de Fuca. We also construct schedules
of sexual maturity, weight-at-age, and fishery selectivity from the
literature and tile data. We use these estimates to drive an age-
based equilibrium yield model that predicts YPR and SPR over a
range of fishing mortality rates. We also explore the limitations of
the model and conduct sensitivity tests to determine which param-
eters most influence the model's predictions. Finally, we use this
information to recommend research aimed at refining the most
important parameter estimates.

METHODS

Sampling Sites and Procedures

Geoducks were sampled between 1979 and 1981 at 14 previ-
ously unharvested sites in Puget Sound and the Strait of Juan de

63

64

Bradbury and Tagart

Fuca to n'jiain information on age distribution (Fig. 1 ). The sites
span fcuf of the current six management regions, with six sites in
the Hood Canal region, two sites in the Central Sound region, one
site in the Strait region, and two sites in the South Sound region.
Sample.s were taken randomly within each site at depths of 10-20
m by washing geoducks from the substrate with a commercial
water jet. Age was determined from annual growth increments in
the hinge plate using the acetate-peel method (Shaul and Goodwin
1982).

The instantaneous rate of natural mortality (A/) was estimated
from the geoduck age-frequency distribution using two different
catch curve models (Robson and Chapman 1961, Ricker 1975).
Both models assume that mortality is constant for all ages used in
the catch curve. The Robson and Chapman model is based on a
geometric distribution and assumes that year-class survival and
recruitment are constant and all ages are equally selected. Geo-
ducks are extremely long-lived, so that the number of animals
observed in each l-y age class is typically low. even for sample
sizes in which n > 1,000. Despite this problem, we chose to pre-
serve the data in l-y age classes rather than aggregating ages, a
procedure that potentially ignores real variability in the original
data and may slightly inflate estimates of M (Noakes 1992). It was
not possible to estimate site-by-site mortality rates, because no
individual site contained enough data to construct reliable catch
curves. Age frequencies were therefore pooled from all 14 sites in
order to create the catch curve.

To avoid arbitrary choices of the upper and lower ages used in

Figure 1. Sampling sites for geoducit natural mortality and growth.
Also shiiwii are boundaries lor four of Washington's geoduck man-
agement regions: the t«o not shown contained no sampling sites and
no surveyed geoduck hiomass.

the catch curve "right limb," we established a protocol for data
inclusion: The initial upper age limit for the catch curve was the
first age at which our sample contained no geoducks (i.e.. the first
gap in frequency). We then excluded younger age frequencies if
they were identified as outliers by Wei.sberg"s (1985) outlier test.
Two methods were used to select the lower age limit for the catch
curve: ( I ) The Chi-square procedure described in Robson and
Chapman (1961) was used to differentiate partially .selected ages,
and (2) catch curve regressions were calculated for all possible
lower age limits, and we used an ad hoc procedure to optimize the
coefficient of determination (r") and the linearity of positive and
negative residuals plotted against age. Once the lower and upper
age limits for the catch curve were identified, a Chi-square formula
was then used to test goodness of fit of fully selected ages to a
geometric distribution (i.e.. the Robson and Chapman model). The
von Bertalanffy growth parameters estimated at 1 1 Washington
sites from Hoffmann et al. (2000) were used as site-specific growth
inputs. Sexual maturity, weight-at-length. and fishery selectivity
parameters were derived on the basis of published literature from
Washington and British Columbia.

Yield Model

Geoduck yield was modeled using a deterministic, age-
structured equilibrium yield model. Given a set of parameter es-
timates for mortality, maturity, growth, and selectivity, the model
collapses the number of geoducks at age for all cohorts in the
population to a single cohort, assumed to represent the stable age
distribution of the population. Population size was based on an
initial unfished spawning population, by a declining exponential
function for survival at age, and by the Baranov catch equation
(Ricker 1975). Baranov's catch equation says that annual catch is
a simple linear function of instantaneous fishing mortality and
mean population size. The derivation of Baranov"s catch equation
is presented in Seber (1982). Seber cites Baranov (1918) as the
origin of the catch equation, hence its common name. The model
assumed continuous recruitment, the magnitude of which was
based on a Beverton-Holt stock-recruitment relationship (Ricker
1975). The Beverton-Holt stock-recruitment relationship, com-
monly used with marine fish, is an asymptotic function that esti-
mates annual recruitment based on parent stock size. The impli-
cation of this relationship is that over a broad range of parent stock
size, recruitment is stable, but as parent stocks reach critically low
levels, recruitment drops precipitously. A maximum age (o,,,^^) in
the model served as an "accumulator age" category that encom-
passed all ages a > rt„,,,^. The assumption implicit in this formu-
lation is that no significant changes in growth, weight, maturity, or
selectivity occurred beyond «,„;,„. In the case of geoducks. this
assumption was reasonable and is addressed below. For other ap-
plications, the model could be simply extended to accommodate an
unlimited nimiber of older age classes. The model was constructed
as a QuattroPro for Windows (version 5.0) spreadsheet.

Table I lists the user-supplied inputs required by the model.
These include estimates of the natural mortality rate, the growth
rate, the stock-recruit (S-R) relationship, the unfished spawning
hiomass. fishery selectivity, sexual maturity, and the population
sex ratio. Table 2 shows the parameters derived from the user
supplied inputs, listed in computational order. To run the model,
fishing mortalily (/'I was stepped from 0 to a specified upper limit
while computing YPR and SPR for each value of /■".

The model is capable of returning a suite of fishing mortalily

Modeling Geoduck P. abkupta Population Dynamics

65

TABLE 1.

Geoduck life history parameter estimates iield constant for all
study sites.

Parameter Description

Parameter
Symbol

Value, Notes

Unfished ("virgin")

spawning stock biomass
(in kg); the spawning
biomass when F = 0

Instanlaneous natural
mortality rate (assumed
constant for all ages)

Weighi-at-age (in g) based
on length-at-age as
derived from the von
Bertalanffy growth
function

Maturity-at-age; the
proportion of female
geoducks of age a (in
years) that are sexually
mature

Fishery selectivity-at-age;
the proportion of
geoducks of age a (in
years) selected by the
fishery

Beverton-Holt

spawner-recruit shape
parameter (Kiinura 1988)

Proportion of males in
population

Maximum (accumulator) age

BO,

M

*.

100.000 kg (only required
to scale absolute
biomass)

0.0226

»\, = "L/

L^ = length (cm) at age a

X = 0.349127

y = 2.972807

cj)^ = 1/(1 +exp"*'')
X = -1.9 y = 9.5

V, = 1/(1 +exp~"'')
X = -1.5 y = 8.0

0.5

2^

benchmarks, such as f „„^, fo.,- and F„,;. . For example, the fishing
mortality rate that produces, over the long run. the maximum YPR
corresponds to the F,„„^ strategy, whereas F„ , represents a rale of
harvest less than f,„„, (Deriso 1987, Gulland 1968).

The fraction of the unfished spawning weight per recruit re-
maining at a given level of fishing mortality was calculated as
SPR/SPRO and is achieved at a corresponding fishing mortality
rate F„..,^ where .v.v represents the ratio (SPR/SPRO)IOO. Model
predictions of this fraction formed the basis for SPR-based fishing
strategies. For example, the fishing mortality rate that resulted in a
value of SPR/SPRO = 0.35 corresponds to the F,v; strategy.

RESULTS

Natural Mortality

Sampled geoducks from 14 previously unfished sites ranged in
age from 2 to 131 y (Fig. 2a). The mean age of geoducks was 46
y (standard error [SE] = 0.56. n = 2.157). The initial upper age
limit for the catch curve was 1 10 y, because no 1 1 l-y-old geo-
ducks were in our sample. Examination of residuals showed a
single large negative residual at the 99-y age class (only one geo-
duck of this age was in our sample), and this age class was elimi-
nated from the analysis as an outlier, based on the test given in
Weisberg (1985). Both the Robson and Chapman (1961) Chi-
square procedure and our ad hoc optimization procedure identified
age 28 as the lower age limit for the catch curve. A Chi-square was
used to test goodness of fit of fully selected ages (28-98) to a

geometric distribution. The resulting Chi-square was highly sig-
nificant (X" = 326.56. degrees of freedom = 68). indicating that
the age frequency was not geometric in distribution and that data
requirements for the Rob.son and Chapman model were not met.
Ricker (1975) pointed out that in most stocks, difference in year-
class strength is the major source of variability, in which case the
best estimate of survival would be obtained from a catch curve
analysis with equal weighting. The Ricker catch curve based on
ages 28-98 (Fig. 2b) produced an estimate of M = 0.0226 y"'
(±0.0018 SE. /! = 71. r- = 0.70).

Other Model Parameters

Goodwin (1976) calculated an allometric length-weight rela-
tionship for Washington geoducks in log-log form. We converted
this to the more familiar power curve form h'„ = .vL„', where w.^
= weight (in g) at age a. L^ = shell length (in cm) at age a (Table
1 ). The proportion of males (/),„) in the geoduck population was set
to /J„, = 0.5 based on a 50:50 sex ratio for geoducks older than 10
y (Goodwin and Pease 1989).

The proportion of sexually mature geoducks at age (<l>) was
estimated by fitting a simple logistic curve to maturity data from
published sources. Anderson (1971 ) found that 50% of his sample
of geoducks was mature at 75 mm and an age that he estimated to
be 3 y. The Washington growth curves described above suggest
that this length would be attained in roughly 5 y. depending on the
site. Sloan and Robinson (1984) reported that geoducks mature at
5 y and reproduce for at least a lOO-y period with no "reproductive
senility." They stated that "unequivocally mature geoducks" were
6-103 y old (late-active males) and 12-95 y old (late-active fe-
males). On the basis of these two sources, we fit a logistic curve
with the least-squares method and two data points, whereby 50%
of the female geoducks would mature at 5 y and 100% by 12 y
(Table 1).

The proportion of geoducks at age a selected by the fishery (i'„)
was based loosely on Harbo et al. (1983), who reported that re-
cruitment to the British Columbia geoduck fishery begins at 4 y
and is complete by 12 y. To more conservatively model fishery
selectivity, we fit a simple logistic curve using the least-squares
method and two data points, whereby geoducks enter the fishery at
roughly 4 y and are fully selected by 8 y (Table I ).

Nothing is known about the form or steepness of the S-R re-
lationship for geoducks. We therefore set the Beverton-Holt shape
parameter (A) equal to 1.0 for all model runs. In other words, we
assumed that recruitment was independent of spawning stock
abundance. This assumption is reviewed below in Discussion.

As a practical convenience, the equilibrium yield model uses an
"accumulator age" category («„„,) as the final age category, en-
compassing all ages a > n,„^^. For this study, we set a^^^ = 25,
which implicitly assumes that there are no significant changes in
growth, selectivity, or maturity beyond age 24. This assumption is
reasonable for geoducks, which reach asymptotic size between the
ages of 10 and 20 y (Hoffmann et al. 2000).

Fishing Mortality Rates for Five Harvest Strategies

We ran the model for each site, varying only the growth pa-
rameters based on the analysis of growth presented in Hoffmann et
al. (2000). The only sites where growth parameter estimates (spe-
cifically, the growth constant A) could be pooled were five of the
six Hood Canal sites. In all other cases, site-specific growth pa-
rameters could not be pooled, and therefore separate model outputs

66

Bradbury and Tagart

TABLE 2.
Description of derived parameters used in tiie geoducli equilibrium yield model.

Description

Derived Parameters

Notes

Number of geoducks surviving to the first age

class (a = 1 year)
Instantaneous rate of fishing morlahty at age u

Instantaneous rate of total mortality at age a

Annual rate of survival

Number of geoducks surviving to age a for a > 1

Average number of geoducks at age u

Average biomass (in kg) of geoducks at age a

Yield per recruit (in kg) at age a

Total yield per recruit (in kg) for all ages

Spawning weight per recruit (in kg)

Fraction of unfished spawning stock biomass
remaining at a given level of fishing mortality

Spawning biomass (in kg) when F > 0

Recruitment (in numbers)

Yield (in kg)

Harvest rate for fully selected age classes (iv, = I )

W, = /),„ for males

N^ = I - /),„ for females

F^ = Fv.^

Z^ = M^ + F,

S^ = exp(-ZJ

Wj = W„(l -SJ/Z, for o<a^,„
^ = ;^/Z„ for a = o„„,
B. = N,w, _ _

YPR„ = v.,f B., = F,B„

YPR = X'',/B: = fXi'A

SPR, = W,<t>, for age a

SPR = ^'B^,<i>„ for all ages

P = \ - (1M)(1 - SPR/SPRO)

B.

P BO.

R = (BySPRO)/[l - A(l - P)]

Y = YPR(R)

|x = F/Z[\ - exp(-Z)]

/),„ = proportion of males in the population (see

Table 1)
F = instantaneous rate of fishing mortality for

fully selected age classes (v^ = 1);

user-supplied. \\, = fishery selectivity at age a

(see Table 1 )
M^ = instantaneous natural mortality rate (see

Table 1)

maximum (accumulator) age (see Table 1)

<i>, = proportion of mature females at age a (see
Table 1)

A = Beverton-Holt shape parameter (see Table 1).

SPRO = unfished spawning weight per recruit

(total SPR when F = 0)
BOs = unfished spawning stock biomass (see

Table 1)
Reference: Kimura ( 1988)

Reference: Ricker (1975)

were calculated for each site. All inputs except growth paratnelers
were identical for each model run (Table I ). Growth parameters
used as site-specific input are shown in Table 3.

Values of the instantaneous fishing mortality rate (F) for five
commonly used constant harvest rate strategies are shown in Table
3. fn,^^ is the fishing mortality rate that produces, over the long
run. the maximum YPR. f,, , is a common alternative to F„„^ and
is the rate of fishing mortality at which the marginal YPR is 10%
of the marginal YPR for a lightly exploited fishery (Deiiso 1987).
F,5,;j, ^409^, and F^,,,. are SPR-based harvest rates that reduce SPR
to either 35%. 40%, or 50% of the unfished level (Clark 1991 ).

F,„,,^ ranged from 0.053 to 0.100 depending on the site (Table
3). These rates correspond to annual harvest rates ((jl) of 5.1-9.4%
of the exploitable geoduck biomass. The Strait of Juan de Fuca
region, represented by the single sampling site at Dallas Bank,
produced the lowest value, whereas Fishermans Point in Hood
Canal produced the highest value. The F,„,^ strategy reduced SPR
to 15-21% of the unfished level, depending on the site. Values for
F,| I ranged from 0.028 to 0.037, corresponding to annual harvest
rates of 2.7-3.67r. This strategy reduced SPR to 35-37% of the
unfished level, depending on the site.

Values for F,,,,, were, predictably, nearly identical to the F,, ,
rates, ranging from 0.30 to 0.36 ((jl = 2.9-3.5%). F values for the
F4o-;^ strategy ranged from 0.025-0.030 ((x = 2.4-2.8%), whereas
those for the Fs,„ strategy ranged from 0.0 1 8-0.020 ((jl = 1.8-
2.0'/r ).

Model Sensitivily to Parameter Estimates

All of the parameter estimates used to drive the model arc
subject to varying degrees of uncertainty. It is therefore reasonable

to ask what might happen to our predictions if the true values of W
or A:, for example, were much lower or higher than our estimates.
We tested the sensitivily of the model by running it with a range
of values for each parameter in turn while holding all other pa-
rameters constant. Values ranging from one-tenth the "best" pa-
rameter estimate (from Tables 1 and 2) to three times the estimated
value were used in the analysis. Only the fishing mortality rates
corresponding to the F_^^y,, strategy were calculated, but the trend
for other strategies would be similar.

The model was most sensitive to the estimate of M, with F^^^,
values ranging from 0.003 to 0.068 as M was increased from
one-tenth to three times our "best" estimate of A^ = 0.0226 (Fig.
3). The model was far less .sensitive to the other parameter esti-
mates, as evidenced by the relatively flat Fjii,^ trajectories for
values of the growth coefficient k. the selectivity constant y, and
the maturity constant y. For example, varying the value of A- from
one-tenth to three times our best estimate resulted in Fj,,,, values
that ranged only from 0.02 1 to 0.033.

Use of Model Results to Set Annual Fishing Quotas

The model results presented above, together with an estimate of
exploitable biomass, may be used to set annual fishing quotas. The
first step in such a process is for managers to recommend one of
the five harvest strategies described above, or an alternate strategy;
the model is capable of returning /-'-values for any desired level of
equilibrium spawning biomass or yield. The decision process in-
volved in recommending a particular harvest strategy is by no
means clear-cut, but some guidelines on risk-averse strategies from
the recent fisheries literature are reviewed below in Discussion.

Modeling Geoduck P. abrupta Population Dynamics

67

70

^60
■| 50

3
C

•-^40

>-

Z 30
LU

O 20
lU

£ 10

1979-1981
mean age = 46.27 yr
SE = 0.56
n = 2157

I

.ijlliiliilillillliihl:]

LMlIk

5 ' 10 20 30 40 5b 60 70 80 90 lOOTio

AGE (yrs)

mis

130T4bT50

4

>-
O

Z 3

o

LU 2
OH

B

' m

^^?^

■

1979-1981
M = 0.0226
ages 28 - 98
r'^2 =0.70
n = 71

■ 1

■ ■

■■

■ ■ ■

-■ — 1 — I — h —

-r- ^ 1

■^^ "P '

0 10 20 30 40 50

60 70

AGE

80 90

(yrs)

100 110 120 130 140 150

Figure 2. (A) Age frequency of geoducks sampled at 14 sites in Wash-
ington. (B) Catch curve used to estimate the instantaneous natural
mortalit> rate (M) of geoduclis.

Once managers reach a decision on the "best" harvest strategy, the
corresponding F-value may be taken directly from the mean values
in Table 3. This mean F-value is then converted to the harvest rate
(jjl) for fully selected age classes (Ricker's equation from Table 2).
To produce the recommended annual fishing quota, the harvest
rate is then simply multiplied by the estimate of harvestable bio-
mass. For example. Washington managers have recommended and
adopted an F^„r; strategy for geoducks in all six management
regions. This strategy is achieved with an instantaneous fishing
mortality rate of F = 0.028 (mean value for all sites. Table 3): the
corresponding annual harvest rate for fully selected age classes (|j.)
is 0.027. or 2.79^ of the exploitable biomass. Annual dive survey
data provide an estimate of exploitable biomass for each of six
management regions. As an example, exploitable geoduck biomass
in the Hood Canal Region in 1999 was estimated to be 18.185 t
(Sizemore and Ulrich 1999). and the resulting annual quota was
{0.027)( 18.185 t) = 491 t.

DISCUSSION

Our primary objective in equilibrium modeling was to simulate
the long-term results of various geoduck fishing strategies, both in
terms of yield and SPR. Before discussing our results, it is perhaps
necessary to explain why we attach such importance to geoduck
harvest rate strategies, particularly since the differences between
many of the modeled options may appear trivial.

In many fisheries, especially those in which biomass is small or

estimated with great uncertainty, debating a 1% difference be-
tween annual harvest rate options would indeed be trivial. But in
Washington's geoduck fishery, where the exploitable biomass is
large (73.843 t in 1999; Sizemore and Ulrich 1999) and the price
is high, even tiny incremental differences in the recommended
harvest rate have tremendous economic significance. Moreover,
because geoducks have a low M (and presumably a low intrinsic
rate of increase), small differences in annual harvest rates can have
profound cumulative effects on stock size, especially if the harvest
rate is set too high. This is not to discount the importance of good
biomass estimates, but we believe there are several reasons why
Washington managers should place the greatest emphasis on im-
proved harvest rate strategies rather than improved biomass esti-
mates. First, biomass estimates for individual geoduck beds in
Washington have coefficients of variation (CVs) averaging about
11%. Simulation tests suggest that biomass estimation errors of
this magnitude are unlikely to result in substantial degradation of
long-term harvest performance (Frederick and Peternian 1995).
Second, even greatly increased sampling is not likely to improve
biomass estimate CVs very much. Third and most importantly,
errors in biomass estimation are assumed to be reasonably unbi-
ased. An error in setting the annual harvest rate, on the other hand,
will have a persistent and cumulative effect on stocks in only one
direction, either underharvest or overharvest. We therefore believe
that, given reasonable estimates of stock size, choosing a harvest
strategy remains the most critical aspect of geoduck management.

In this study, we evaluated five common harvest strategies. Our
model predicts that fishing at F,„ ,^ will eventually reduce SPR to
less than 20% of the unfished level, a threshold below which many
fish stocks are assumed to collapse (Thompson 1993). Therefore,
F^^^ should be considered a high-risk strategy for geoducks.

Less risky are the SPR-based strategies, three of which were
evaluated here. In this study, we assumed that recruitment was
independent of stock size at all levels of fishing (Beverton-Holt
parameter /4 = 1.0). Although this is the common default assump-
tion in cases in which the S-R relationship is unknown, the risk
inherent in this assumption is that given an existing but undetected
S/R relationship. F^^,,, can be greater than F^,sy (the preferred
fishing rate with a known S/R function; MSY, maximum sustain-
able yield). As an alternative to F„^^, SPR-based strategies seek to
preserve some minimum level of spawning biomass and at the
same time produce yields that are close to the MSY. In an attempt
to find fishing strategies that are robust for any likely S-R rela-
tionship, recent modeling studies have simulated groundfish yields
using a range of typical life history parameters and realistic S-R
models. Clark ( 1991 ) showed that fishing at F,5^; would achieve at
least 75% of MSY for a wide range of detemiinistic S-R relation-
ships. On the basis of his results, F^^^,;, has been adopted as a target
rate for a number of fish stocks in Alaska and the U.S. Pacific
coast. Clark (1993) later revised his recommendation to Fj,,,- after
considering variability in recruitment, but remarked that "it would
be silly to argue very hard for or against any specific rate between
F,5<7, and F^^.^^,." Mace (1994) also recommended Fj^^j. which she
claimed was a modest improvement over Fjj.^^. She states that
Fjii^j represents a risk-averse fishing strategy in the common situ-
ation in which there is adequate information to place bounds on all
relevant life history parameters except the S-R relationship. Quinn
and Szarzi (1993) modeled clam fisheries in Alaska and recom-
mended SPR-based strategies equivalent to a range of F309j,-f45*-

As noted earlier. Washington managers have adopted an Fj,,^^
strategy for geoducks. which corresponds to F = 0.028 (averaged

68

Bradbury and Tagart

TABLE 3.
Benchmark instantaneous fishing mortality rates for fully selected geoducks (v^ = 1.0) from seven sites in Washington.

n

I.=

Region

Site

(sites)

(cm)

k

'ii

fm.v

fo.i

F,sr,

''40%

^^50%

South Sound

Hunter Point

1

16.4

0.23

0.72

0.090

0.036

0.036

0.029

0.020

Herron Island

1

13.2

0.15

0.42

0.064

0.031

0.032

0.027

0.018

Central Sound

Agate Passage

1

15.8

0.20

0.18

0.085

0.035

0.035

0.029

0.020

Blake Island

1

14.6

0.16

0.81

0.064

0.031

0.032

0.027

0.019

Hood Canal

Five sites pooled

5

12.8

0.16

0.47

0.067

0.032

0.033

0.027

0.019

Fishermans Point

1

16.8

0.24

0.55

0.100

0.037

0.036

0.030

0.020

Strait

Dallas Bank

1

12.0

0.11

0.33

0.053

0.028

0.030

0.025

0.018

Mean of all sites

0.075

0.033

0.033

0.028

0.019

Model inputs except growth parameters are from Table 1. Growth parameter estimates are from Hoffmann et al. (2000).

over all sites) and annua! harvest rate (fj,) of 2.7% of cuirent
exploitable biomass. British Columbia managers calculate annual
quotas using a fixed harvest rate of Wc (Campbell et al. 1998), but
this rate is applied to the estimated virgin biomass rather than
current biomass estimates, as is done in Washington.

A secondary objective of our study was to detertnine which of
the estimated geoduck life history parameters were most influen-
tial in predictions of yield and SPR. The model was most sensitive
to the estimate of natural mortality (AT), whereas growth, selectiv-
ity, and maturity parameters had relatively little effect on SPR-
based fishing mortality rates. This suggests that future research
monies are best spent making more reliable estimates of M.

Because our model is an equilibrium model and admittedly
sensitive to the estimate of M, one could ask how it might cope
with time varying natural mortality. If it were possible to construct
a functional relationship between specific, measurable categorical
variables — such as predator density, or sea temperature and natural
mortality rates — and if these categorical variables were themselves
predictable, one could estimate the expected changes in M. With a
credible estimator, the equilibrium model could be conxerted to a
dynamic pool model and revised estimates of F could be derived
for a specific future time interval of interest. Such an application
would be highly dependent on the accuracy and precision of the
predictive functions, not only the functions related to M but also
the expected annual recruitment. We are doubtful that this ap-

0.08

0.06

<i>

0.04

o

li.

0.02

0.00

matunty y

0.5 1 1.5 2 2.5 3

multiple of parameter estimate

Figure .3. The effect of difTerenl paranieter estimates on model-
derived /•■411.. values. Numbers (m the \-a\is represent nuilliples of the
"hesi" parameter estimates from fable 1 (inortalitv. selectivity, and
maturity) and Tahle 3 (growth parameter k).

proach would become profitable. Alternatively, annual or fixed
interval updates of the equilibrium F could be computed using
revised estimates of M.

If natural mortality varies over time, the true F^f^,-^ would
rise and fall proportionately with the change in M (Fig. 3). We
would err in the application of our equilibrium F dependent on the
trend in M. If M fluctuates around some normally distributed
mean, then on average our equilibrium F is probably reasonable. If
there is a significant periodicity in the trend in M (a long duration
decline, for example) and it goes unrecognized, application of the
equilibrium F risks overharvest of the resource. Managers could
impose a safety valve by creating a harvest policy that reduces the
exploitation rate below that derived from the preferred F (e.g., 0.75
F). but it would be speculative whether this precaution was suffi-
cient to account for real variability in M. Models of sto-
chastic variability in recruitment have led scientists to suggest
maintaining a larger spawning biomass and therefore adoption of
a lower prefeired F (e.g., Fj^'; rather than ^,5,,) (Mace 1994,
Clark 1993).

Our estimate of M = 0.0226 is similar to estimates from British
Columbia. Sloan and Robinson (1984) estimated M = 0.035 at a
single site, while Breen and Shields (1983) reported M = 0.01-
0.04 in five populations. Noakes (1992) estimated M = 0.03-0.04
at three sites. Both our estimate and the British Columbia estimates
relied on the catch curve method, which assumes that mortality
rate is unifortn with age and that recruitment has been constant
over the range of age groups analyzed. There is some suggestion
in our age-frequency data that a shift in geoduck recruitment has
occurred that could have biased the estimate of M. Age frequencies
did not begin to decline until about age 25. a pattern in catch
curves that is often due to inefficient sampling of younger age
classes. But for geoducks, which grow quickly and are fully se-
lected by the commercial fishery at half this age (Harbo et al.
1983). sampling inefficiency is not a plausible explanation for the
low numbers of geoducks in ihe l()-25-y age group. Instead, low
numbers of IO-25-y-old geoducks may indicate poor recruitment
during the 15-y period before sampling. This suggests that recruit-
ment declined during the period 1955-1970 (before the advent of
a fishery) and perhaps more recently. Sloan and Robinson ( 1984)
suggested Ihe possibility of a similar decline in recruitment during
the same time period in British Columbia.

Thus, catch curve estimates of M for geoducks based on older
age classes may not accurately represent current (rends in natural
mortality. They likewise reveal nothing about M for younger geo-
ducks. In either case, our results indicate that biases in the estimate

Modeling Geoduck P. abrupta Population D>namics

69

of M will have a major influence on model-based predictions of
yield and SPR. Independent estimates of M should therefore be a
high priority for research. Given the fact that geoducks are entirely
sedentar) . direct or "known fate"" estimates of M may be possible
if a reliable and noninvasive tag can be developed. Such straight-
forward measurements of annual mortality would rely on fewer
assumptions than the catch curve method and might also provide
age-specific and area-specific estimates M.

A final caveat related to the use of simple yield models such as
ours is that they do not take into account the spatial distribution of
harvested animals. Spatial structure is frequently ignored in the
management of finfish stocks, because it is assumed that survivors
are being continually mixed by movement. Under this "dynamic
pool" assumption, it does not matter whether the annual quota is
taken in small amounts over the entire fishing area or taken en-
tirely within a tiny comer of that area. But as Orensanz and Jamie-
son (1998) point out. the dynamic pool assumption may be risky
when applied to sedentary benthic species such as geoducks. More
research should therefore be devoted to the long-terin effects of
various spatial harvesting strategies on yield and spawning bio-
mass of geoducks. An experiment of this sort is underway in

Washington, where geoduck densities at 15 commercial beds are
being monitored before and after fishing to estimate an empirical
rate of population recovery. If it is based on a long span of time,
an empirically determined turnover (i.e.. recruitment) rate for com-
mercially fished geoduck beds could be used to validate, improve,
or replace the harvest rate strategies on the basis of structural
models.

ACKNOWLEDGMENTS

We thank Tom Jagielo and Dr. Annette Hoffmann for statistical
advice and reviews of an earlier draft. Lynn Goodwin. Warren
Shaul. and Conrad Budd collected and read the age samples. Don
Rothaus and Bob Sizemore provided extensive reviews of earlier
drafts. Michael Ulrich drew the site map. Don Flora, Dr. Bob
Conrad (Northwest Indian Fisheries Commission), and Dr. J. M.
("Lobo"") Orensanz (University of Washington) reviewed earlier
drafts and made helpful suggestions. We thank Dr. W. G. Clark for
providing the original FORTRAN-coded equilibrium yield model.
Finally, we thank the anonymous reviewers who made suggestions
on the final draft.

LITERATURE CITED

Anderson. A. M., Jr. 1971. Spawning, growth, and spatial distribution of
the geoduck clam, Panope generosa (Gould) in Hood Canal. Wash-
ington. PhD Thesis. University of Washington. Seattle. 133 p.

Baranov. T. I. 1918. On the question of the biological basis of fisheries, pp.
81-128. Report of the Division of Fish Management and Scientific
Study of the Fishing Industry, vol. 1.

Breen. P. A. & T. L. Shields. 1983. Age and size structure in five popu-
lations of geoduck clams (Panope generosa) in British Columbia. Can.
Tech. Rep. Fish. Aquat. Sci. No. 1 169. 62 p.

Campbell. A., R. M. Harbo & C. M. Hand. 1998. Harvestmg and distri-
bution of Pacific geoduck clams. Panopea abniphi. in British Colum-
bia, pp. 349-358. In: G. S. Jamieson and A. Campbell (eds.). Proceed-
ings of the North Pacific Symposium on Invertebrate Stock Assessment
and Management. Can. Spec. Publ. Fish. Aquat. Sci. 125.

Clark, W.G. 1991. Groundfish exploitation rates based on life history pa-
rameters. Can. J. Fish. Aquat. Sci. 48:734-750.

Clark. W.G. 1993. The effect of recruitment variability on the choice of a
target level of spawning biomass per recruit, pp. 233-246. In: G. Kruse.
D. M. Eggers, R. J. Marasco, C. Pautzke, and T. J. Quinn II (eds).
Proceedings of the International Symposium on Management Strate-
gies for Exploited Fish Populations. Alaska Sea Grant College Program
Report No. 93-02. University of Alaska Fairbanks.

Deriso. R. B. 1987. Optimal f o , criteria and their relation to maximum
sustainable yield. Can. J. Fish. Aquat. Sci. 44(Suppl. 2):339-349.

Frederick, S. W. & R. M. Peterman. 1995. Choosing fisheries harvest
policies: when does uncenainty matter? Can. J. Fish. Aquar. Sci. 52:
291-306.

Goodwin. C. L. 1976. Observations of spawning and growth of subtidal
geoducks {Panope generosa. Gould). Proc. Nat. Shellfish. As.soc. 65:
49-58.

Goodwin, C. L. 1978. Puget Sound subtidal geoduck survey data. Wash.
Dept. Fish. Prog. Rep. No. 36. 107 pp.

Goodwin, C. L. & B. C. Pease. 1987. The distribution of geoduck (Panope
abrupta) size, density, and quality in relation to habitat characteristics
such as geographic area, water depth, sediment type, and associated
flora and fauna in Puget Sound. Washington. Wash. Dept. Fish. Tech.
Rep. 102. 44 p.

Goodwin. C. L. & B. Pease. 1989. Species profiles: life histories and
environmental requirements of coastal fishes and invertebrates (Pacific
Northwest): Pacific geoduck clam. 14 pp. U.S. Fish. Wildl. Serv. Biol.

Rep. 82( 1 1 .120). U.S. Army Corps of Engineers (TR EL-82-4), Wash-
ington, D.C.

Gulland. J. A. 1968. The concept of maximum sustained yield and fisheries
management. FAO Fish. Tech. Paper 70. 30 pp.

Harbo. R. M., B. E. Adkins, P. A. Breen & K. L. Hobbs. 1983. Age and
size in market samples of geoduck clams (Panope generosa). Can. MS
Rept. Fish. Aquat. Sci. No. 1714. 77 pp.

Hoffmann. A., A. Bradbury & C. L. Goodwin. 2000. Modeling geoduck,
Panopea abrupta (Conrad 1849) population dynamics. I. Growth. J.
Shellfish Res. (in press).

Jamison, D., R. Heggen & J. Lukes. 1984. Underwater video in a re-
gional benthos survey, pp. 15-17. In: Proceedings of the Pacific Con-
gress on Marine Technology. Marine Technology Society. Honolulu,
Hawaii.

Kimura, D. K. 1988. Stock-recruitment curves as used in the stock-
reduction analysis model. / Cons. Int. Explor. Mer. 44:253-258.

Mace. P. M. 1994. Relationships between common biological reference
points used as thresholds and targets of fisheries management strate-
gies. Can. J. Fish. Aquat. Sci. 51: 1 10-122.

Noakes, D. J. 1992. On growth and mortality of geoduck clams (Panope
abrupta). pp. 22-34. In: G. Thomas (ed.). Shellfish stock assessments
for the west coast of Canada in 1991 as reviewed by the Pacific Stock
Assessment Review Committee (PS.ARC). Can. Manuscr. Rep. Fish.
Aquat. Sci. 2169.

Orensanz, J. M. & G. S. Jamieson. 1998. The assessment and management
of spatially structured stocks: an overview of the North Pacific Sym-
posium on Invertebrate Stock Assessment and Management, pp. 441-
459. In: G. S. Jamieson and A. Campbell (eds.). Proceedings of the
North Pacific Symposium on Invertebrate Stock Assessment and Man-
agement. Can. Spec. Publ. Fish. Aquat. Sci. 125.

Quinn. T. J. II & N. J. Szarzi. 1993. Determination of sustained yield in
Alaska"s recreational fisheries, pp. 61-84. In: G. Kruse, D. M. Eggers.
R. J. Marasco. C. Pautzke, and T. J. Quinn II (eds.). Proceedings of the
International Symposium on Management Strategies for Exploited Fish
Populations, Alaska Sea Grant College Program Report No. 93-02.
University of Alaska Fairbanks.

Ricker. W.E. 1975. Computation and interpretation of biological statistics
offish populations. Fish. Res. Bd. Can. Bull. No. 191. 382 pp.

Robson, D. S. & D. G. Chapman. 1961. Catch curves and mortality rates.
Tratis. Am. Fish. Sac. 90:181-189.

70 Bradbury and Tagart

Seber. G. A. F. 1982. The Estimation of Animal Abundance and Re- the geoduck clam /"anope oftrapfa (Conrad) from southern British Co-

lated Parameters. 2nd ed. MacMillan Publishing Co., New York. 654 pp. lumbia. Canada. / Shellllsh Res. 4:131-137.

Shaul. W. & C. L. Goodwin. 1982. Geoduck (Panope generosa: Bivalvia) Thompson, G. 1993. A proposal for a threshold stock size and ma.ximum

age as determined by internal growth lines in the shell. Can. J. Fish. fishing mortality rate. pp. 303-320. In: S. J. Smith, J. J. Hunt, and D.

Aqiiat. Sci. 39:632-636. Rivard (eds.). Risk Evaluation and Biological Reference Points for

Sizemore, B. & M. Ulrich. 1999. 1999 Geoduck Atlas: Atlas of Major Fisheries Management. Can. Spec. Piihl. Fish. Aiiuat. Sci. 120: 303-

Geoduck Tracts of Pugel Sound. Washington Department of Fish and 320.

Wildlife, Olympia, 40 pp. Weisberg, S. 1985. Applied Linear Regression. 2nd ed. John Wiley and

Sloan, N. A. & S. M. C. Robinson. 1984. Age and gonad development in Sons, New York. 324 pp.

Joiirihil ofSlwllfhli Resfiiirh. Vol. 19, No. 1, 71-75. 2U00.

MICROSPORIDIOSIS IN QUEEN SCALLOPS (AEQUIPECTEN OPERCULARIS L.) FROM

U.K. WATERS

KARIN B. LOHRMANN,' ^ ' STEPHEN W. FEIST," AND
ANDREW R. brand'

' Universidad Catolica del Norte. Fucultad de Ciencias del Mar. Coquimbo.

Chile
'CEFAS Weymouth Laboratory. Barrack Road. The Nothe. Weymouth.

Dorset DT4 SUB. UK
^The Uuiversitx of Liverpool. Port Erin Marine Laboratoiy. Port Erin. Isle

of Man IM9 6JA. UK

ABSTRACT Spores of a microsporidian parasite were found in the queen scallop, Aequipecten opercularis (L.), collected from
several coastal sites around the United Kingdom. Developing spore stages were detected in contact with the host cell cytoplasm.
Infected host cells formed aggregates in the connective tissue of the digestive gland. Fully mature spores were found free within blood
vessels. These spores exhibited a dome-shaped polaroplast. a diplokaryon. a posterior vacuole, and an isofilar polar tube, with seven
to eight coils in a single row . In immature spores, the number of coils in the polar tube varied, with some having 7 to 8 coils and others
having 10 to 12 coils.

KEY WORDS: Microsporidian, spores, scallop, Aequipecten opercularis. parasite

INTRODUCTION

MATERIALS AND METHODS

Scallop species are of commercial interest in many parts of the
world. In Europe, the main species exploited is the great scallop
(Pecten maximits), which is the subject of important fisheries in
France, Ireland, the United Kingdom, and Norway, where it is
cultivated on a small scale. There is also a significant natural
fishery for the queen scallop. Aequipecten opercularis. This spe-
cies is distributed from northern Norway and the Faroe Islands to
the Mediterranean and Adriatic Seas, It lives in depths ranging
between 18 and 46 m on fine sand, fine gravel, or sandy gravel.
The main areas of fisheries for this species lie along the western
coasts of the British Isles and France but also include the Shetland
Isles and Moray Firth in the northeast and both sides of the English
Channel (Anseil et al, 1991, Brand 1991),

Since little is known on the natural disease status in these two
species, a survey was undertaken to collect baseline data on para-
sites and possible pathogens in natural, apparently healthy popu-
lations. During this study, a microsporidian was found in the di-
gestive gland of the queen scallop, and electron microscopy (EM)
studies were undertaken for characterization and identification of
this parasite.

Microsporidians are eukaryotic, obligate intracellular parasites
of almost all animal phyla. The most common hosts are arthropods
and fish (Canning 1990), Only a few microsporidians have been
reported from bivalves. Comps et al. (1975) found an unidentified
species in Cardium edule and Jones (1981) described Microspo-
ridium rapua from the oyster Ostrea lutaria in New Zealand. In
mytilids, a microsporidian parasitizing the oocytes has been de-
scribed by Figueras et al. (1991a. 1991b) who found Steinhausia
mytilnvum in Mytilus galloprovincicdis from Spain and in Mytilus
edulis from the United States, Villalba et al. (1997) observed the
same parasite in ova of Mytilus galloprovincialis. and Sagrista et
al. (1998) described the developmental cycle and ultrastructure of
this protistan in M. galloprovincialis.

In this study, the spores of a microsporidian parasite from the
queen scallop are described.

A total of 454 adult queen scallops (A. opercularis) were
sampled during the period July 1997 to April 1998, The sampling
localities are shown in Figure 1, The localities and dates of sam-
pling are shown in Table 1.

For histology, transverse tissue sections were taken that in-
cluded the digestive gland, kidney, gills, gonad and mantle, and
they were fi.xed in Davidson's fixative (Shaw and Battle 1957) for
24 h. The tissues then were dehydrated, cleared, infiltrated with
paraffin wax, and sectioned at 5-6 \i.m. The routine stain used for
all samples was Gill's hematoxylin and aqueous eosin. Selected
samples were stained with special stains: Farley-Feulgen (Farley
1969) for identifying DNA. and Giemsa for staining presumptive
parasites. For each sample, one section was cut, and the slides were
examined using a Reichert Polyvar microscope. Photographs were
taken with a photomicroscope (E800 Eclipse, Nikon. Tokyo. Ja-
pan),

For transmission EM. l-mm' pieces of digestive gland from
each specimen sampled for histology were fixed in 3% glutaral-
dehyde in 0.2M cacodylate buffer with 1.75% NaCI, for 2 h at
room temperature, and were washed in the same buffer. After
histological assessment, those samples found to harbor the mi-
crosporidian were further processed for EM, Tissues were washed
another three times in 0,2M cacodylate buffer with 1.75% NaCl,
and were postfixed for I h in 1% osmium tetroxide in the same
buffer. After washing twice with buffer, they were rinsed in dis-
tilled water, stained for I h en bloc with 2% aqueous uranyl ac-
etate, dehydrated in ethanol, washed in propylene oxide, and em-
bedded in Epon 812 (premix. BDH). Semithin sections. 1 p.m
thick, were cut on a Reichert Ultracut S microtome and were
stained with toluidine blue, Ultrathin sections were cut with a
diamond knife and were stained with aqueous uranyl acetate and
lead citrate. The sections were viewed and photographed with an
electron microscope (EM 900. Zeiss) at 50 kV.

Measurements from stained histological or semithin sections
were made using a Nikon E800 microscope with LUCIA screen

71

72

LOHRMANN ET AL.

SiteD

Site A

TABLE 2.
Prevalence of microsporidiosis in A. opercularis.

Site C

Figure 1. Map of England and Wales showing the sampling sites.

measurement system. The number of longitudinally sectioned
spores that were measured is indicated in each case.

RESULTS

The prevalence of microsporidiosis in A. opercularis is given in
Table 2.

Spores were found in two different locations in the digestive
gland o{ A. opercularis. Immature spores were found in the cyto-
plasm of connective tissue cells, and mature spores were observed
free in blood vessels. In some scallops, both kinds of spores were
seen together in the same section; in others, only one kind of spore
was found (Fig. 2).

The cells with maturing spores formed aggregates measuring
approximately .^00 (xni in diameter. They were found in two of the

TABLE I.
Localities and dates of sampling.

Site

Number

Date of

Locality Name

Reference

Sampled

.Sampling

Isle of Man. Bradda Offshcire

.Sile A

.5

05/08/97

Isle of Man. Douglas

Site A

150

18/11/97

Red Wharl Bay

Site B

59

1 WO 1/98

West ol Portland Bill

Site C

33

24/09/97

West of Portland Bill

SiteC

117

14/10/97

West of Portland Bill

Site C

50

24/04/9,8

Hunibcr Rough

Site D

40

08/01/98

Isle of
Man (A)

Red Wharf

Bay (B)

Portland
Bill (C)

Humber
Rough (D)

4.5%

10.2%

I2.57f

20%

80 resin blocks examined. The whole aggregate (Fig. 2) as well
as each infected host cell was surrounded by layers of fibroblast-
like cells, as shown at the EM level (Fig. 3). In infected cells, the
nucleus could be observed in some sections. The cytoplasm gen-
erally was degraded, although mitochondria could still be recog-
nized. The presence of a sporophorous vesicle was not confirmed,
the spores being in direct contact with the host cytoplasm. The
spores measured 2.3 (range 1.8-2.8) x 1.3 (range 1.1-1.9) (i.m (h
= 20). had an elongate-ovoid shape, and showed different degrees

Figure 2. Histological section shoeing nialure spores (msl, free in a
blood vessel (l)\ », and Iho aggregates of host cells containing immature
spores (isl. The upper aggregate is less mature, and both aggregates
are surrounded h\ I'lhrohlast-like cells of host origin (arrows): dl =
digestive gland tuhule ((ilenisa stain: bar = 50 pm).
Figure 3. Transmission electron micrograph of one infected cell v»ith
immature spores (isl. surrounded h\ nbroblast-like cells of host origin
( Fl. fhe cell membranes of these cells form layers around each infected
cell (arrows) (bar = 1 ^ml.

MiCROSPORIDIOSIS IN QliEEN SCALLOPS

73

Figure 4. Mature spore viewed in longitudinal section. The exospore (Ex) and the endospore (En) can be observed. Internally, the polaroplast
(P) and oblique sections of the polar tube can be seen (*) (bar = 0.1 fini).

Figure 5. Electron micrograph of a slightly oblique section of a mature spore. The outer covering consists of an exospore (Ex), endospore (En),
and the cell membrane (arrow). The polaroplast can be observed at the anterior end of the spore, with the two nuclei (N) forming a diplokaryon
in the central region of the spore. The coils of the polar tube in transverse section (*), the posterior vacuole (Pv), and in close proximity Golgi-like
membranes (G) can also be seen (bar = 0.1 jim).

of maturation. It was not possible to determine whether the im-
mature spore contained a single nucleus or a diplokaryon, since
areas considered to be nuclear did not appear to be delimited by an
envelope. The polar tube was isofilar, with 10 to 12 coils in a
single row. Some spores showed a shorter polar tube, with seven
to eight coils. The spores were limited by an inner electron-lucent
endospore and an outer electron-dense exospore.

Mature spores were found in only one of the resin blocks ex-
amined. They were located in blood vessels and were elongate-
ovoid in shape, measuring 2.3 (range 1.9-3.2) x 1.2 (range 0.8-
1.7) |jLm (/! = 9). They were limited by an outer exospore and an
electron-lucent endospore covering the plasma membrane (Figs. 4,
5). The polar tube was inserted into the anterior anchoring disc
(Fig. 6). passing through the center of the spore, and then, in the
posterior two thirds of the spore, were wound in most cases into 7
to 8 coils (Fig. 7), and exceptionally into 9 coils (Fig. 5), and were

aligned in a single row. The polar tube was isofilar. measuring 83.5
nm in diameter. A conspicuous, dome-shaped polaroplast occupied
the anterior third of the spore, enclosing the straight region of the
polar tube and terminating close to the coiled polar tube (Fig. 4).
Two spherical nuclei were closely apposed, forming a
diplokaryon. Each measured up to 0.88 |jim in its longest axis and
was flattened in the zone of contact with the other nucleus (Fig. 7).
The diplokaryon was located in the central third of the spore,
between the polaroplast and the posterior vacuole. The latter was
limited by a single membrane, with Golgi-like membranes often
present in close association (Fig. 5).

DISCUSSION

This is the first time that a microsporidian infection has been
reported in any scallop species.

74

LOHRMANN ET AL.

Figure 6. Two electron micrographs showing the anterior pole of the spore. In these sections the anchoring disc (Ad) with the polar tube (*)
attached can be seen. The polaroplast (P) is also apparent (bar = 50 nm).

Figure 7. Electron micrograph from a spore sectioned through the nuclei (Nl of the diplokaryon. The nuclear envelope can be clearly observed
(arrows). The polar tube (*) has seven coils, and the posterior vacuole (Pv) is also present in this section (bar = (1.5 nm).

Spores were found in two dift'erent locations in the digestive
gland. Immature spores were located intracellularly. and mature
spores were located within blood vessels.

We were unable to find any of the earlier stages of this parasite
in the scallops examined. Unlike in other microsporidian species,
where developmental stages and spores are present concurrently
(Comps et al. 1979, Amigo et al. 1996, Johnson et al. 1997. Larsson
et al. 1997), in this species only one developmental stage, the spore,
could be observed. It is possible that the early stages were present
in a tissue other than that of the digestive gland. .Since the cells
infected by the microsporidian appear to be hemocytes, the poten-
tial for infection in tissues apart from the digestive gland, such as
the intestine, stomach, and gills should be recognized. Additional
EM studies are needed to investigate this possibility. That the
microsporidian has an intermediate host within which the earl\
developmental stages could be present should also be considered.
Although most microsporidians have only one host, there are sev-
eral examples of the requirement for an intermediate host (i.e.,
Amhlyiyspora) (Andreadis 19S.'S. Beciiel 1992).

The immature spores differed slightly in the length of the polar
tube, some having 7 to 8 coils, and others showing 10 to 12. The
presence of two types of spores differing mainly in the length of
the polar tube has been described for other microsporidian species,
i.e., Noseinii spp. (Iwano and Ishihara 1991 ) and Noseiua miiscidi-
fiiracis (Becnel and Geden 1994). These authors suggest that a
shorter polar tube is characteristic of spores involved in infection
of other cells in the same host, the longer polar tube belonging to
spores that are involved in transmission from host to host. All the
mature spores examined in the present study had a short polar tube,
but they were all from one specimen and from the same resin
block. We presiMiie that mature spores with a long polar tube also
exist, because ue found them in the immature spores. Despite the
large number of resin blocks examined, we did not succeed in
finding the mature spores with 10 to 12 coils of the polar lube.

Fully mature spores showed clear evidence of a diplokaryon.
This feature, together with a polar tube consisting of eight coils in
a single row, the overall dimensions, and the fact that it is infecting
an invertebrate host, places this microsporidian near to the genus

MlCROSPORIDlOSlS IN QUEEN SCALLOPS

75

Pseiidopleistophora (Sprague et al. 1992). a microsporidian first
described as Pleistophora sp. parasitizing eggs of the annelid Ar-
mandia brevis by Szollosi (1971).

One important point that needs to be explained is the mecha-
nism by v\ hich immature spores contained in individual cells later
appear as free, mature spores in blood vessels. No transitional
forms were seen in the current study, but in some reports, as the
maturation of the spores progresses, the host cells start to loose
their plasma membranes and become a syncytium (Weiser 1976).
In this way. the spores would be released to reinfect adjacent cells
or to become phagocytosed and perhaps migrate to other tissues.

No host reaction against this microsporidian was seen, other
than a thin capsule made by fibroblast-like cells. This protistan
does not seem to be a threat to queen scallops, as those sampled
showed no evidence of poor condition. However, if these scallops
become stressed due to changes in temperature, salinity, or crowd-
ing, as occurs in culture situations, the parasite could potentially
become harmful to the host (Sindermann 1990). Despite the high

prevalence of microsporidiosis in animals from a variety of loca-
tions around the United Kingdom, the impact of this parasite on
wild populations oi A. opercidaris remains unknown. Further stud-
ies are needed to investigate the pathogenicity of the microsporid-
ian in A. openidwis held in laboratory conditions under different
temperatures and stocking densities. In addition, the identification
of potential interinediate hosts and early developmental stages of
the parasite are required for a specific identification of this mi-
crosporidian.

ACKNOWLEDGMENTS

K.B.L. thanks The British Council for a fellowship that allowed
her to work for one year in the United Kingdom, and also MAIT
funding, which contributed to this work. The authors also want to
thank Dr. Eduardo Couve for access to the electron microscope at
the Universidad de Valparaiso. Valparaiso, Chile, and Mr. Fidel
Vargas for his skillful technical assistance.

LITERATURE CITED

Andreadis. T.G. 1985. Experimental transmission of a microsporidian
pathogen from mosquitoes to an alternate copepod host. Proc. Natl.
Acad. Sci. U.S.A. 82:5574-5577.

Amigo. J.M.. H. Salvado. M.P. Gracia & C.P.Vivares. 1996. Ultrastructure
and development of Microsporidium ovoidewn (Thelohan. 1895) Spra-
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Amblyospora califomica ( Microsporida: Amblyosporidae) in the in-
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coast of the United States. ./. Shellfi.di Res. 10:89-94.

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cultured cell. / Invertebr. Pathol. 57:211-219.

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quence in Edhazardia aedis (Microsporidia: Culicosporidia), a parasite
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Pathol. 70:69-75.

Jones. J.B. 1981. A new microsporidian from the oyster Ostrea lutaria in
New Zealand. J. Invert. Pathol. 38:67-70.

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dosporidae fam. Nov.), a new microsporidian parasite of Daphnia ma-
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digestive tract of the oyster Crassostrea virginica (Gmelin). Can. J.
Zool. 35:325-347.

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eggs of the polychaete Armandia brevis. J. Invertebr. Pathol. 18:1-15.

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and diseases of farmed mussels Mytilus galloprovincialis throughout
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Jourmil of Shellfish Reseurch. Vol. 19, No. 1. 77-83. 2000.

EVALUATION OF THREE METHODS OF BOTTOM CULTURE OF THE TROPICAL SCALLOP

EUVOLA (PECTEN) ZICZAC (L. 1758)

LUIS FREITES V,' ANIBAL VELEZ AND CESAR LODEIROS

Depuruimeiuo de Biologia Pesqitera

Instituto Oceanogrdfico de Venezuela

Universidad de Oriente

P.O. Box 245

Ciimaiui 6101

Venezuela

ABSTRACT Three methods were used to study the growth and survival of juvenile Euvola ziczac (initial shell height of 40.4 mm
SD = 4.21. and initial dry mass tissues of 0.35 g (SD = 0.01). which were set out at a density of 15 individuals m"- on a sandy bottom
at Turpialito in the Golfo de Cariaco. Venezuela. The first method was applied on an area of 3 x 5 m (15 m*) with minimum
demarkation (0.20-m low walls) on the bottom, the second method was applied on 1 x 1 m corrals with 1-m high walls, and the third
method on 1 x 1 x 1 m cages with bottom and top covers. Both treatments with high walls were conducted with 15 replicates. We could
not quantify growth and survival in the first treatment, because the rate of escape was >80'7f month"' (12 scallop m~- month"' ). In the
corrals, the escape rate increased progressively from 4% ( 1 scallop m"- month"') to 36% (5 scallop m"- month"'), suggesting that the
swimming ability of Euvola ziczac increased with size from an initial 40.4 mm to final 69.7 mm in shell height obtained in this study.
No scallops escaped from the cages, but survival was less than in the corrals. Our observations suggest that the most appropriate bottom
culture method would be corrals with walls higher than 1 m.

KEY WORDS: Euvola ziczac. bottom culture, scallop, enclosure, grow-out

INTRODUCTION

Euvola ziczac is a functional hermaphrodite scallop present
from Cape Hatteras, North Carolina, throughout the Gulf of
Mexico and the Caribbean Sea to southern Brazil off Santa Cata-
rina (Abbott 1974). Although Euvola ziczac does not form dense
natural banks able to support commercial fisheries activity, the
species is considered to have great potential for commercial aqua-
culture activity off the Bermudas, Columbia, Venezuela, and Bra-
zil (Hernandez 1990. Velez and Lodeiros 1990, Waller 1991, Cas-
tellanos et al. 1997). In Venezuela, several studies have determined
aspects of biological feasibility for culture in the marine environ-
ment under hanging culture conditions (Freites et al. 1993, Freites
et al, 1995, Freites et al. 1996, Lodeiros and Himmelman 1994). In
this manner, rapid growth (up to 30-35 mm) and high survival rate
have been attained. However, in larger sizes, diverse factors in-
trinsic to suspended culture, such as fouling (Lodeiros and Him-
melman 1996), wave action (Freites et al. 1999). and food quality
(Hunaulth et al. unpublished data), linked with unfavorable periods
of high temperature, low available food, and reproduction effort in
this species, generating stressful conditions, which lead to a de-
crease in growth and survival, have been noted (Lodeiros and
Himmelman 1994, Lodeiros and Himmelman 2000). However,
when Euvola ziczac is cultured in contact with the sandy substra-
tum on the seabed (its natural habitat), high growth and survival
rates have been noted, considering bottom culture as the most
appropriate for the grow-out stage of the species (Velez et al. 1995,
Hunaulth et al. unpublished data).

Studies of the feasibility of various bottom culture techniques
have been made for numerous pectinid species including Chlamys
farreri (Wang et al. 1992), Placopecten magellanicus (Kleiman et
al. 1996). Pecten maximus (Cliche et al. 1994, Dao et al. 1995).

'Address correspondence to: E-mail: lfreites@cumana.sucre. udo.edu. ve or
lfreites@iim.csic.es

Patynopecten yessoensis (Aoyama 1989, Ito 1991), Argopecten
circularis (Caceres-Marti'nez et al. 1986: Maeda-Marti'nez et al. in
press), and Pecten novaezelandicie (Bull 1991 ). So. bottom culture
is an alternative that has shown important levels of profitability in
other scallop species. This is because of a lower investment in
equipment, consumables, and maintenance than with the hanging
method (Frishmand et al. 1980, Felix-Pico et al. 1991, Gilbert and
Leblanc 1991. Wang et al. 1992, Kleinman et al. 1996).

In this manner, the aim of this study was to evaluate the growth
and survival of scallop Euvola ziczac applying two bottom culture
grow-out methods: with barriers in the cage and corral enclosures
and with no barriers, to obtain market size.

MATERIALS AND METHODS

This study was conducted over a 6 month period (February
27-September 7, 1994) off the south coast of the Golfo de Cariaco,
eastern area of Venezuela (Fig. 1 ). The individuals used in the
experiment were obtained from a hatchery under controlled con-
ditions at the end of August 1993, following the methodology
described by Velez and Freites (1993). Scallops were held in sus-
pension for intermediate culture following the methodology de-
scribed by Freites et al. (1993, Freites et al. 1995) until the initial
mean shell height for the study of 40.4 mm (SD = 4.20) and initial
dry mass tissues of 0.35 g (SD = 0.01) was obtained. A total of
720 individuals oi Euvola ziczac were divided into three batches of
240 individuals each and thereafter, we took 15 individuals for
each batch to the initial sample. Later, the remaining 225 individu-
als of each batch were allotted to the cages, corrals, and the barrier-
free method. In the case of the enclosures. 15 replicates were
introduced. 12 of which were experimental and three replace-
ments. The latter were introduced to maintain density of the indi-
viduals reduced by the effects of mortality and escape. In the case
of the barrier- free method, a total area of 15 m" was evaluated.

The cages measured 1 x 1 x 1 m, built with galvanized iron bars
8 mm in diameter, lined on the six sides by a galvanized wire mesh

77

78

Freites et al.

100° 90°

40°

30°

20°

10°

0°

Figure 1. Geographical location of the study area.

with a 30-mm diameter opening (Fig. 2a). The corrals were built of
the same size and with the same materials as the cages, except that
the galvanized mesh was not fitted on the top and bottom parts
(Fig. 2b). Both types of enclosures were buried 7-8 cm into the
sand to allow the scallops in cages also to bury, and in case of the
corrals, to avoid the escape of individuals under the enclosure and
at the same time, to avoid entry of such predators as gastropods
and crabs. The individuals in the bamer-free method were distrib-
uted ill the area marked out beforehand by galvanized mesh, but
with an edge of 20 cm. This was used to mark out the original area
and thus enabling control of the density but not to act as a barrier
(Fig. 2c). Both cage and corral methods were randomly placed at
a depth of 7-8 m by a SCUBA diver. Density was a common
parameter ( 15 individuals m"^) both for the enclosures and for the
barrier-free method.

Growth of individuals in the enclosures was followed by sam-
pling the three replicates of five individuals ( 1 5 individuals) taken
randomly, by previously allotting them random numbers. These
samples were obtained over appro.ximately 60 days. Also, the
number of dead and live individuals was quantified monthly in all
the experimental replicates in terms of determining moilality. es-
cape, and monitoring the density of individuals.

The paramclers for evaluating growth were shell height (dis-
tance between the anterior-posterior margins taken with a Vernier
calliper with 0.01 accuracy) and the dry mass of ihe shell, gonad,
muscle, digestive gland, and remaining somatic tissues (dried at
80 ' C for 72 h).

Because there was an initial escape rate on the order of 84% of
the individuals placed in the original area with the barrier-free
method and because these could not be recovered, the evaluation
of Ihis method could no! continue. Moreover, because of scallop in

corrals escaping, we could not continue the evaluation of methods
for longer than the 6 months of the study. To evaluate the results
on the enclosures, cages, and corrals during the experimental pe-
riod, the paired student's t test was applied to all growth param-
eters. Also, in terms of evaluating the masses and heights attained
at the end of the study, the nonpaired student's ; test was applied.
To evaluate the survival rate, because the data were incompatible
with assumed normal levels, analysis was conducted by nonpara-
metric tests not correlative to those previously noted (Wilcoxon
and Mann-Whitney range tests, respectively, following the re-
comendations in Zar ( 1984). For all test a a = 0.05 was applied.

RESULTS

Escape

At the start of the experiment, an 84'7f escape rate was found
(12 scallops m"~ month"') from the original area using the barrier-
free method (Table 1). Furthermore, despite having searched an
approximate area of 2500 m" taking the original area as the center,
none of the individuals (C/r recovery) was recovered, so that we
were unable to continue with the evaluation. In the corrals, a
progressive increase in monthly escapes was noted, from 4% (1
scallop m"- month"') rising to TibVc (5 scallops m"" month"'),
observed at the end of the experiment (Table 1 ). In this way, the
ratio of growth in shell height with the percentage increase in
escape of individuals reared in cage was directly proportional (P <
0.05, r" = 0.89: b = 2.15). No scallops escaped from the cages.

Survival Rate

Monthly survival rates in the two enclosures showed similar
trends (Fig. 3a) (Wilcoxon test, P = 0.679). At the end of the
study, however, accumulated mortalities result in a significantly
lower survival rate of the individuals in cages (51%); whereas, in
the corrals, it was in the order of 78% (Fig. 3b) (Mann-Whitney
test, P < 0.05).

Shell Size and Mass

Growth curve trends in shell height, both for indisiduals in
cages and in corrals, were similar throughout the study period
(paired student's /-test, P = 0.912). with the exception of the last
sampling, where a reduction in the growth rate of cage-reared
individuals was observed (Fig. 4a). At the end of the experimental
period, the individuals reared in conals had an average of 73.1 ±
2.34 mm; whereas, the average for the cages was 69.7 ± 3.93 mm.
These differences, however, were not significant (nonpaired stu-
dent's /-test, P = 0.082). The dry mass of the shell showed a
growth pattern similar to that of shell length during almost the
entire study period (Fig. 4a. b). but in this case, significant differ-
ences were noted (paired student's /-test. P < 0.05). So, there were
significant differences (nonpaired student's /-test, P < 0.05) noted
in the shell growth rates at the end of the study between scallops
in corral (26.5 ± 2.61 g) compared to the indi\ iduals maintained in
cages (23.9 ± 3.72 g).

Somatic Tissue Mass

The groulh trend for somatic tissues muscle, digestive gland,
and the remaining somatic tissues observed during the study period
in cages and corrals, (Fig. 4c, d, e), showed no significant differ-
ences (paired student's /-test, P = 0.719, 0.679, and 0.369, re-
spectively), despite the fact that these showed divergences in the

Bottom Culture of Euvola zkzac

b

79

Galvanized net

1 m

1 m

Cage

1 m

close ' f

bottom

Galvanized iron rod

open top

open
bottom

Corral

yyyyy^yyyyyy^vvyyywyyyyyvf^yvyyyv^ | 0.20 m in height

»-

5.0 m

Barrier-free

Figure 2. Design of enclosure cages (a) and corrals (b) and tlie barrier-free method (c).

latter period of the sampling, particularly in the remaining somatic
tissues of both groups of individuals. Also, the decrease in growth
of the mass of remaining tissues and gonads of individuals main-
tained in cages, observed at the end of the experimental period,
contrasted with the increase in mass of these tissues in the corral-
reared individuals (Fig. 4d, f). in such a manner that these were
significantly greater at the end of the experimental period (non-
paired student's ?-test, P < 0.05). In the case of the muscle mass in
individuals reared in cages and corrals, no significant differences
were shown at the end of the study period (nonpaired student's
r-test. P = 0.947).

DISCUSSION

Our results showed that by applying the bottom culture method,
in the course of approximately I year (from fertilization of the
oocytes), sizes and biomasses considered as commercially feasible

TABLE 1.

Monthly escape ( % ) of the scallop Euvola ziczac observed during the
study in the methods evaluated: barrier-free and cage.

Month

Methods

Shell Height

% Escape

April

Barrier-free*

84

Corrals

54 mm

4

May

Corrals

59 mm

10

June

Corrals

65 mm

18

July

Corrals

67 mm

33

September

Corrals

70 mm

36

In the cages, the percentage of escape was always of 0%.
* We were unable to continue with the evaluation.

80

Freites et al.

100

80 9*^

-©- Corrals
-O- Cages

J J

Month

Cages Corrals

Figure 3. Monthly (a) and cumulative survival (b) of Euvola ziczac in bottom culture (vertical bars represent the standard deviations of the
values).

for the scallop Euvola ziczac may be obtained. Thus, the size range
in adults located on natural banks is between 65 and 95 cm (Him-
melman and Lodeiros, unpublished data), and sizes for individuals
reared both in corrals (73 mm) and in cages (70 mm) were within
this size interval. These sizes of scallop had wet muscle weights of
7-8 g. which are considered excellent for scallop commercializa-
tion (Dore 1991).

The rate of growth observed in this study (approx. 0.16 mm
d"') was similar to that observed in Euvola ziczac (approx. 0.15
mm d~') for Velez et al. ( 1995) in the same locality, for a similar
period of year (70% of the same period) and in the same period of
190 days, but with a higher initial high density (64 individuals
m"~) than in this study ( 1 5 individuals m""). These similar growth
rates, despite the different densities in both .studies, suggest that the
growth observed in this study was not more influenced by the
density used. This also suggests that the bottom culture produc-
tions of this species can be increased with the use of higher den-
sities in the methods studied.

The high escape rate of individuals with the barrier-free method
led to the discontinuation of this method. This suggests that Euvola
ziczac has a high dispersion capacity, which would lead to a low
recovery rate of the stock originally used for cultivation with no
barriers. Therefore, we considered that it is necessary to develop a
new experiment with a more adequate scale before suggesting the
use of this barrier-free method. In any ca.se, in other countries, such
as Canada, Japan, and France, the use of the barrier-free bottom
culture method in more adequate scales had a low recovery rate of
the initial stock because of the high escape rates of scallops (Wild-
ish et al. 1988. Aoyama 1989, Cliche et al. 1994, Dao et al. 1995).
Nevertheless. Wang et al. (1992) showed that even when the re-
covery rate of the initial bottom stock was on the order of 54'/r, the
low production costs exerted an influence on the high profitability
of scallop cultivation of Chlaniys farrcri. In our case, the recovery
rate of individuals with the barrier-tree method was 16%. This may
be considered as very low it ue take into account thai it was
obtained after only .^0 study days. This escape capacity was also
evident in the corrals where, despite l-m high barriers, escape
gradually increascil unlil the end of the study (.^6% escape). Fur-

thermore, it was observed that some specimens cultured with this
method, when unintentionally disturbed for the purpose of taking
samples, showed a clearly evident capacity to escape beyond the
1 -m high barriers. In these observations, we noted the increase in
the vertical displacement capacity of Euvola ziczac as size in-
creases. However, we do not exclude the possibility of the increase
in the rate of depredation by some fish, octopus, and crab decapods
during the time of the experinient. Nevertheless, this phenomenon
was not noted in the course of our frequent observations.

At the end of the study, the cage-reared individuals presented a
significantly lower survival rate than those reared in corrals. In the
cages, greater protection from predators was expected because of
the presence of netting on all sides that, theoretically, would im-
pede their entrance of the same. In the cages, however, several
fragmented shells were collected, a fact that indicated the action of
predators. For this reason, a detailed search was conducted, and the
presence of decapod crab juveniles Calappa cinerea was discov-
ered. These had gone unnoticed until that point because of their
strategy of burying themselves in the substrata. This decapod has
strong chela that allow it to fragment Euvola ziczac shell. Judging
by the condition of the shells. This ability has also been noted in
the species Calappa ocelluia. as a result of its preying action on the
bivalve Brcuhidonlcs doiuiitf^cnsis (Hughes and Finer 1989). This
suggests that the decapod C. cinerea apparently entered the cages
at its juvenile stage, when the opening in the mesh still made this
possible, so that it was also able to take advantage of the protection
afforded by the cage. This situation helped avoid competition for
food and being preved upon. One observation that supports this
hypothesis is that in the corrals, where there was no upper netting,
the dead individuals of Euvola ziczac showed no shell fragmenta-
tion and nor were any detected C. cinerea. These observations
differ from those for the cultivation of temperate water scallop
species where the use of nets substantially decreased predation
(Morgan et al. 1980, Quayle and Newkirk. 1990).

Because this study was conducted in a certain season of the
year, possible biocontrol of decapod C. cinerea juveniles may not
be present throughout the year. Furthermore, one of the predators
that nui\ possibly exert a dramatic effect on scallop survival under

Bottom Culture of Euvola ziczac

81

8-1

a

Shell height

7-

Cages,^

V^

^

^ 6-

B

u

"-^ 5 J

/

Ct>rrals

4-

/

3-

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F M A M J J A S

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f'm' a'm' J ' j' a' s'

1.6-]

C

Dry

mass muscle

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1.2-

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••

^ 1.0-

130
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/

K

0.6-

/

0.4-

/

f

0.2-

1 1 1 1 T I

1

bo

l.On

d

Dry mass remaining tissues

0.8-

y.

fi

0.6-

.^'^^

0.4-

A

0.2-

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/^

0.0-

— 1

1 1 1 1 1 1 1

F M A M J J A S

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(30

0.5

0.4-1

0.3

0.2
0.1

0.0^

Q Dry mass digestive gland

F ' M ' A 'm

J
month

— I 1 —

J A S

1.0'

f

Dry mass gonad

0.8-

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z

^0.6-
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0.4-

A

y

0.2-

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— 1 1 1 r— 1 r

1

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month

Figure 4. Growth in siieli iieiglit (a) and dry mass of the shell (b) muscle (c), remaining soft tissues (d), digestive gland (e), and gonad (f) of the
cultivated specimens oi Euvola ziczac from bottom culture (vertical bars represent the standard deviations of the values).

bottom culture conditions, as noted on natural scallop banks, are in future studies aimed at determining the effect of predators on the

the cephalopods Octopus spp. (Freites, personal observations), survival of bivalves in corrals, it is advisable to cover different

Nevertheless, despite the fact that the period of greatest influence periods or seasons of the year.

by these predators fell within the experimental period of this study Also, in the Golfo de Cariaco. hanging culture of these bivalves

(from June to September), they were not observed. For this reason. does not guarantee a lesser impact of predation compared to some

82

Freites et al.

temperate water species (Quayle and Newkirk 1990, Hickman
1992). This is because of the recruitment of some predatory de-
capod and gastropod species during their planktonic larval stage,
which allows them to gain access to the hanging baskets. Once
inside these baskets, if uncontrolled, their growth is so fast that, in
some cases, they have caused substantial mortality rates (>60%) in
the cultivation of several bivalve species with culture potential,
including Euvola ziczac (Freites et al. 1995, Freites et al. 2000).
Pinna carnea (Narvaez 1999) and pearl oyster Piiutcula inihricala
(Pico D., unpublished data).

The growth pattern for the corral and cage-reared individuals
was similar, except in the latter sampling period, when the cage-
reared individuals showed lower growth rates. These differences
may not be attributable to a differential colonization by fouling
organisms in nets of the enclosures or on shell that may, in the long
term, affect food availability for the scallops, because the nets in
both enclosures were cleaned throughout the experimental period
because of the action of "grazing" of some fish and benthonic
invertebrates on the net of the corrals and cage (personal obser-
vation), and because virtually no organisms colonized the shells of
scallops in both enclosure, probably because the scallops were
usually recessed in the sand. This together with maintaining the
same density of individuals in the enclosures suggests that food
availability was not a factor in the decreased growth observed in
the cages.

One possible explanation is based on the fact that, as the Euvola
ziczac individuals reared in cages increased in size and even when
new C. cinerea decapod recruits were observed, they were physi-
cally unable to prey on the scallop because of the larger, more
resilient shell, as evidenced by the subsequent lack of fractured
shells. We do not rule out the fact, however, that the decapod
juveniles may cause some disturbance leading to a defensive be-
havior, so that the bivalves close their valves, thus restricting fil-
tration time and, con.sequently, affecting growth.

As we have seen earlier, the growth of juvenile scallops reared

in the two types of enclosures may not be used as a selection
criterion for recommending the use of dismissal of one of these
two types of enclosures studied, particularly if we take into ac-
count that at the end of the experimental period, no significant
differences were noted in muscle weight. Survival, however, may
be used as a selection criteria, because, in the case of corrals, the
rate was 27% higher. This difference would significantly affect the
production level of the culture in favor of corrals. Furthermore,
corrals involve a lower investment cost, and it is likely that op-
erational costs would also be lower, because of a need for less
material to construct the enclosure, and while seeding, supervision
and harvesting tasks are easier. Taking the above into account, the
use of corral-type enclosures is advisable, with a height of over I
meter, to minimize escape.

Finally, during this study, an average growth rate of 6 mm
month"' was found. This is high if we compare it to growth rates
of other scallop species of commercial importance, such as Pecten
maxinnis (2 mm month"'), P. siilsicostatus (2 mm month"'), P.
albicans (3 mm month"' ), and P. novaezelandiae (4 mm month"')
(Mottet 1 979). Only Amusimn halloti (Williams and Dredge 198 1 ),
Clilaiiiys piiipiiratiis (DiSalvo et al. 1984), and Arf>opecren ciicu-
laris (Felix-Pico 1991 ) scallops attained similar rates of growth. In
this manner, the growth rate of bottom-reared Euvola ziczac. its
survival and relatively low cost with this culture method (Ventilla
1982) offer clear possibilities for further investigations in the de-
velopment of commercial culture of this species.

ACKNOWLEDGMENTS

We thank the valuable cooperation of the personnel at the Tur-
pialito Hydrobiology Station of the Instituto Oceanografico de
Venezuela, Universidad de Oriente: Maximiano Nfmez, Antonio
Sotillet, Aquiles Rojas and Eduardo Gonzales. This research work
was funded by grants from the Consejo de Investigacion de la
Universidad de Oriente. Finally, we thank Ian Emmett for trans-
lation of this article.

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Journal of Shellfish Reu'iiich. Vol, I*). Nd. I. 85-8S. 2000.

ALLOZYME AND BIOCHEMICAL VARIATION AT THE OCTOPINE DEHYDROGENASE

LOCUS IN THE SCALLOP EUVOLA ZICZAC

J. E. PEREZ,' O. NUSETTI,- N. RAMIREZ," AND C. ALFONSi'

Instinito Ocecmogrdfwo de Venezuela, and
'Departamento de Biologia
Esciiela de Ciencias
Universidad de Oriente
Nucleo de Sucre, Venezuela

ABSTRACT High activities of octopine dehydrogenase (Odh) in the adductor muscle of bivalve molluscs are associated with a
dependence on anaerobic glycolysis during swimming. The Odh locus is polymorphic in the scallop EiivoUi ziczcic. Estimated apparent
Odh A',„s for arginine were not different among nine Odh genotypes; however. A'„,s for pyruvate differed significantly (P < 0.001)
between heterozygous and homozygous scallops. The estimated apparent A'„, values of Odh for arginine and pyruvate are dependent
on their respective cosubstrate concentrations. Possible mechanisms for this overdominance include null alleles, aneuploidy. and higher
fitness of the heterozygous. Our data suggest that heterozygous superiority in fitness is the most likely explanation for the apparent
o\erdominance at the Odh locus.

A'£}' WORDS: Arginine. bivalves, molluscs, octopine dehydrogenase, overdominance. pyruvate, scallops

INTRODUCTION

Numerous studies demonstrate how allozymes influence vari-
ous components of fitness. For example, allozymes differ in their
catalytic properties, including kinetic parameters (K„^ and V,„^^),
and heterozygous genotypes may show overdominance (exceeding
the two corresponding homozygous genotypes) (Sarver et al.
1992); be intermediate in catalytic efficiency between homozy-
gous genotypes (Hoffmann 1981, 1983): or exhibit dominance,
having catalytic efficiencies similar to the most efficient genotype
(Hillbish and Koehn 1985; Nirchio et al. 1991).

Allozyme heterozygosity and growth rate are positively corre-
lated in many bivalve species (Beaumont and Zouros 1991 ; Zouros
et al. 1992; Hedgecock et al. 1996). Higher levels of heterozygos-
ity are associated with a lower basal metabolic rate that allows
heterozygous individuals to devote more of their aerobic scope to
growth and reproduction (after meeting basal requirements)
(Koehn and Shumway, 1982). Recently, Hedgecock et al. (1996)
found in the Pacific oyster Crassostrea gigas (Thunberg) that not
only are oxygen consumption rates lower for hybrid compared
with the inbred larvae, but also the net efficiency of protein syn-
thesis is much higher for the hybrids. Several authors (Carton et al.
1984; Rodhouse et al. 1986: Volckaert and Zouros 1989) have
suggested that organisms use this energy surplus for functions that
increa.se fitness. In sedentary molluscs such as mussels and oys-
ters, metabolic energy would be better invested in growth during
the juvenile stages and in reproduction in adults. However, scal-
lops are active bivalves that avoid predation by vigorous swim-
ming; thus, selection of an allozyme could result in an increased
locomotion capacity.

Scallops display sudden bursts of muscle activity, initially sus-
tained by arginine phosphate breakdown, followed by the activa-
tion of glycolytic pathways that result in rising levels of octopine
(Chih and Ellington 1983: Bricelj and Shumway 1991). Octopine
is produced by the reductive condensation of arginine and pyruvate
catalysed by octopine dehydrogenase (Odh, EC 1.5.1.11). in the
presence of NADH. High activities of Odh occur in the adductor
muscles of scallops (Chih and Ellington 1983: Alfonsi et al. 1995).
The advantage of octopine formation in adductor muscles may be

that oxidation of NADH removes arginine and thereby facilitates
the formation of ATP from arginine phosphate.

The energy needs among molluscs vary, as scallops, which
swim, require higher instantaneous rates of ATP production than
sedentary bivalves such as mussels and oysters. Phosphoarginine is
the principal fuel during valve snapping, and octopine accumulates
during the subsequent recovery phase under functional anaerobio-
sis (Bricelj and Shumway 1991 ). Genetic effects on glycolitic ATP
production are correlated with increased ability for burst activity in
pectinids. Volckaert and Zouros (1989) found in the scallop Pla-
copecten magellanicus (Gmelin) that heterozygosity and octopine
accumulation after burst activity are correlated. The degree of
heterozygosity and the maximal activity of pyruvate kinase and
Odh are positively correlated in the adductor muscle of the scallop
Eitvoki ziczac (Alfonsi et al. 1995).

In scallops, the primary function of Odh is to maintain the
redox balance of the muscle during exhaustive exercise. The
present study was designed to determine whether allozymes of
Odh. which is polymorphic (Coronado et al. 1991), differ in cata-
lytic properties. We determined this by measuring the apparent A"^
of Odh in the scallop. E. ziczac, under varying concentrations of
pyruvate and arginine.

MATERIALS AND METHODS

Adult scallops. E. ziczac {n = 103), were collected in 1998
during their sexual resting period from the waters of the Gulf of
Cariaco (Chacopatica) on the northeastern coast of Venezuela
(10°30'I0"N. 64°13'06"W). They were maintained in running aer-
ated seawater.

To determine the genotype at the Odh locus, the adductor
muscle from each individual was excised, minced, and centrifuged,
and the supernatant was analyzed by horizontal 12% starch gel
electrophoresis. The activity of Odh was identified using the stain-
ing procedures described by Morizot and Schmidt ( 1990). Allelic
variants were designated by letters, with "a" being always the most
anodic. To prepare the enzyme extracts, the frozen adductor
muscle of each specimen was chopped and homogenized in 20%
w/v cold 50 niM imidazole-HCI buffer in ice, pH 7.5, with 2 mM
ethylenediaminetetra-acetic acid. The homogenized tissue was

85

86

Perez et al.

centrifuged at 27,000 ,1; for 20 min at 4 °C. Solid ammonium
sulphate was added to the supernatant to reach 70% saturation. The
resulting suspension was stirred at 4 'C for 30 min and then cen-
trifuged at 20,000 g for 20 min. The pellet was dissolved in a small
volume of the homogenizing medium, applied to a Sephadex
G-lOO column equilibrated with 50 mM Tris-HCl (pH 7.6) at
24 °C, and eluted with the same buffer. The eluted fraction with
highest Odh activity was used for kinetic analyses.

Odh activity was measured by recording changes in optical
density (OD; 365 nm) that were caused by the oxidation of NADH.
Reactions were run using 25 |j,L of the enzyme preparations in
1.25 mL of incubation mixture. The routine enzyme assay for
maximal activity was 0.2 mM NADH, 2.5 mM pyruvate, and 5.0
mM arginine in a 50 mM imidazole buffer at pH 7.5. All of the
assays were run at 24 °C. The enzyme activity was expressed as
spectrophotometric units (OD). Maximal activity was recorded
between pH 6.0 and 7.5 in pilot enzymatic assays.

Odh followed Michaelis-Menten kinetics for both arginine and
pyruvate, at saturation concentrations of the other substrate and of
NADH. Substrate inhibition by pyruvate was observed at concen-
trations over 2.5 mM. Accordingly, the apparent Michaelis con-
stants (apparent AT,,,) for the substrates arginine and pyruvate were
estimated from the Michaelis-Menten equation, according to Chur-
chill and Livingstone (1989):

Estimated apparent K„

(V„,^,yV) - 1/S

where V represents the initial reaction velocity at either a pyruvate
or arginine subsaturating concentration, when the respective co-
substrate was at a saturating level. Before applying this formula,
the maximal velocity was calculated from the Lineweaver-Burke
plots (Segel 1975), in which the concentration of one substrate (A)
was varied and the concentration of the other substrate (B) kept
constant. The data were fitted to the following equation:

lA'

0/V„_(l +^„;VA) I/B+ 1/V„

The initial velocity was recorded against the pyruvate concentra-
tion (0.10, 0.20, 0.83, and 2.50 mM) at fixed arginine concentra-
tions (0.5, 1.5, 3.0, and 5.0 niM). The inverse of the initial velocity
(lA') was plotted against the inverse of the pyruvate concentration
(l/S) for each arginine concentration. The Y intercepts of the
Lineweaver-Burke lines estimated by linear regression analysis
were plotted against the inverse of arginine concentration. The
maximal velocity was determined from the value of the resulting Y
intercept, which was essentially similar to that estimated by the
routine enzyme assay. Likewise, the data were plotted as a func-
tion of the arginine (0.5, 1.5, 3.0, and 5.0 niM) concentration. The

V,„^^ value estimated agrees with that obtained using varying con-
centrations of pyruvate at fixed concentrations of arginine. Sub-
strate inhibition of V,,,^^ was observed for pyruvate concentrations
over 2.5 mM at each arginine concentration.

Deviations from expected values of allele frequency for Hardy-
Weinberg equilibrium were tested by using a Chi-square analysis.
The deficiency or excess of heterozygotes (analyzed by the F
statistic) and the effective number of alleles at this locus (N^, the
reciprocal of the sum of squares of the allele frequencies) were
calculated by using the statistical program Genes in Populations,
version 2 (Perkins and Paul 1995).

RESULTS AND DISCUSSION

The sample of 1 03 individuals from the population of Chaco-
patica contained nine Odh genotypes: c/c, d/d, e/e, b/c, c/d, c/e, c/f,
d/e, and d/f, determined by five alleles Odh'', Odh'. Odh'', Odh^
and Odh'. Because genotypes that include alleles a and b are very
rare (Table 1), it was not possible to obtain sufficient samples to
study their catalytic properties of these rare alleles. Allele frequen-
cies have been stable since the first sample was examined in 1984.
All three samples were in Hardy-Weinberg equilibrium. Hetero-
zygote superiority probably provides the best explanation for the
maintenance of the polymorphism.

Apparent A',„ for arginine and pyruvate were related to varia-
tions in the concentrations of the respective cosubstrates, because
K,„ decreased as the concentration of cosubstrate increased (Table
2). This suggests a mechanism that favors the formation of oc-
topine when the concentration of the two substrates increases si-
multaneously, as is seen in active individuals. The availability of
arginine and pyruvate could be the two limiting factors in the
regulation of Odh activity for maximal glycolytic capacity during
the escape response and recuperation of E. ziczac. In addition,
specific genetic influences affect the regulatory properties of the
enzyme by acting on their relative substrate affinities.

Results for the K^ of pyruvate and arginine at different cosub-
strate concentrations were separated into two groups: homozygotes
and heterozygotes. Table 3 indicates no significant differences for
the A",,, of arginine (pyruvate as cosubstrate) (F = 0.017; P>Q.05),
whereas highly significant differences were detected between ho-
mozygotes and heterozygotes for the K^ of pyruvate (arginine as
cosubstrate) (F = 29.33; P < 0.00 1 ). These results indicate that the
affinity of the Odh enzyme for pyruvate was predominantly greater
in heterozygous than in homozygous individuals. Similar results
were observed by Walsh ( 1981 ) for three phenotypes of Odh in the
anemone Metridium senile (L.), in which (he heterozygotes
showed a higher affinity for pyruvate. Sarver ct al. (1992) mea-

TABLE 1.

Allele frequencies, effeclive number of alleles (A'^), observed (W,,) and expected (//,.) values for hetero/.vsi'sitv, and values of 7' are tests of
goodness of fit to Hardy-VVeinberg proportions for the Odh locus in samples of C'hacopatica collected in 1984, 1994, and 1998.

Allele Frequency

N

N,

"„

H.

F;.

y'

a

b

c

d

e

f

P

1984*
1994t
1998

0.0 II
0.000

o.ooo

0.033
0.000
0.005

0.456
0.500
0.495

0.244
0.310
0.3 1 1

0.244
0.1.50
0.165

0.01 1
0.0.50
0.024

45
113
103

3.05
2.75
2.66

0.533
0.655
0.62 1

0.609
0.636
0.63 1

0.12
0.03

0.014

8.82
4.76
3.72

>0.l
>0.1
>0.3

N = sample size. F,^ indicaies deficiency or excess of heterozygotes.
*Coronadoet al. 1991.
t Fernande/ 1 995.

Polymorphism at the Odh Locus in Euvola ziczac

87

TABLE 2.

Means and standard deviations for the estimated apparent A„, for both substrate arginine and p.vru>ate for the different genotypes, at the

different concentrations.

N

Cosubstrate Arginine (mM)

Cosubstrate Pyruvate (mM)

Genotypes

1.5

3.0

5.0

0.025

0.83

2.5

be

1

0.25

0.18

0.11

0.91

0.83

0.42

cc

7

1.70± 1.15

0.58 + 0.14

0.35 ± 0.09

2.80 ± 1.38

1.61 ±0.60

0.82 ±0.29

cd

7

1.20 ±0.33

0.57 ±0.1 7

0.19 ±0.07

1 .50 ± 0.59

1.18 ±0.47

0.61 ±0.21

ce

7

1.52 ±0.62

0.48 ±0.1 5

0.25 ± 0.08

4.60 ± 1 .60

1.99 ±0.61

0.99 ± 0.35

cf

1

0.19

0.13

0.06

2.39

1.44

0.76

dd

7

2.33 ± 1.44

1.04 ±0.27

0.42 ±0.11

2.48 ±1.65

1.71 ±0.93

0.83 ±0.33

de

7

0.67 ± 0.34

0.35 ± 0.09

0.17 + 0.04

2.41 ±1.15

1.31 ±0.46

0.67 ±0.19

df

4

0.49 ±0.21

0.26 ±0.11

0.16 ±0.06

1.86 ±0.63

1.40 ±0.43

0.59 ±0.16

ee

3

0,69 ± 0.33

0.22 ± 0.05

0.14 ±0.02

3.56+ 1.24

1.71 ±0.52

0.53 ±0.11

N is the number of animals examined.

sured specific Odh activities in a large number of individuals of the
mussel Mytilus tiossulus Gould for Odh and found that the mean
Odh activity was greater in heterozygotes than homozygotes.

Multiple range analysis (least-significant difference) indicate
three groups in increasing order of A",,,: (1) d/f, e/e, d/e. c/d, and
c/e: (2) c/d, c/e, and c/c; and (3) d/d. Genotypes c/d and c/e be-
longed to the groups with higher and medium affinities [ 1 1 and [2],
Table 3). By increasing Odh affinity for pyruvate, heterozygous
individuals could enhance the ability of the muscle to maintain the
NADH/NAD* redox balance during the glycolytic flux which can
occur during high-intensity muscle work. This affinity would be
particularly useful during the initial phase of glycolysis when py-
ruvate concentration is low and arginine levels begin rising (argi-
nine phosphate pool is depleted). Moreover, the shunting of pyru-
vate to mitochondrial metabolism or cytoplasmic synthesis of ala-
nine could be inhibited to rapidly meet the energy demands under
functional anaerobiosis of the contracting fibers. On the other
hand, it appears that arginine does not represent a control or lim-
iting factor for the anaerobic glycolitic capacity for the fast muscle
contraction of E. ziczcic. This assertion, however, does not exclude
the possibility of other genetic influences on the enzymatic con-
version of arginine into arginine phosphate, which would assure a
faster recuperation after a burst exercise.

Heterozygotes show an apparent overdominance in A',,, for py-
ruvate thai may be a fitness component if the concentration ot

TABLE 3.

Means and standard deviations from the estimated apparent A„, for
pyruvate for heterozygous and homozygous individuals.

A'

Cosubstrate Arginine

(mM)

Genotype

L5

3.0

5.0

Heterozygous
Homozygous

27
17

0.72 ±0.49 0.32 ±0.1 6 0.16 ±0.06
1.57±0.68 0.61 ±0.33 0.30±0.12

Cosubstrate Pyruvate (mMl

0.205

0.83

2.5

Heterozygous

Homozygous

27
17

2.84 ± 1 .30
2.64 ± 1.16

1.49 ±0.36
1.66 ±0.33

0.75 ±0.1 6
0.83 ± 0.2

N is the number of animals examined. Means are different (P < 0.001).

pyruvate is low. Apparent overdominance (heterozygous geno-
types are phenotypically superior to homozygous genotypes), in
fitness components such as growth, viability, and fecundity, has
been observed in many species of marine bivalves (Sarver et al.
1992). Possible explanations for overdominance, as well as the
commonly reported deficiencies of heterozygotes. include null al-
leles (Foltz 1986) and aneuploidy (Thiriot-Quievreux et al. 1988).
Several studies of allozyme inheritance have found substantially
higher frequencies of null alleles in bivalves than found in other
organisms, suggesting that null alleles or segmental aneuploidy
may play a role in the apparently lower fitness of allozyme het-
erozygotes (Gaffney 1994). Null alleles or missing chromosomes
(and therefore missing alleles) could contribute to fitness advan-
tages of heterozygotes. In Mylihis ediilis (L.), Hoare and Beaumont
(1995) found not only heterozygotes, but also homozygotes for a
null Odh allele. We believe this situation unlikely to occur an
active species, such as E. ziczac.

Considering an alternate explanation of heterozygote superior-
ity in fitness or other phenotypic attributes to explain our results,
there are two possible scenarios, as follows.

( 1 ) Individuals that appear single banded (homozygous), which
were numerous in our sample, may really be heterozygous (active/
null alleles); if so. we would expect a bimodal distribution, with a
resulting higher variance of activity in homozygous compared with
heterozygous. However, this was not the case for our samples of £.
ziczac (see Table 3).

(2) Results obtained in the analysis of other enzymes (pyruvate
kinase, glucose 6-phosphate dehydrogenase, isocitrate dehydroge-
nase, and malate dehydrogenase) in E. ziczac indicate that their
specific activity is correlated positively with heterozygosity. Be-
cause the Odh activity in homozygotes and heterozygotes scallops
increases with heterozygosity at multiple loci, it seems highly
unlikely that null alleles (or missing chromosomes) would occur at
all of these loci (Alfonsi et al. 1995).

Therefore, our results can be best explained by assuming an
overdominance at the Odh locus, which could enable the heterozy-
gotes to increase their ability to escape from predators.

In conclusion, the pyruvate affinity of the adductor muscle Odh
allozyme in £. ziczac appears to be a catalytic target upon which
genetic influences act to determine the tissue's capability for main-
taining a steady NADH/NAD ratio, that would support the rate of
anaerobic glycolysis at the burst working muscle, such as the

88

Perez et al.

sudden escape behavior commonly observed among scallops when
they tlee from predators. During routine work, we have observed
that E. ziczac scallops are easily induced to vigorously snap their
valves (swimming) when approached by gastropods, crabs, star-
fish, and human divers. This predator-avoidance behavior may be
repeated for several minutes before entering in a variable resting
period. However, the nature of the relationship that exists between
the capability to sustain muscle contraction in response to preda-
tory stimulus and the Odh genotypic variants in E. ziczac is not
clear.

Finally, further research is required on some biochemical and
physiological events associated with activity in different genotypic
variants of E. ziczac, both under laboratory bioassays and field
work conditions, because predation is a significant component in
the life history of adult scallops, we are currently searching for a

possible relationship between the escape reaction and the different
genotypes, and because polymorphism at the Odh locus in E. zic-
zac seems to be important for understanding genetic variation in
molluscs, we are also searching for the presence of polymorphism
in other, sedentary and motile, species of molluscs. Additionally,
we are examining stronibine and alanopine dehydrogenase (which
also serve as hydrogen and carbon sinks in maintaining redox
balance) in several species of molluscs during anaerobic metabo-
lism, for possible polymorphisms and their maintenance mecha-
nisms.

ACKNOWLEDGMENTS

We thank Dr. Kent Rylander, Texas Tech University, as well as
two anonymous reviewers, for critically reading the first version of
the manuscript.

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Walsh, P. J. 1981. Purification and characterization of two allozymic form
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Joiinwl of Shellfish Rfsearch. Vol. 19. No. 1. 89-93, 2000.

ENVIRONMENT AND POPULATION ORIGIN EFFECTS ON FIRST SEXUAL MATURITY OF
CATARINA SCALLOP, ARGOPECTEN VENTRICOSUS (SOWERBY II, 1842).

PEDRO CRUZ. CARMEN RODRIGUEZ-JARAMILLO, AND
ANA M. IBARRA

Centre) de Investigaciones Bioldgicas del Noroeste, S.C..

Km. I Can: a San Juan de la Costa,

El Comitdn, A. P. 128.

La Pa: B.C.S. 23000. Mexico

ABSTRACT Two populations of catarlna scallop, and their cross ( F 1 ). were evaluated for the age at first se.\ual maturity, and for their
gonadal development in both populations' native environments. All experimental groups were hatchery produced. Differences in mean
gonad index (MGI) were seen between the different environments. In Bahia Magdalena. a site characterized by high productivity and
lower average water temperatures, the MGI was higher than for scallops grown in Bahia Concepcion, a bay with lower productivity
and higher average water temperatures. Differences in age and size at first sexual maturity, defined as tho,se when 50% of the scallops
in any group were sexually mature, were seen between the populations when grown at Bahia Magdalena but not when grown at Bahia
Concepcion. At Bahia Concepcion. none of the groups had reached their first sexual maturation after the experimental period of 7 mo.
At Bahia Magdalena. the Magdalena population and the Fl reached sexual maturity at an eariy age of 4 mo, whereas the Concepcion
population reached maturity when 5 ino old. Also for the Bahia Magdalena environment, the gonad index (GI) estimated at first sexual
maturity for the Magdalena population and the Fl was significantly higher than that estimated for the Concepcion population at that
same age. There were no significant differences in GI values when the groups were grown at Bahia Concepcion. The differences
between populations in age at first sexual maturity suggest that a triggering mechanism exists in catarina scallop for the initiation of
sexual maturation, whereas the differences between environments suggest that regardless of that mechanism, environmental conditions
have a significant role in further maturation processes.

KEY WORDS: Argopecten venlricosus, environment, gonad index, populations, maturation

INTRODUCTION

The catarina scallop, Argopecten ventricosus (Sowerby II,
1842), which is a functional hertnaphrodite species, is an important
fishery and aqiiaciilture resource on both coasts of the Baja Cali-
fornia Peninsula, Mexico. The geography of the peninsula results
in this species distributing and growing in different environmental
conditions: semitropical to temperate on the Pacific Ocean side
and tropical on the Gulf of California side. Because of this, as well
as the presumed isolation caused by the Peninsula barrier itself,
natural populations existing on both sides are expected to be ge-
netically different, that is, to have evolved differently in response
to environmental conditions on each side. In fact, we have dem-
onstrated that there are differences between these two populations
in growth and survival (Cruz and Ibarra 1997, Cruz et al. 1998).

An additional important trait to compare in populations on both
sides of the peninsula is the age and size at which each population
reaches its first sexual maturity. It has been stated that the repro-
ductive cycle of scallops is a genetically controlled response to
environmental conditions (Sastry 1970, Sastry 1979, cited by Bar-
ber and Blake 1991). which depends on the optimum interactions
between exogenous and endogenous factors. When the appropriate
combination of exogenous and endogenous factors occurs, a mini-
mum age (or size) has to be reached before the beginning of
gametogenesis (Barber and Blake 1991). Differences in the onset
of sexual maturity and reproductive cycle have already been re-
ported for other bivalve species (Dalton and Menzel 198.3. Knaub
and Eversole 1988. Barber et al. 1991. Mackie and Ansell 1993).
Among different bivalves studied simultaneously at different sites.
or through transplantation, there are differences in the onset of
gametogenesis (Newell et al. 1982. Barber and Blake 1983.
Walker and Heffernan 1994). spawning time (Brousseau 1987.

Emmett et al. 1987. Paulet et al. 1988). fecundity (Bricelj et al.
1987). and gatnetogenic cycle (Wilson 1987, Thorarinsdottir 1993.
Sbrenna and Campioni 1994). Some of the previously reported
differences are not necessarily caused by genetic factors but by
different environmental conditions at each site studied.

Different studies with catarina scallop have been peiformed
regarding sexual maturation and gametogenic cycles (Baqueiro et
al. 1981. Tripp-Quezada 1985. Villalejo-Fuerte 1992. Felix-Pico
1993. Villalejo-Fuerte and Ochoa-Baez 1993). However, differ-
ences between populations in age at first sexual maturity or in the
effects of different environments on gametogenic cycles and matu-
ration have not been investigated.

In this study, we evaluated the onset of first sexual maturity and
the gametogenic cycles for two populations of catarina scallop and
their cross (Fl). The two populations were Concepcion in Bahia
Concepcion on the Gulf of California side of the Baja California,
and Magdalena in Bahia Magdalena on the Pacific Ocean side of
the Baja California peninsula. All groups, Magdalena, Concep-
cion. and their Fls. were simultaneously evaluated in both envi-
ronments.

MATERIALS AND METHODS

Populations and Fl

Spawners used, conformation of the experimental groups, and
larvae rearing have been described by Cruz and Ibarra (1997). In
summary, four groups were produced by mass spawning;
Magdalena. Concepcion, and both reciprocal Fls. At a spat size of
1.5 cm length and 2.5 mo old. 9 (pseudo) replicates, each with 100
scallop spats, were formed within each group by randomly sam-
pling 900 spats from the total group pool.

89

90

Cruz et al.

Grow-Out

Statistical Analyses

The spat contained within each of the nine replicates per group
were simultaneously transported to each of the experimental field
areas. Bahia Magdalena and Bahia Concepcion, where they were
maintained for 5 mo. At each site, the scallops contained in each
replicate were kept in a Nestier tray suspended from a long-line for
45 days and then were transferred to bottom culture to avoid po-
sition effects on growth caused by water temperature stratification.
Nestier trays were attached to a metal structure anchored to the
bottom. Maintenance was performed monthly. Densities were the
same for all replicates within groups and in both environments
(Cruz et al. 1998).

Gonad Sampling and Histology Analysis

Sampling for gonad tissue began after 1 .5 mo of grow-out. at 4
mo age. Three individuals were sampled per replicate (27 per
group) at ages 4. 5. 6, and 7 mo. Samples were fixed in Davidson's
fixative and were preserved in 70% alcohol. The hematoxylin-
eosin staining technique was used. Sexual maturity was evaluated
with a modified Villalejo-Fuerte (1992) scale for this hermaphro-
dite scallop, where seven stages are included for the female portion
of the gonad (Stage 0 = undifferentiated or virginal; Stage I =
resting: Stage II = start of gametogenesis; Stage III = advanced
gametogenesis; Stage IV = maturity; Stage V = spawned; Stage
VI = postspawned).

Age-Size at First Sexual Maturity

Age and size at first maturity were established by a different
criterion than that commonly used when populations are evaluated
following a field-born cohort (Nikolsky 1969). Under that meth-
odology, age-size at first maturity is estimated when the cumula-
tive frequency of mature individuals reaches 50% in the cohort.
Field-born individuals of a population are of different ages because
spawning of the whole population usually last from days to weeks.
In the present study, all individuals were the same age. Therefore,
in this study, age and size at first sexual maturity were defined as
the age when 50% of the organisms within any group were in the
"maturity"" gonadal stage, or Stage IV as defined above, or when
the sum of individuals in Stage IV (maturity). Stage V (spawn).
and Stage VI (post-spawn) was 2 50%. Only the female gonad
portion was used for the establishment of the age and size at sexual
maturity.

Gonad Indices

Gonad indices (GIs) were calculated for each replicate within
each group based on a calculation by Seed (1976) by using the
number of individuals and the stage at each age (4, 5, 6. and 7 mo)
to find u GI at age for each group as follows:

GI„K = [(0*N„) + ( 1*N,) + (2*N„) -h (.V'^N,,,) -I- (4*N,v)
-K5*Nv)-H(6*Nv,)]/N,„„„„

where 01,^,^ is the GI for replicate / (/ = 1,2,. ^,...9), of the group /
(/ = 1,2,3), in the environment k {k = 1,2): N^uhsiMpi 's 'he number
of individuals in that gonadal stage for replicate /: and N,,,„,||^, is
the total number of individuals in that replicate of that group in that
environment.

The GIs estimated for each replicate within the groups were
analyzed by a complete two-factor. Model I, analysis of variance,
where age was taken into consideration as a covariable. After
establishing the lack of differences between the reciprocal Pis {P
> 0.05). for all further analysis the Fls were pooled into what is
defined as the Fl between these two populations. The effects of
group (Magdalena, Concepcion. and Fl), environment (Bahia
Magdalena and Bahia Concepcion), and their interaction on GI
were analyzed. Effects means were compared with a Tukey test,
setting a = 0.05. Additionally, at the age when first sexual matu-
ration was observed, as defined above, a second partial Model I
analysis of variance was made. This was performed to establish the
effect of groups and environments on GIs at the age of first sexual
maturity and to find out whether there was a group by environment
interaction for GI. All statistical analyses were performed using a
computer software (Statistica, version 5; StatSoft, Inc.; Tulsa,
OK), and significance for all analyses was set to P < 0.05.

RESULTS

First Sexual Maturity

At Bahia Magdalena. the age at first sexual maturity (Stage IV)
for the Magdalena population was 4 mo. However, at this age,
which corresponds to the first sampling time during grow-out (1.5
mo of grow-out), 56% of the individuals were scored as matured,
but 9% were spawned, and 13% postspawned. This indicated that
first sexual maturity occurred slightly before this time. Shell height
at 4 mo of age was 20.0 mm (SD ± 0.88 mm). At this same age,
the Fl also reached sexual maturity, as defined in this study, since
it had 41% individuals in the maturity stage, 19% spawned, and
5%^ postspawned (Table 1). Shell height was 21.2 mm (SD ± 0.85
mm). At the age of 4 mo, the Concepcion population had no
mature or spawned individuals, but 4% were postspawned (Table
1 ). At 5 mo of age, the Concepcion population had 75% mature
individuals, reaching sexual maturity (Fig. 1) at a shell height of
32.9 mm (SD ± 1.34 mm).

At Bahia Concepcion, sexual maturity was not reached by any
group during the experimental period (Fig. 1 ). Although sexual
maturity was not detected in this environment, a differential pat-
tern between groups was evident from 5 mo to the end of the study:
a larger percentage of individuals from the Magdalena population
and the Fl were postspawned than the percentages seen for the
Concepcion population. Also, at 7 mo of age, corresponding to the
last sampling date, 16%- of the individuals within the Magdalena
group and 4% of the Fl were already matured, v\hercas within the
Concepcion group there were no mature, spaw ned, or postspawned
individuals (Fig. 1 ).

GIs

Bolh main effects (group antl en\ ironmentl were significant for
both analyses, the whole grow-out period and the age (4 mo) at
sexual maturity in the Magdalena population and the Fl. There
was no interaction between groups and environments (Table 2).
For the whole grow-out period, mean GIs (MGIs) for all groups at
Bahia Concepcion (MGI 1 .63) were significantly less than those at
Bahia Magdalena (MGI 3.72) (Table 3). These MGI values indi-

Environment and Population Effects on First Sexual Maturity of Catarina Scallop

91

TABLE 1.

Frequencies (in percentages), of A. ventricosus at 4 mo of age. in each gametogenic stage within each experimental group when grown at

Bahia Magdalena and Bahia Concepcion.

Bahia Magdalena

Bahia Concepcion

Magdalena

Concepcion

Magdalena

Concepcion

Stage

Population

Fl

Population

Population

Fl

Population

0 Undifferentiated

0

5

22

75

89

100

I Resting

0

5

0

25

6

0

II Initial gametogenesis

0

5

48

0

5

0

III Advanced gametogenesis

22

17

26

0

0

0

IV Maturity

56"

4r

0

0

0

0

V Spawned

9

19

0

0

0

0

VI Postspawned

13

5

4

0

0

0

' Indicates whether sexual maturity of female gonad portion was reached for that group at this age.

cated that, over the grow-out period, scallops at Bahia Magdalena
were between the advanced gametogenesis (Stage III) and
spawned (Stage V) stages, whereas those at Bahia Concepcion
were between resting (Stage I) and initial (Stage II) gametogenic
stages. Within environments and for the whole grow-out period,
there were significant differences between groups only when
grown at Bahia Magdalena. where the two populations were dif-
ferent, and the Fl was in an intermediate maturity stage, which is
not different from either population. The largest GI was that of the

Magdalena population (GI 4.02), followed by the Fl (GI 3.81 ) and
the Concepcion population (GI 3.33) (Table 3).

At 4 mo of age. when sexual maturity had occurred, the MGl
at Bahia Concepcion was lower (MGI 0.5) than that at Bahia
Magdalena (MGI 3.17). There were significant differences be-
tween groups only in Bahia Magdalena. with no significant dif-
ferences in GI between the Magdalena population (GI 4.08) and
the Fl (GI 3.57). whereas the Concepcion population had the
lowest GI ( GI 1.85) (Table 3 ). At Bahia Concepcion. GIs were not

Magdalena

BAHIA MAGDALENA

Fl

Concepcion

Age (months)

Age (months)

CD 1

Age (months)

Magdalena

BAHIA CONCEPCION

Fl

Concepcion

100
90

^ V.

■^S5'

£13 » w

ES3 V

EST

r'.-'i-'A

ES3 s

5 6

Age (months)

Figure. 1. Frequencies of gonadal developmental stages in A. ventricosus at the ages of 4, 5, 6, and 7 mo, for each experimental group at each
environment. Stage 0 = undifferentiated: Stage I = resting; Stage II = start of gametogenesis; Stage III = advanced gametogenesis; Stage IV =
maturity; Stage V = spanned; and Stage VI = postspawned.

92

Cruz et al.

TABLE 2.

Results of the analyses of variance testing significant effect on

female GIs of A. venlricosiis for the complete model during the

grow-out period, and the partial model only at 4 mo of age (see

Materials and Methods section).

Source of Variation

Full Model

Partial Model

Environment

Group

Interaction

0.0000-'
0.0015"
0.8373

O.OOOO"
0.0069-'
0.3340

' Indicates significance at the pre-established level of P < 0.05.

different between groups ( 1 .0. 0.5, and 0.0, respectively, for the
Magdalena population, Fl, and Concepcion population).

DISCUSSION

Differences between the two populations in age and size at
sexual maturity were clearly evident when grown at Bahia
Magdalena but not at Bahia Concepcion. At Bahia Concepcion.
sexual maturity was not reached by any of the groups. However, at
7 mo, some mature individuals were already present for the
Magdalena population, but not for the Concepcion population.
Previous work by Villalejo-Fuerte and Ochoa-Baez (1993) indi-
cates that the native population at Bahia Concepcion reaches sex-
ual maturity at the age of 1 y and a 58-mm shell height. For other
Argopecten species, as for example Argopecten irradians, the
maximum gonad weight was reported to be at 57 mm shell height
(Bricelj et al. 1987), whereas 'tax Argopecten gihhits. ripe individu-
als as small as 20 mm shell height have been reported (Miller et al.
1979). In fact, precocious individuals such as those seen in the
population of A. ventricosus from Bahia Magdalena have only
been reported for A. gihhus. which reaches sexual maturation when
only 71 days old (see review by Barber and Blake 1991 ).

The failure of all groups to reach sexual maturity during our
experimental period when grown at Bahia Concepcion can be ex-

TABLE 3.

GIs (SD) in A. ventricosus, for the whole grow-out period and for

the age at sexual maturity (reached when grown at Bahia

Magdalena at 4 mo of age I for each envinmment and for each

experimental group."

Whole

Age 4 mo

grow-out

(Sexual

Environment

(■roup

period

Maturity)

Bahia Magdalena

Magdalena GI

4.02(0.4.5)"

4.08 (0.75)"

Fl ni

3.S1 (O.0)2)-'''

3.57 (0.94)"

Concepcion Gl

3.33(1.14)"

1.85(1.28)"

BM MGI

3.72 (O.Xl)'^

3.17(1. .32)^

Bahia Concepcion

Magdalena Gl

1.81 (1.26)'-

1.0(2.24)'-

Fl GI

1.75(1.-30)'

0.5(1.58)'

Concepcion Gl

!..34(0.96)'

0.0 (0.00)'

BC MGI

l.fi3(1.22)"

0.5(1. .54)"

MGI (SD) is the average female Gl of all groups within that environment.
GIs for gonad by group are given. Means with the same letter wilhin gonad
part (female or male) are not significantly dilTerent. Group means dilTer-
ences within environment and sc\ in lower case. Capital case lellers tor
dilTcrences between environments.

plained by the environmental conditions which characterize this
bay; low productivity (chlorophyll-o 0.38-1.63 mg/m') and high
average annual temperature (24.9 °C) with a wider range (17.7-
32.1 °C) (Martinez-Lopez and Garate-Lizarraga 1994, Reyes-
Salinas 1994). Bahia Magdalena is characterized as a more benign
environment. Average temperature is 22 "^C, with a small range
(20-26.6 °C) (Hemandez-Rivas et al. 1993), and a high chloro-
phyll-a concentration (1.5-5.1 mg/m') (Acosta-Ruiz and Lara-
Lara 1978). Poor environmental conditions are known to affect
gonad development (i.e.. decreases in reproductive output seen in
Placopecten inagellaiucus) (Macdonald and Thompson 1985).
Barber and Blake (1991) proposed that the oocyte reabsorption
seen in different species of Pectinids could be caused by unfavor-
able temperatures that inhibit full gonad development. This was
probably the case in the Bahia Concepcion population, where de-
spite the fact that mature individuals 4-6 mo old were not detected,
there were some classified as postspawned. Furthermore, rather
than in the undifferentiated stage, most scallops in Bahia Concep-
cion were in a resting stage during most of the experimental pe-
riod, which could have been caused by attempted maturation fol-
lowed by follicular atresia because of high temperatures and low
productivity.

The mechanism that detains the maturation process under in-
adequate environmental conditions is not known. However, it is
known that in A. irradians, the regulation of the gametogenic cycle
is controlled by a neurosecretory cycle with a checkpoint that
seems to act as a switching mechanism, allowing or delaying oo-
cyte growth depending on food and temperature (Barber and Blake
1991). When scallops are subjected to prolonged threshold tem-
peratures after the neurosecretory cycle enters the neurosecretory
stage (NS) corresponding to cytoplasmic growth phase (NS III) or
vitellogenesis (NS IV), scallops do not regress in NS, and vacu-
olization of cytoplasm and lysis of oocytes can occur (Sastry,
1966a, 1968, cited by Barber and Blake 1991). Whether a similar
mechanism exists in the catarina scallop is not known, but it could
explain the presence of atresias in scallops when they are grown in
Bahia Concepcion.

The differences between populations in age at sexual maturity
suggest that a genetic triggering mechanism might exist for the
onset of sexual maturity in the catarina scallop. When grown in
Bahia Magdalena. an environment characterized by high produc-
tivity and lower temperatures, the mechanism of early maturation
in the Magdalena population and the Fl is triggered, and because
the prevailing environmental conditions at this site (low tempera-
ture and abundant food), full development is reached at an early
age. A suggestion that the mechanism is genetically controlled
comes from the age and size at which the two populations reached
their first sexual maturity. The Concepcion population reached
sexual maturity in this environment at least 1 mo later than the
Magdalena population. Inheritance (from the Magdalena popula-
tion to the Fl) in the dominant fashion of an early triggering
mechanism is suggested by the Fl reaching sexual maturity at the
same age as the Magdalena population and by the fact that the GIs
of the Fl showed no significant differences with the Magdalena
population at first sexual iT)aturity. However, the GI of the Fl
group was intermediate to the GIs of the two populations for the
whole grow-out period, indicating more of an additive form of
inheritance for this trait. Furthermore, whereas at Bahia Concep-
cion sexual maturity was not reached for any group in this study.
at 7 mo of ane there were 16'/f mature indi\'iduals wilhin the

Environment and Population Effects on First Sexual Maturity of Catarina Scallop

93

Magdalena population, but only 4"^^ mature and 2'7r post spawn for
the Fl (6';*-). Further research, with segregation studies included, is
required to provide a definitive answer to the inheritance of this
trait. Whereas the inheritance of reproductive traits has been sug-
gested in other mollusk species, no study has attempted to dem-
onstrate it at the genetic level. For example. Knaub and Eversole
(1988) found that the Fl between two populations o{ Mercenaria
mercenaria resembled the paternal population in some reproduc-
tive traits and the maternal population in others. Also, each of two
lines of Crassosirea virginica. derived from two populations 5-6

generations before, still followed the reproductive pattern of their
original populations (Barber and Blake 1991).

ACKNOWLEDGMENTS

We thank MAZAVI enterprise for help during field mainte-
nance of the experimental groups, and M. Romero from SEMAR-
NAP, Guy. A. Garcia and Jose L. Ramirez from CIBNOR for
technical support during this research. This research was partially
supported by CONACyT grants 720-N9204 and 1473PB to A.M.
Ibarra. Dr. Ellis Glazier edited the English-language text.

LITERATURE CITED

Acosta-Rui7. J. & J. R. Lara-Lara. 1978. Resultados tisico-quiniicos en un
estudio de variacion diurna en el area central de Bahia Magdalena.
B.C.S. Ciemias Mar. 5:37-t6.

Baqueiro. C. E.. I. Pena. & J. A. Masso. 1981. Analisis de una poblacion
sobreexplotada de Argopecten circularis (Sowerby. 1835) en la
Ensenada de La Paz.B.C.S.. Mexico. Cieiiciu Pesqiieni. Inst. Niil.
Pesca. Depro. Pesca. Mexico. Y(2):57-65.

Barber. J. B. & N. J. Blake. 1983. Growth and reproduction of the bay
scallop. Argopecten irradians (Lamarck) at its Southern distributional
limit. / E.xp. Mar. Biol. Ecol. 66:247-256.

Barber J. B. & N. J. Blake. I99I. Reproductive physiology. In: S. E.
Shumway (ed.). Scallops: Biology. Ecology and Aquaculture. Elsevier.
Amsterdam. The Netherlands, pp 377^28.

Barber. J. B.. S. E. Ford, & R. N. Wargo. 1991. Genetic variation in the
timing of gonadal maturation and spawning of the eastern oyster, Cras-
sosirea virginica (Gmelin). Biol. Bull. 181:216-221.

Bricelj. V. M., J. Epp, & R. E. Malouf. 1987. Intraspecific variation in
reproductive and somatic growth cycles of bay scallops Argopecten
irradians. Mar. Ecol. Prog. Ser. 36:123-137.

Brousseau. D. J. 1987. A comparative study of the reproductive cycle of
the soft-shell clam. Mya arenaria in Long Island Sound. J. Shellfish
Res. 6:7-15.

Cruz, P. & A. M. Ibarra. 1997. Larval growth and survival of two catarina
scallop (Argopecten circularis. Sowerby, 1835) populations and their
reciprocal crosses. / E.xp. Mar. Biol. Ecol. 212:95-1 10.

Cruz. P., J. L. Ramirez. G. A. Garcia. & A. M. Ibarra. 1998. Genetic
differences between two populations of catarina scallop {Argopecten
venlricosiis) for adaptations for growth and survival in a stressful en-
vironment. Aquaculture. 166:321-335.

Dalton. R. & W. Menzel. 1983. Seasonal gonadal development of young
laboratory-spawned southern (Mercenaria campechiensis) and north-
em [Mercenaria mercenaria) quahogs and their reciprocal hybrids in
northwest Florida. J. Shellfish Res. 3:1 1-17.

Emmett. B.. K. Thompson & J. D. Popham. 1987. The reproductive and
energy storage of two populations of Mytiliis edulis (Linnel from Brit-
ish Columbia. / Shellfish Res. 6:29-36.

Felix-Pico. E. F. 1993. Estudio biologico de la almeja catarina. Argopecten
circularis (Sowerby. 1835) en Bahi'a Magdalena. B.C.S.. Mexico. (Bio-
logical study of catarina scallop, Argopecten circularis (Sowerby.
1835) in Magdalena Bay. B.C.S.. Mexico). Tesis de Maestria (M.Sci.
Thesis). CICIMAR-IPN. La Paz B.C.S. 89 pp.

Hemandez-Rivas. M.. J. Gomez-Gutierrez. C. A. Sanchez-Ortiz. R. J.
Saldierna-Martinez & G. R. Vera-AIejandre (eds.). 1993. Atlas de tem-
peratura superficial en el complejo lagunar de Bahi'a Magdalena-Bahia
Almejas. Baja California Sur. Mexico 1980-1989. CICIMAR. La Paz
B.C.S., Mexico.

Knaub, R. S. & A. G. Eversole. 1988. Reproduction of different stocks of
Mercenaria mercenaria. J. Shellfish Res. 7:371-376.

Macdonald. B.A. & R. J. Thompson. 1985. Influence of temperature and
food availability on the ecological energetics of the giant scallop Pla-
copecten magellanicus. II. Reproductive output and total production.
Mar. Ecol. Prog. Ser 25:295-303.

Mackie. L.A. & A. D. Ansell. 1993. Differences in reproductive ecology in
natural and transplanted populations of Pecten imuimus: evidence for
the existence of separate stocks. / E.\p. Mar. Ecol. 169:57-75.

Marti'nez-Lopez. A. & 1. Garate-Lizarraga. 1994. Cantidad y calidad de la
materia organica particulada en Bahia Concepcion. en la temporada de
reproduccion de la almeja catarina /^. circularis (Sowerby. 1835). Cien-
cias Mar. 20:301-320.

Miller. G. C. D. M. Allen, T. J. Costello & J. H. Hudson. 1979. Maturation
of the calico scallop. Argopecten gibbus. determined by ovarian color
changes. Northeast Gulf. Sci. 3:96-103.

Newell. R. E.. T. J. Hillbish. R. K. Koehn & C. J. Newell. 1982. Temporal
variation in the reproductive cycle of Mytihis edulis L (Bivalvia. Mytil-
idae) from localities on the east coast of the United States. Biol. Bull.
162:299-310.

Nikolsky. G. V. 1969. Fish Population Dynamic. P.A. Constable Ltd..
Edimburgh. Scotland.

Paulet. Y. M.. A. Lucas & A. Gerard. 1988. Reproduction and larval
development in two Pecten ma.\imus (L.) populations from Brittany. J.
Exp. Mar. Biol. Ecol. 119:145-156.

Reyes-Salinas. A. 1994. Relacion entre estructura hidrografica y la abun-
dancia. distribucion y origen de diferentes expresiones de biomasa del
seston organico en Bahia Concepcion. Golfo de California. Tesis Lie.
en Biologi'a. UNAM, Mexico.

Sbrenna G. & D. Campioni. 1994. Gametogenic and spawning patterns of
Manila clams Tapes philippinarum (Bivalvia: Veneroida) in two la-
goons of the river Po Delta. Italy. J. Shellfish Res. 13:37^6.

Seed. R. 1976. Ecology. In: B. L. Bayne (ed.). Marine Mussels: Their
Ecology and Physiology. Cambridge University Press. Cambridge. UK.
pp 13-60.

Thorarinsdottir. G. G. 1993. The Iceland scallop, Chlamys islandica (O.F.
Muller). in Breidatjordur, West Iceland. II. Gamete development and
spawning. Aquacidture. 1 10:87-96.

Tripp-Quezada, A. 1985. Explotacion y cultivo de almeja catarina Ar-
gopecten circularis en Baja California Sur. (Fishery and culture of
catarina scallop Argopecten circularis in Baja California Sur). Tesis de
Maestria (M.Sci. Thesis), CICIMAR-IPN. La Paz B.C.S.

Villalejo-Fuerte. M. T. 1992. Aspectos reproductivos de la almeja catarina
[Argopecten circularis Sowerby. 1835) en Bahia Concepcion. B.C.S..
Mexico. (Reproductive aspects of catarina scallop [Argopecten circu-
laris Sowerby. 1835) in Concepcion Bay, B.C.S.. Mexico). Tesis de
Maestria (M.Sci. Thesis). CICIMAR-IPN, La Paz B.C.S.

Villalejo-Fuerte, M. & R. I. Ochoa-Baez. 1993. The reproductive cycle of
the scallop Argopecten circularis (Sowerby. 1835) in relation to tem-
perature and photoperiod in Bahi'a Concepcion. B.C.S., Mexico. Cien-
cias Mar. 19:181-202.

Walker. R. L. & P. B. Heffernan. 1994. Temporal and spatial effects of
tidal exposure on the gametogenic cycle of the Northern quahog. Mer-
cenaria mercenaria (Linnaeus, 1758). in coastal Georgia. J. Shellfish
Res. 13:479^86.

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Aquaculture. 61:99-111.

Journal of Shellfish Research. Vol. 19. No. 1. 95-99, 2000.

REPRODUCTIVE CYCLE OF THE RUGOSE PEN SHELL, PINNA RUGOSA SOWERBY, 1835

(MOLLUSCA: BIVALVIA) FROM BAHIA CONCEPCION, GULF OF CALIFORNIA AND ITS

RELATION TO TEMPERATURE AND PHOTOPERIOD

BERTHA PATRICIA CEBALLOS-VAZQUEZ,
MARCIAL ARELLANO-MARTINEZ,
FEDERICO GARCIA-DOMINGUEZ, AND
MARCIAL VILLALEJO-FUERTE

Centra Interdisciplinario de Ciencias Marinas

Institiito Politecnico Nacional

Apartado Postal 592

La Paz. B.C.S. 23000. Mexico

ABSTRACT This study describes, through monthly histological examinations of gonadal tissue samples, the reproductive cycle of
Pinna nigosa and relates gametogenesis to temperature and photoperiod. Monthly gonadal samples were obtained from February 1993
to February 1994. in Bahi'a Concepcion. Gulf of California. Mexico. Five stages of gonadal development were characterized: indif-
ferent, developing, ripe, partially spawned, and spent. Histological evidence revealed hermaphroditism in 20.9% of animals sampled.
Gametogenesis commenced in March, with ripe and spawning stages occurring from April to November, and no gametogenic activity
occurring from December to February. From March to November, water temperature ranged from 20 °C to 31 °C. with an average
range of light of 650-820 min/day. P. nigosa had a seasonal gametogenic cycle directly related to water temperature and photoperiod.

KEY WORDS: Reproduction, bivalve. Pinna, histology. Gulf of California

INTRODUCTION

The rugose pen shell. Pinna nigosa Sowerby, 1835, is com-
monly known in Mexico as "hacha" (hatchet). This bivalve is of
commercial importance and supports a fishery in the northwestern
area Gulf of California. Mexico. P. nigosa is greatly appreciated
by consumers because of its tasty, large adductor muscle, com-
monly refeired to as "callo." The pen shell fishery has been an
important economic activity in Mexico for many years. Production
trends, however, have drastically declined over the past years, and
some populations have been depleted (Reynoso-Granados et al.
1996). Few biological studies of P. nigosa have been conducted
(Arizpe and Felix 1986, Arizpe and Covairubias 1987, Mazon-
Suastegui and Aviles-Quevedo 1988. Rui'z-Verdugo and Caceres-
Martinez 1990, Arizpe 1995).

Documentation of the reproductive biology of P. nigosa in the
Gulf of California is extremely scarce. Noguera and Gomez-
Aguirre (1972) described the reproductive cycle of P. nigosa in
Laz Paz Bay, B.C.S.. Mexico, and they showed that gametogenesis
commenced in mid-spring and that the animals spawned in late
summer.

Because of the economic importance and high price obtained
by the callo. efforts have recently been under way to cultivate this
species. Therefore, studies of its reproductive biology are essential
to achieve reproduction in a laboratory setting. This study docu-
ments the reproductive cycle of P. nigtKsa from Bahi'a Concepcion.
Gulf of California, Mexico, and examines the relationship of ga-
metogenesis to temperature/photoperiod.

MATERIALS AND METHODS

Bahi'a Concepcion. Mexico, is located on the western coast of
the Peninsula of Baja California, between 26'55' and 26°30'N and
1 12° and 1 1 l°40'E. The bay is approximately 40 km long and 10

km in its widest part and oriented in a NW-SE direction (McFall
1968).

Monthly, between 13 and 35 specimens of rugose pen shell
were collected by a scuba diver at a 2- to 8-ni depth from February
1993 to February 1994. Animals were collected from a wild popu-
lation located off Santispac Beach in Bahia Concepcion. Gulf of
California. The individuals were collected and fixed in 10'7f for-
malin solution. When the biological samples were collected, water
temperature at the collection site was recorded.

The visceral mass (gonad included) was dissected from each
pen shell and stored in 70% alcohol. Later, a slice of tissue of the
dorsal area of the visceral mass was cut. This tissue samples were
dehydrated in an ethanol series of progressive concentrations,
cleared in toluene, and embedded in paraffin. Serial sections 7-9
|jLm thick were obtained with a rotary microtome. Preparations
were stained with hematoxilyn and eosin. The gonad structure was
examined under a microscope, and the sex was determined for
each animal by the presence of egg or sperm in the tissue section.

Each tissue section of P. nigosa was categorized on the basis of
the qualitative characteristics of five stages of maturation (indif-
ferent, developing, ripe, partially spawned, and spent) as described
by Villalejo-Fuerte and Garcia-Domi'nguez (1998). The monthly
relative frequencies of the stages of gonadal development through-
out the annual cycle were obtained. This enabled the description of
the reproductive cycle. The spawning season is defined as the time
period containing ripe and partially spawned individuals.

To obtain a quantitative value that represents the reproductive
activity, a monthly gonad index (GI) was computed (Heffernan et
al. 1989) utilizing a numerical grading system. Three categories
were established according to the degree of development of the
gonad, with 1 = indifferent and spent. 2 = developing, and 3 =
ripe and partially spawned. The monthly GI was determined by
multiplying the number of specimens ascribed to each category by
the category score, summing all such values, and dividing the
resulting value by the total number of pen shells analyzed. The

95

96

Ceballos- Vazquez et al.

Figure I. Photimiicrographs of gonadnl stsiRcs of P. nif-osa. (a) (ionad classified as developing female; small oocytes groHing attached to the
follicle wall, male spent, lb) Developing male; thick layer of spermatocytes developing, (c) Mature female; large oocytes free In the lumen of
follicles, (dl Mature male; large (juantity of spermalo/oa tilling the follicles, (el Partially spawned female; empty follicle with some residual
oocytes. (f| Partially spawned male; a marked decrease in the nunihcr of spermatozoa lllling the lumen, igl Indifferent goniid; follicles with total
absence of gametes, (h) Gonad spent; follicles collapsed, aniebocytes phagocytizing residual gametes. Scale bar = ?l) pm.

Reproductive Cycle of Pinna rugosa

97

values obtained permit us to realize the correlation analysis of
reproductive activity with temperature and photoperiod.

Data for photoperiod for this study were not determined di-
rectly by the authors. Data from nautical almanacs of the Secretaria
de Marina of Mexico were used to define the photoperiod. The
data correspond to the daily period of illumination, and an average
in minutes of illumination was calculated for each month, between
February 1993 and February 1994, for the latitude corresponding
to Bahi'a Concepcion.

A Spearman rank order correlation analysis was used to inves-
tigate the relationship between GI, temperature and photoperiod.
Correlation analysis were carried out with the monthly values {n
= 13).

RESULTS

A total of 3 1 1 specimens was collected, 33 females (10.6%), 55
males (17.7%). 65 hermaphrodites (20.9%). and 158 indifferenti-
ated (50.8%). The range in shell length of pen shells was from 134
to 366 mm (258 mm average, 29 mm standard deviation).

In the hermaphrodite gonads, the development of both sexes
was not synchronous. On the contrary, one sex was always in a
more advanced stage of development {i.e.. the female phase was
developing, whereas the male phase was spent) (Fig. la).

To describe the reproductive cycle, all of the organisms were
considered, including the hermaphrodites. In the case of hermaph-
rodites, they were each considered as one individual accordingly
with the more advanced developing stage. The similar range of
gonadal development for small to large individuals indicated that
all pen shell sampled were reproductively active. All five stages of
gonadal development were observed (Fig. 1).

The reproductive cycle of P. rugosa from Bahi'a Concepcion,
Gulf of California, is summarized in Figure 2. Indifferent indi-
viduals were observed all year, except in June. In February 1993
and from December 1993 to February of 1994, most pen shell were
indifferent staged (94.1%, 100%, 93%, and 100%. respectively).
Gametogenesis commenced in March. Maturation was continuous
through November. Ripe stage was present from April to Novem-
ber, except in September. The partially spawned stage was present
in May and from July through November. Spent specimens oc-
curred from May to September, except in June.

Monthly quantitative assessments of histological reproductive
condition are illustrated in Figure 3a. From these data, it is appar-
ent that the GI has a seasonal tendency along the year, with high
values coinciding with ripe individuals and the fall of values co-
inciding with spawning activity. The values of GI were higher in
April, June, and October and were lower from December to Feb-
ruary. The GI values indicated that the gametogenesis started in
March and continued until November, with pen shell quiescent
from December to February.

Water temperature showed considerable seasonal variation
(Fig. 3b) with extreme values of 31 °C in August and 19 °C in
February.

The photoperiod (minutes of daily illumination) is illustrated in
Figure 3c. The longest monthly average daily illumination in the
study area occurred during May to July, with the highest in June
(820 min). The minutes of daily illumination presented a decreased
tendency during July through November. The shortest time of
illumination occurred in November/December and January (640
min).

100

80

> 60

z

UJ

O 40

UJ

cc

u.
20

17 26 13 25 30 35 18 27 25 20 19 29 27

M A

M J J
1993

J F
1994

H INDIFFERENT \
■ PART SPAWN [

1 DEVELOPING ^RIPE
! SPENT

Figure 2. Reproductive cycle of P. rugosa from Bahia Concepcion,
Gulf of California, Mexico. Relative frequency of gonadal stages be-
tween February 1993 and February 1994. Observations of males and
females are combined. Numbers at top indicate the sample sizes for
each month.

In all cases significant correlation (P < 0.05) was found. The GI
presented a positive correlation with temperature (/; = 13: r =
0.85: P = 0.000192) and photoperiod (n = 13: r = 0.69: P =
0.008980). Temperature and photoperiod were positively corre-
lated (n = 13: '• = 0.59: P = 0.031929).

DISCUSSION

In Bahfa Concepcion, the rugose pen shell exhibits an annual
gametogenic cycle, which commences in March with rapid prolif-

o
o

o E

D. .5-
O ro

o -s

Figure 3. Monthly variation of GI (a), water temperature (b). and
photoperiod (c) in Bahia Concepcion, B.C.S., Mexico.

98

Ceballos-Vazquez et al.

eration of gametes and ends by December. According to the his-
tological analysis, the spawning occurs from May to November,
except in June. Our results are in agreement with the reproductive
cycle of P. rugose occurring in La Paz Bay. B.C.S.. Mexico, as
described by Noguera and Gomez- Aguirre (1972). who docu-
mented that sexual maturation begins in mid-spring, with spawn-
ing occurring by late summer.

The characteristics of gametogenesis in P. rugosci from Bahfa
Concepcion were similar to those described for Spondyliis leuca-
canthus from Isla Danzante (Villalejo-Fuerte and Garci'a-
Dominguez 1998). The gonad off. nigosa has oocytes with the
same degree of development, common for bivalves with a syn-
chronic development. The histological examination additionally
showed that P. nigosa is a hermaphrodite species; in this study
20.9% of pen shell presented this condition. Hermaphroditism is
common in bivalves (Tranter 1958, Garcfa-Domi'nguez et al. 1996,
Villalejo-Fuerte and Garcia-Dominguez 1998).

There are two basic types of reproductive pattern exhibited by
marine bivalves in the Gulf of California waters. Many bivalve
species have no seasonal reproductive cycle, and their spawning
activity is continuous, for example. Megapitaria aiinintiaca (Gar-
cfa-Donii'nguez et al. 1994) and Pinctada mazatlanica (Garcia-
Domi'nguez et al. 1996). Other bivalve species exhibit distinct
seasonal reproductive cycles, such as Dosinia ponderosa (Arreola-
Hernandez 1997). Chione undatella (Baqueiro and Masso 1988),
and M. sqiialida (Villalejo-Fuerte et al. 1996). which usually are
related to temporal variations of environmental factors such as
food availability, water temperature, and/or photoperiod.

The reproductive activity of P. nigosa was significantly corre-
lated to the water temperature and photoperiod. The protracted
period of reproductive activity (March to November 1993) of P.
nigosa from Bahi'a Concepcion coincides with the gradual increase
of sea-surface temperature (from 20 °C until a maximum of 31

°C), and with increased values of photoperiod (720 min/day). The
period of reproductive inactivity was clearly distinguished in win-
ter (November 1993 to February 1994), and coincides with an
abrupt decrease of 3.5 °C in the sea-surface temperature (26 °C)
and with the photoperiod minimum values (640-650 min/day).

The Spearman correlation analyses indicated that the major
environmental factor that directly influences the gonadal growth is
the water temperature, suggesting that the production of gametes is
stimulated by increases in temperature. The same has been ob-
served for other bivalve species, such as Spondyliis leiicacanthus
(Villalejo-Fuerte and Garcia-Domi'nguez 1998) and Argopecten
circiilaris (Villalejo-Fuerte and Ochoa-Baez 1993). However, in
other bivalves from the Gulf of California, no clear relationship
exists between gonadic development and water temperature (e.g..
M. aiirantiaca [Garci'a-Domi'nguez et al. 1994] and P. mazatlanica
[Garcia-Dominguez et al. 1996]). Although water temperature af-
fects reproduction, other environmental factors may well play an
integral role in determining the pattern of annual gonad activity for
species in a given geographical area (Sastry 1970).

Giese and Pearse ( 1974) have reported photoperiod as a factor
that influences spawning of invertebrates. However, it has not been
widely studied in bivalves (Villalejo-Fuerte and Ochoa-Baez
1993). The temperature and photoperiod are positively coiTelated.
But it may not be possible to separate the effects of these two
factors with the data presented in this paper.

ACKNOWLEDGMENTS

We are grateful to Direccion de Estudios de Postgrado e In-
vestigacion del Instituto Politecnico Nacional for funding this
work and to Comision de Operacion y Fomento de Actividades
Academicas for the fellowships to F. Garci'a-Dominguez and M.
Villalejo-Fuerte. Thanks to Ma. Consuelo Gonzalez Ordonez for
her editorial help on English manuscript.

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the bivalve. Pinna nijio.sa (Sowerby), in suspended and boltoni culture
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Arizpe, CO. & O. Covarrubias. 1987. Reclutamiento y mortalidad de
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Arizpe. C. O. & U. R. Felix. 1986. Crecimientode Pinna itigosa (Sowerby.
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Arreola-Hernandez, F. 1997. Aspectos reproductivos de Dosinia ponder-
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CICIMAR, La Paz, Mexico, 85 pp.

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Joiirnai of Shi-ilfish Resfiirch, Vol. 19. No. 1. 101-105. 2000.

CHROMOSOME SEGREGATION IN FERTILIZED EGGS FROM ZHIKONG SCALLOP
CHLAMYS FARRERI (JONES & PRESTON) FOLLOWING POLAR BODY 1 INHIBITION

WITH CYTOCHALASIN B

HUIPING YANG, HUAYONG QUE, YICHAO HE, AND
FUSUI ZHANG

Experimental Marine Biology Laboraton-

Institute of Oceanology, Chinese Academy of Sciences,

Qingdao, Shandong 266071. China

ABSTRACT Chromosome segregation in fertilized eggs of the zhikong scallop. Chlamys farreri. following polar body 1 (PBl)
inhibition with cytochaUisin B (CB) was studied. The fertilized eggs were treated with CB (0.75 mg/L) at 7-10 min postfertilization
until polar body 2 (PB2) was released in control groups. The embryos were sampled every 5-10 min after fertilization and fixed in
Carney fixative. Chromosome segregation in both control groups and treated groups were analyzed using a hematoxylin stain method.
In fertilized eggs of control groups, the 19 tetrad chromosomes went through meiosis I and II, and released PBl and PB2, finally
reaching 19 chromatids. In CB treated groups, meiosis I proceeded normally and produced two groups of dyads, 19 in each group. With
the CB treatment, both of the two dyad groups were retained in the eggs and entered meiosis II. The segregation in meiosis II had four
patterns: bipolar, tripolar, tetrapolar, and unsynchronized segregation. When the two groups of dyads from meiosis 1 united, the treated
eggs entered meiosis II through tripolar (40.9'7f) and bipolar (11.4%) segregation patterns. Otherwise the two groups of dyads
segregated separately and formed tetrapolar segregation ( 15.7%). Also a small proportion of treated eggs (4.0%) underwent meiosis
II in an "unsynchronized segregation" pattern, which means that the two groups of dyads from meiosis I did not segregate synchro-
nously. There were 28.0% of treated eggs that could not be classified. The four segregation patterns produced different ploidies of
embryos in CB treated groups, such as triploids, tetraploids. pentaploids, and aneuploids.

KEY WORDS: Zhikong scallop, Cliluiiiys farreri. chromosome segregation, triploid, tetraploid, polar body

INTRODUCTION

Triploids can be induced by blocking the first polar body (PB I )
in some mollusk species, such as American oyster, Crassostrea
virginica (Stanley et al. 1981), Pacific oyster. Crassostrea gigas
iThunberg) (Quillet and Panelay 1986). Pacific abalone. Haliotis
discus hamuli (Aral et al. 1986). pearl oyster, Pinctada martensii
(Jiang et al. 1987). and blue mussel. Mytiliis ediilis (Yamamoto
and Sugawara 1988). Tetraploids were also reported among trip-
loids in American oyster (Stanley et al. 1981 ) and in other mollusk
species (Arai et al. 1986. Yamamoto and Sugawara 1988).
Stephens and Downing (1988) reported that 917f tetraploid at 24-h
postfertilization (PF) was produced by inhibiting PBl in fertilized
eggs from the Pacific oyster. In similar work, Guo et al. (1992a)
reported that many aneuploids embryos (57.6%) were also pro-
duced. All of these results indicate that PB 1 inhibition results in
complicated chromosome segregation. Observation of chromo-
some segregation in the Pacific oyster explained the mechanism
for formation of different ploidies when PB 1 was blocked (Guo et
al. 1992b). Three different types of segregation, including "tripolar
segregation," "united bipolar .segregation." and "separated bipolar
segregation" were evident. Later, the chromosome segregation in
triploid Pacific oysters was also studied when eggs from triploids
were fertilized with diploid sperm and PBl was blocked with CB
(Que et al. 1997). The observation showed that there were also
three types of segregation patterns, confirming the mechanism by
which viable tetraploid Pacific oysters can be successfully induced
through blocking PBl in fertilized eggs from triploids (Guo and
Allen 1994).

In the zhikong scallop. Clilamys farreri. blocking PBl in fer-
tilized eggs from normal diploids can result in triploid, tetraploid,
pentaploid, and aneuploid embryos. We have also found that both
triploids and tetraploids can survive to 2-3 mm juvenile stage
(unpublished). In this paper, the behavior of chromosome segre-

gation was observed in fertilized eggs from normal diploids when
PBl was blocked with CB, offering an explanation for the forma-
tion of embryos with different ploidies.

MATERIALS AND METHODS

Gametes

Parent scallops were from Rizhao and Qingdao, Shandong,
China. The scallops were conditioned indoors to accelerate gonad
maturity. Gametes were obtained through natural spawning. Eggs
were collected with a 25-[j, screen and resuspended into 2 ~ 3 L
seawater at 20 °C, ready for fertilization. Sperm were prepared by
screening sperm suspension through a 25-p. nylon screen. For
fertilization, sperm were added to the egg suspension at a final
density of 5-7 sperm per egg. Fertilization, treatment, and embryo
culture were all conducted at 20 °C.

Treatment and Sampling

PBl in fertilized eggs was blocked with 0.75 |jLg/mL CB dis-
solved in dimethyl sulfoxide (DMSO-final concentration 0.1%).
CB treatment began at 7-10 min PF and ended when the second
polar body (PB2) in control groups was observed under micro-
scope. Fertilized eggs in both control groups and CB-treated
groups were .sampled every 5-10 min during development until 75
min PF. Samples were directly fixed in Carnoy fixative (methanol:
acetic acid = 3:1), which was changed twice, and the samples
were then stored at 4 °C before analysis. The experiment was
repeated four times using different parent scallops.

Chromosome Observation

Slides for observing chromosomes were made by a modified
squashing method. The staining solution was made by dissolving
0.5%' hematoxylin in 45% acetic acid, with ammonium iron sulfate

101

102

Yang et al.

dodecahydtate as a mordant (about 0.5%). Embryo samples were
dropped and spread on clean slides. Excessive fixative was al-
lowed to run off the slides, and then drops of staining solution were
added onto the samples just before the fixative evaporated. A clean
cover glass was placed gently on the samples. Before squashing on
filter paper, slides were warmed slightly by passing them across an
alcohol burner. Then, the cover glass was sealed on all four sides.
Alternatively, the whole cover glass was sealed with neutral bal-
sam after removal by icing the slides.

Slides were examined with a Nikon compound microscope.
Photographs were taken using LUCKY black and white film (ASA
100 and 400).

RESULTS

Initially, normal diploid eggs were observed in prophase of
meiosis I (Fig. la). Zhikong scallop has a diploid number of 38
chromosomes (Wang et al. 1990). Nineteen tetrads were observed
in the unfertilized eggs.

In control groups, the 19 tetrads began to segregate at about
9-10 min PF, then the tetrads in the majority of fertilized eggs
segregated into 38 dyads, and then divided into two groups, 19 in
each group (Fig. lb). Later, one group of dyads condensed and
released as PBI (Fig. Ic). The remaining 19 dyads continued
meiosis II and segregated into two groups of chromatids (Fig. Id).
One of the groups of chromatids was released as PB2 in most
fertilized eggs at 40^3 min PF. Normally, the two polar bodies
were positioned next to each other (Fig. le). As for the chromatids
from sperm, at first, they could only be observed as dark-stained
material. Only during mitosis I, did the chromatids from egg and
sperm unite, yielding 38 chromosomes.

In CB-treated groups, chromosome segregation was compli-
cated. After fertilization, the 19 tetrads segregated into 38 dyads
(Fig. Ig). Under the microscope, no PBI was released in the ma-
jority of treated, fertilized eggs during CB treatment. Thus, 38
dyads in fertilized eggs entered meiosis II, and chromosome seg-
regation differed greatly from that in control groups. Four patterns
of segregation were observed: bipolar, tripolar. tetrapolar, and un-
synchronized. Some segregations could not be classified. When
the 19 dyads from PBI united with the other 19 dyads, the chro-
mosome segregation in meiosis II proceeded with bipolar or tri-
polar segregation.

Bipolar Segregation

The 19 dyads from PB 1 united with the remained 19 dyads, and
went through meiosis II together (Fig. Ih). All 38 dyads segregated
in a bipolar pattern just like normal meiosis II, and divided into
two groups of sister chromatids, 38 in each group (Fig. 1 i). One of
the two groups of chromatids was released as PB2 after CB treat-
ment. This pattern of chromosome segregation could result in Irip-
loids.

Tripolar Scungalioii

The 38 united dyads divided into three groups, apparently al
random (Fig. Ij), and the dyads in each group .segregated in two
directions. Finally, the chromatids migrating in one direction
united with the chromatids from its neighboring group at a pole,
forming three groups of chromatids (Fig. Ik). The number of chro-
matids in the three groups varied considerably, apparently depend-
ing upon random distribution of dyads before meiosis II. Rarely,
one of the three groups had exactly 19 chromatids. In this pattern

of chromosome segregation, the three groups of chromatids had
probability of being released as PB2 after CB treatment.

Sometimes the 19 dyads from the unreleased PBI fail to unite
with the remaining 19 dyads in the fertilized eggs (Fig. 11). They
entered meiosis II independently, resulting in two patterns of chro-
mosome segregation: tetrapolar and unsynchronized segregations.

Tetrapolar

In both dyads groups, the chromosomes segregated in a normal
bipolar pattern. The final result was that four groups of chromatids
formed, 19 chromotids in each group (Fig. Im).

Unsynchronized Segregation

The unreleased dyads and the remaining dyads in the fertilized
egg went through meiosis II asynchronously. Sometimes one
group of dyads did not go through meiosis II. but remained un-
changed, and another group of dyads went through meiosis II and
underwent bipolar segregation to anaphase (Fig. In).

Unclassified

In addition to the above segregation patterns, there were also
other patterns that could not be classified. In fertilized eggs with 38
united dyads, only some began meiosis II segregation; whereas,
some were left as dyads. In some treated fertilized eggs, the 38
dyads went through meiosis II. but the 76 chromatids distributed
themselves randomly. No segregation poles were observed (Fig.
lo).

The frequencies of the four segregation patterns were calcu-
lated from embryos where PBI had been blocked (Table I). On
average, the majority of treated eggs went through meiosis II as
tripolar segregations (40.9%), 11.4% were bipolar, and 15.7%
were tetrapolar. Only a small proportion of treated eggs (4.0%)
went through unsynchronized segregation. Finally, 28.0% of seg-
regations in treated eggs could not be classified.

DISCUSSION

We made slides for observing chromosome segregation by a
modified squashing method using a hematoxylin stain. For fertil-
ized eggs and embryos from the zhikong scallop, this procedure
was quite useful. In fertilized eggs of the Pacific oyster, orcein
dissolved in 60% acetic acid employed chromosome observations
(Guo et al. 1992a, Que et al. 1997). In the zhikong scallop, we have
tried orcein staining, but it produced poor contrast. Hematoxylin is
a typical chromosome stain (Sharma and Sharnia 1980). Normally
hematoxylin solution must be made in advance to ripen for a few
weeks. In this experiment, the stain solution was modified as 0.5%
dissolved in 457r acetic acid with ammonium iron sulfate as mor-
dant and could be used instantly. In addition, this method produced
satisfactory results for observing chromosomes in the fertilized
eggs and embryos from the jinjiang oyster, Cni.\si'strc'ii ariakensis
(unpublished). We suggest this new staining method for chromo-
some observations in bivalve mollusk.

Chromosome Segregation

Unfertilized eggs of normal diploid zhikong scallops are ar-
rested al late prophase of meiosis I. Only after fertilization, did the
eggs continue meiosis I and II. releasing PBI and PB2. In the end,
19 maternal chomatids remained in the lertili/ed eggs. This pattern
of chromosome segregation is common among bivahe mollusks
(Longo and Anderson 1969).

Chromosome Segregation in PB 1 Blocked Eggs

103

> V

. V

J h

m

^2

ri*i

j^

1

**

,*■'. 1' tM',

r-'f

> >•

•** 4

\e

(

^

r

^\ *

il

n

•^ if-'

^^'

Figure 1. Segregation patterns observed in fertilized eggs from diploid zhikong scallop, CMamys farreri. following normal fertilization (a-e) and
PBl blocking with Cvtochalasin B (f-ol. a-e: meiosis in normal fertilized eggs; f: two polar bodies positioned side-by-side in fertilized eggs
following PBl blocking with CB: g: the united 38 dyads, h-i: bipolar segregation pattern: j-k: tripolar segregation pattern; 1-m: tetrapolar
segregation pattern. 1 and 2 indicated two separate poles; n: unsynchronized segregation pattern. 3 indicated dyad groups; o: undassifled
segregation pattern.

104

Yang et al.

TABLE 1.
Chromosome segregation patterns (%) in fertilized eggs wlien PBl was blocl^ed with CB in zhikong scallop Chlamys farreri.

Replicate

Chromosome Segregation

Patterns

(Number)

n

Bipolar

Tri polar

Tetrapolar

Unsynchronized

Unclassiried

1

109

11.0

4.'i.9

17.4

1.8

23.9

2

162

7.4

46.9

17.3

4.3

24.1

3

106

13.2

46.2

11.4

2.8

26.4

4

85

14.1

24.7

16.5

7.1

37.6

Average

11.4

40.9

15.7

4.0

28.0

In CB-treated groups, chromosomes in fertilized eggs, follow-
ing PBl blocking, segregated in four patterns: bipolar (11.4%),
tripolar (40.9%), tetrapolar (15.7%), and unsynchronized (4.0%).
Bipolar, tripolar, and tetrapolar segregation patterns were similar
to those reported in diploid (Guo et al. 1992b) and triploid Pacific
oysters (Que et al. 1997) when PBl was blocked.

In addition, in the zhikong scallop a small proportion of treated
eggs (4.0%) went through meiosis II asynchronously. Blocked
dyads from PBl failed to unite and segregated asynchronously
from the remaining dyads. Sometimes the remaining dyads went to
anaphase of meiosis II and divided into two groups of chromatids,
while the dyads from blocked PBl remained paired and skipped
meiosis II. leaving three chromatin groups. In eggs of triploid
Pacific oysters, asynchronous segregation was also observed when
crossed with a normal sperm of diploid followed by PBl inhibition
by CB (Que et al. 1997). In the Japanese pearl oyster, Pinctada
fiicala inanensii. Komaru et al. (1990) reported that three groups
( 20.6% ) and four groups ( 1 7.6% ) of maternal chromatin were pro-
duced by blocking PB 1 . The observation of three groups of chro-
matin might be explained in two ways: asynchronous segregation
or tripolar chromosome segregation, both resulting in three chro-
matin groups. The percentage of fertilized eggs with three groups
of maternal chromatin (20.6%) as observed by Komaru was much
lower than our observations of tripolar segregation (40.9%) and
asynchronous segregation (4.0%) in the zhikong scallop. This is
possibly caused by differences in chromosome segregation be-
tween the different species or because the conditions of CB treat-
ment were different. In diploid Pacific oysters, chromosome seg-
regation following PBl inhibition was observed to pass through
meiosis II synchronously (Guo et al. 1992b).

In addition to the described tour segregation patterns, there was
a large proportion of chromosome segregations (28.0%) that could
not be classified, such as 76 chromatids scattered randomly. Un-
classified .segregation patterns have also been observed in both
diploid and triploid Pacific oysters (Guo et al. 1992b. Que et al.
1997). Considering the results of this experiment and those in
diploid and triploid Pacific oysters and pearl oysters, we suggest
that tripolar, tetrapolar, bipolar, and unsynchronized segregation
patterns are the normal ways for fertilized eggs to go through
meiosis II alter PBl blocking.

Observations of chromosome segregation using the st|uashing
method provide an incomplete picture of cytological events, be-
cause compression of the eggs transforms the three dimensionality
of the meiotic plates into a plane, thus rearranging the position of
chromosomes. This method also fails to display centrosomes and
spindles that play an important role in meiosis. Observing cen-
trosomes and spindles might provide a clearer picture of how
chromosomes segregated. Especially for the centrosome. its num-
ber and replication are critical factors in the chroiiiosome segre-

gation. Normally the centrosome from sperm does not participate
in meiosis (Sluder et al. 1993), and the centrosome from maternal
replicates two times with each meiotic stage, resulting in the nor-
mal bipolar segregation. In this experiment, we hypothesize that
centrosome number is the primary factor controlling patterns of
chromosome segregation. With PBl blocked in eggs, centrosome
number could change profoundly, affecting chromosome segrega-
tion in meiosis II. Centrosome numbers could range from 2—4,
depending upon whether centrosomes replicated, and could result
in bipolar, tripolar, or tetrapolar chromosome segregation patterns.
This supposition must be tested by visualization of the cen-
trosomes, spindles, or both.

Ploidy Consequences

In the zhikong scallop, diploid, triploid, tetraploid, pentaploid.
and aneuploid 2—4 cell stage embryos were all produced when PBl
was blocked in fertilized eggs. Both triploid and tetraploid zhikong
scallops survived to juvenile stage (21.3% triploid and 1.9% tet-
raploid in one group, unpublished). The various ploidy conse-
quences of PB I blocking relate to the different chromosome pat-
terns, as observed in this study.

First, bipolar segregation patterns formed two groups of 38
chromatids. Either of the two chromatids group could be released
as PB2, leaving 38 chromatids. No matter which group was re-
leased, triploids would be produced by bipolar segregation v\ith 19
chromosomes contributed by the sperm.

For tetrapolar segregation patterns, four separated chromatids
groups were formed after meiosis II, 19 chromatids in each group.
The ploidy consequences would depend upon how many chroma-
tid groups would be released with PB2. Release of one group
would produce tetraploids; whereas, release of two groups would
produce triploids and release of three would produce diploids.
After CB was washed off, embryo development showed that .some
fertilized eggs in the treated groups released one PB, and some
fertilized eggs released two PBs positioned side-by-side (Fig. If)
or separated from each other on the egg. Rarely were these two
polar bodies positioned next to each other, as in Figure le. It was
impossible by our methods to observe total number of chromatids
in released PBs. This problem might be resolved by using special
staining methods to label chromatids individually, such as //; siiu
fluorescent hybridization.

The ploidy consequences of embryos after tripolar segregation
were the most complicated because of random allocation of chro-
matids at three poles and the random release of PB2. The meta-
pliase and anaphase period In meiosis II were very short, so it was
not practical to count numbers of chromatids at the three poles in
most fertilized eggs. By counting the chromosome of 2-4 cell
embryos, we could infer that chromosome number varied highly.

Chromosomh Segregation in PB 1 Blocked Eggs

105

In tripolar segregations, tetraploids would be produced only when
one pole had exactly 19 chromatids, and the chromatids at this pole
were released as PB2. If the 19 chromatids at one pole remained in
the eggs, and the chromatids at the other two poles were released
as PB2. diploids would be produced. Otherwise, aneuploids re-
sulted. The majority of fertilized eggs proceeded by tripolar seg-
regation (40.9'/(-), explaining why about 23.3% of 2-4 cell stage
embryos were aneuploid (unpublished). In most aneuploids. chro-
mosome numbers were distributed mainly into three groups: 42-
48, 62-69. or 83-89. most likely the result of random allocation of
chromatin from the three poles.

Unsynchronized segregation resulted in three groups of chro-
matin, two with 19 chromatids in each and the other with 19 dyads
from blocked PBl. Diploids, triploids. and tetraploids could pos-
sibly be produced, depending upon which group was relea.sed as
PB2. Supposing one group of 19 chromatids was released as PB2.
tetraploids would be produced. If 19 dyads were released as PB2,
triploids would be produced. If two groups of chromatin were
released as PB2. triploids or diploids would be produced.

Pentaploids were also observed at the 2^ cell embryo stage
(unpublished). The formation of pentaploidy was probably caused
by the failure of PB2 to be released in fertilized eggs after PB 1 was
blocked with CB. Thus, the 76 chromatids from maternal chro-
motids plus the 19 chromatids from sperm formed pentaploids.

Clearly, differences in chromosome segregation resulted in dif-
ferent ploidy consequences and agree with the proposed mecha-

nism to form different ploidies in diploid Pacific oyster when PBl
was blocked (Guo et al. 1992b), PBl blocking is also an effective
way to induce triploids and tetraploids. Both triploid and tetraploid
embryos have been produced through blocking PBl in fertilized
eggs from normal diploid, such as in Pacific oyster (Guo et al.
1992a), American oyster (Stanley et al. 1981). Pacific abalone
(Aral et al. 1986) and blue mussel (Yamamoto and Sugawara
1988).

In summary, the inhibition of PBl in fertilized eggs of zhikong
scallop with CB resulted in complicated chromosome segregation
patterns, including bipolar, tripolar, tetrapolar. unsynchronized.
and unclassified segregations, producing diploid, triploid, tetra-
ploid, pentaploidy, and aneuploid embryos. This study provided
cytological evidence about possible formation of different ploidies
and valuable information on polyploid induction.

ACKNOWLEDGMENT

The authors thank Drs. Ximing Guo and Standish K. Allen Jr.
for their constructive comments on the manuscript. This study is
supported by Chinese postdoc fund (No. 6975), Grant 819-01-07
from China's National High-Tech Development Program (863),
the "100 Scholar" program of the Chinese Academy of Science
and China's Natural Science Foundation (No. 39825121). This is
publication No. 3682 of the Institute of Oceanology, Chinese
Academy of Sciences.

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Quillet. E. & P. J. Panelay. 1986. Triploidy induction by thermal shocks in

the Pacific oyster. Crassostrea gigas. Ac/uaculture 57:271-279.
Sharma. A. K. & A. Sharma. 1980. Chromosome techniques: theory and

practice. 3rd ed. Butterworth. London, pp. 1 1 1-1 13.
Sluder. G.. F.J. Miller & K. Lewis. 1993. Centrosome inheritance in

starfish zygotes 11: selective suppression of the maternal in centrosome

during meiosis. Dev. Biol. 155:58-67.
Stanley. J. G., S. K. Allen Jr. & H. Hidu. 1981. Polyploidy induced in the

American oyster, Crassostrea virginica, with cytochalasin B. Aqiiacul-

ture 12:1-10.
Stephens. L. B. & S. L. Downing. 1988. Inhibiting first polar body forma-
tion in Crassostrea gigas produces tetraploids. not meiosis triploid. /

Shellfish Res. 7:550-551.
Wang. M.. J. Zheng & H. Wang. 1990. The karyotype of Zhikong scallop,

Chlamys farreri. J. Ocean Univ. Qingdao 20:81-85 (in Chinese).
Yamamoto. S. & Y. Sugawara. 1988. Induced triploidy in the mussel,

Mytilus edulis. by temperature shock. Aquaculture 72:21-29.

Journal of Shellfish Research. Vol. 19. No. 1. 107-112. 2U()().

REPRODUCTIVE CYCLE OF ARGOPECTEN VENTRICOSUS (SOWERBY 1842) (BIVALVIA:

PECTINIDAE) IN THE RADA DEL PUERTO DE PICHILINGUE, B.C.S., MEXICO AND ITS

RELATION TO TEMPERATURE, SALINITY, AND FOOD

ANTONIO LUNA-GONZALEZ,* CARLOS CACERES-MARTINEZ,'
CLAUDIA ZUNIGA-PACHECO,' SILVERIO LOPEZ-LOPEZ," AND
BERTHA PATRICIA CEBALLOS-VAZQUEZ"

' Departamento de Ingenieiia en Pesquen'as
Universidad Autonoma de Baja California Sur
Lahnratorio Experimental de Maricultura. Apartado Postal I9-B.
La Paz. B.C.S.. 23081 Mexico.

'Centra Interdisciplinario de Ciencias Marinas
Apartado Postal 592.
La Paz. B.C.S.. 23000 Mexico

ABSTRACT The reproductive cycle of the catarina scallop Argopecten ventricosiis and its relation to temperature, salinity, and
quantity of food was studied in the Rada del Puerto de Pichilingue. B.C.S. Mexico, from April 1995 to March 1996. Organisms were
obtained from a hatchery and grown on the bottom. Ripe organisms occurred throughout the year showing the lack of seasonality in
its reproduction. No consistent relation between reproductive cycle and environmental factors or food was evident. The muscle yield
index showed a significant positive correlation with temperature, but it had no correlation with gonadosomatic index. The relation
between the muscle yield index and seston with the reproductive cycle suggested the transference of energy from the muscle to the
gonad and directly from the seston ingested. This relation suggested that A. ventricosus is a conservative and opportunistic species
depending on the available food. Histochemical analysis revealed the transference of carbohydrates from the intestinal loop to the
gonad and therefore to the oocytes.

KEY WORDS: Argopecten. reproductive cycle, bivalves, histochemistry, seston, food index

INTRODUCTION

The scallop Argopecten ventricosus (Sowerby 1842) is distrib-
uted from Isla Cedros and the Gulf of California to Peru (Keen
1971). A. ventricosus supports an important fishery in northwest
Mexico, especially in Baja California Sur (Chavez- Villalba and
Caceres-Martinez 1992). It is an important resource because of the
high commercial value of its adductor muscle (Villalejo-Fuerte
and Ochoa-Baez 1993).

The necessity of measures for the regulation of the fishery has
prompted several studies about reproduction of the catarina scallop
in Baja California Sur (Baqueiro et al. 1981, Caceres-Martinez et
al. 1990, Villalejo-Fuerte and Ochoa-Baez 1993. Felix-Pico et al.
1995).

The reproductive cycles of scallops are influenced by changes
in environmental variables, such as temperature and food (Mac-
Donald and Thompson 1985. Barber and Blake 1991). and by
genetic characteristics (Barber and Blake 1991). Gametogenesis
needs a lot of energy (Sastry 1979). This energy is obtained di-
rectly from the seston or from storage organs or tissues, like the
digestive gland, mantle, and adductor muscle (Ansell 1974, Gab-
bott 1975, Barber and Blake 1983).

The seston includes live plankton, organic detritus, and inor-
ganic particles (Navarro and Thompson 1995). The quantity and
quality of seston varies in response to physical and biological
factors such as tides, storms, wind, bacteria, fungi, and primary
consumers (Berg and Newell 1986, Mann 1988).

The objective of the study is to examine the reproductive cycle
of A. ventricosus in relation to its condition, histochemical com-
position of somatic and reproductive tissues, temperature, salinity,
and quantity of available food.

MATERIALS AND METHODS

Between April 1995 and March 1996, 30 specimens of A. ven-
tricosus (shell height mean ± SD = 5.25 ± 0.02 cm) were col-
lected randomly per month by diving between 3- and 4-m depth
from a population grown in the Rada del Puerto de Pichilingue,
B.C.S. , Mexico (24°16'N; 1 10°19'W). These organisms were ini-
tially produced in September 1994 at our hatchery at Universidad
Autonoma de Baja California Sur and seeded on the bottom in
February 1995. The surface water temperature and salinity were
recorded at the time of sampling. Total soft body, adductor muscle,
and gonad wet weights were recorded for each specimen.

Reproductive Cycle

The scallops were fixed in 10% formalin. Tissue sections were
taken through the middle of the gonad, dehydrated in alcohol, and
embedded in paraffin wax. Sections (5 |j.m) were placed on slides
and stained with hematoxylin-eosin (Humason 1979). Gametoge-
nesis (either spermatogenesis or oogenesis) of A. ventricosus was
divided into five stages (undifferentiated, developing, ripe, spawn-
ing, and spent) on the basis of the developmental stages defined by
Villalejo-Fuerte and Ochoa-Baez (1993) for the same species and
our own observations.

Undifferentiated Stage

Abundant connective tissue, without germ cells or residual ga-
metes. It was not possible to distinguish the sex.

Developing Stage

In the female, this stage is characterized by the presence of
variable quantities of developing oocytes attached to the follicle

107

108

LUNA-GONZALEZ ET AL.

wall. Some detached ripe oocytes occurred in the lumen of the
follicle. In the male, this stage had variable quantities of germinal
cells, spermatocytes, spennatids, and ripe spermatozoa. Interfolli-
cular connective tissue decreases and follicles increase in area as
the result of the accumulation of ripe gametes.

Ripe Stage

In the female, there were abundant, ripe polygonal-shaped oo-
cytes free within the follicles. Yolk droplets were observed in the
oocyte cytoplasm. Some developing oocytes remained attached to
the follicle wall by a slender stalk. In the male, this stage was
characterized by follicles filled with ripe spermatozoa arranged in
characteristic radial bands with tails pointing toward the center of
the lumen. Almost all the connective tissue has been completely
replaced by follicles forming the gonadic tissue, which is occupied
by gametes.

Spawning Stage

The walls of follicles become broken. Variable quantities of
unspawned oocytes and spermatozoa were observed into the fol-
licles. Free spaces inside the follicles were abundant. Some fol-
licles are completely devoid of gametes.

Spent Stage

The follicles were empty, with the exception of some residual
oocytes and spermatozoa. Connective tissue begins increasing.
The broken follicles are invaded by phagocytes. The relative fre-
quencies of the stages of gonadal developinent throughout the year
were obtained. This enabled the description of the reproductive
cycle.

Gonadosomatic Index (GSI)

This index in wet weight was calculated according to Sastry
and Blake (1971).

GW

Where GSI is the gonadosomatic index, GW is the gonad weight
in grams, and TSBW is the total soft body weight in grams.

Muscle Yield Index (MYI)

The muscle yield index was calculated as an indicator of the
condition of the scallops (Caceres-Martinez et al. 1990).

MW

Where MYI is the muscle yield index. MW is the weight of muscle
in grams, and TSBW is the total weight of the soft body in grams.

Histochemical Analysis

Four .scallops corresponding to each stage of gonadal develop-
ment (twenty in total) were collected in September 1995 for his-
tochemical analysis (qualitative analysis) of gonad, mantle, and
muscle to delcrminc carbohydrate and lipid content. Unfortu-
nately, we did not take samples since April 1995 to get an annual
cycle. Tissue sections were embedded in paraffin wax and O.C.T.
compound (an embedding medium for frozen tissue specimens).
Sections 5-(j.m thick from paraffin wax and sections 16-^m thick
from O.C.T. were placed on slides. The oil red technique (Spann-

hof 1966, Martoja and Person 1970) was used on frozen cuts to
determine unsaturated lipids. Periodic acid of the Schiff-Malt tech-
nique was used to determine glycogen (Martoja and Person 1970,
Sheehan and Hrapchak 1973, Humason 1979), and the blue alzian
technique was used to detect acid mucopolysaccharides (Spanhoff
1966, Martoja and Person 1970).

Seston Analysis

During the study period, every 15 days, 12-L of unfiltered
seawater samples of the scallop-sampling area were collected in
clean plastic containers and transported to the laboratory. The
seawater samples were collected at 3.5-m depth, close (about 15
cm) to the sandy bottom on which the scallops grew. The water
was screened through a 180-|jLm Nitex mesh to eliminate large
zooplankton and debris before analysis.

For dry weight and chemical analysis, 2-L of seawater for each
filter (six filters in total every 15 days) were immediately filtered
under gentle vacuum through washed, precombusted, preweighed
Whatman GF/C filters, 4.7-cm diameter. Three filters for chemical
analysis were stored at -40 °C until the analysis was done. Three
filters for dry weight were dried in an oven at 80 °C for 24 h. Then
they were weighed and combusted at 475 °C for 4 h. Finally, filters
were reweighed after cooling in a desiccator. The particulate or-
ganic matter (organic seston) was obtained by difference of both
weights.

For chemical analysis, two filters per month (one filter per
sampling) with 2-mL of distilled water were ground at 5 '^C in an
ice bath. A 400-|jlL aliquot was used for lipid determination using
the Bligh and Dyer (1959) method. Carbohydrates were analyzed
in a 300-p.L sample by the method of Dubois et al. (1956), modi-
fied by Malara and Charra (1972a). Proteins were analyzed in a
300-(i,L aliquot by the method of Lowry et al. (1 95 1 ), modified by
Malara and Charra (1972b). Results of chemical analysis were
standardized for volume of seawater filtered.

Total Seston ( TS)

The TS was obtained as the sum of inorganic seston and or-
ganic seston (dry weight).

Inorganic Seston/Organic Seston Ratio (IS/US ratio)

This ratio was obtained to relate (monthl) ) inorganic seston to
organic seston.

Food Index (FI)

An evaluation of the nutritional value of the seston throughout
the annual cycle in the Rada del Puerto de Pichilingue was done
using the 3 major biochemical components of the seston (lipid,
carbohydrate, and protein). Thus food quantity was defined as the
sum of these components and a food index was calculated accord-
ing to Widdows et al. (1979) as the percentage of food nialerial
contained in the total seston.

Fl= — * 100

Where 1-1 is the food index, F is the lood material (mg/L), and TS
is the total seston (ma/L).

Reproductive Cycle of A. ventricosus

109

RESULTS

Reproductive Cycle

The scallop A. ventncosiis is a functional hermaphrodite. In the
female and male follicles, the gametes were in the same develop-
mental stage. The gonad showed well-differentiated male and fe-
male areas. Figure 1 summarizes the reproductive cycle of A.
ventricosus. The presence of ripe gonads throughout the year in-
dicated a prolonged reproductive period with a lack of a clear
seasonal pattern. Despite this, there was a major resting period in
June and September 1995 where the undifferentiated stage reached
a maximum (91.3 and 46.15%. respectively). The spawning stage
was observed in 9 of the twelve months sampled but reached the
maximum \alue in August 1995 (50%).

Environmental Parameters

Temperature and salinity fluctuated relatively little (Fig. 2a).
The maximum water temperature was in September 1995
(29.5 °C). and the minimum (20.5 °C) in January 1996. The maxi-
mum salinity was in January. February, and March (37 7cc). and the
minimum in August and September 1995 (34 %o).

Gonadosomatic Index

The GSI supported the results obtained in the histological
analysis (Fig. 2b). The values were at a minimum in April, May.
June. September. January, and increased drastically from January
(4.08%) to February (7.79%) and March (8.75%).

Muscle Yield Index

The MYI was at a maximum in June and September 1995 (45.8
and 46.9%, respectively) and was at a minimum in April 1995
(33.7%) and from December 1995 to March 1996 (Fig. 2c). The
MYI showed a significant positive correlation with temperature (r
= 0.797; P = 0.001; n = 12). and a significant negative corre-
lation with salinity (r = -0.788; P = 0.002; n = 12). With the
GSI, there was no significant correlation (r = -0.405; P = 0.190;
n = 12).

Histochemical Analysis

The results of the histochemical analysis of gonad, adductor
muscle, and mantle are in Table 1. Positive results were found for
glycogen in the female area of the gonad (developing and ripe

I I Undifferentiatec|~p Developing
^■Spawning ^H Spent

I Ripe

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1995

S O N D

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1996

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Figure 1. Reproductive stages of Argopecten ventricosus in the Rada
del Puerto de Pichilingue, B.C.S., Mexico (n = 30).

Figure 2. Water temperature and salinity in the Rada del Puerto de
Pichilingue, B.C.S., Mexico (a) and gonadosomatic (b) and muscle
yield index (c) of Argopecten ventricosus. (Error bars = SD).

oocytes) and in muscle fibers. Unsaturated lipids were found in the
female area of the gonad (developing and ripe oocytes). Acid
mucopolysaccharides were found in the interfollicular connective
tissue of developing stage gonads (male and female areas), epi-
thelium and food content of intestinal loop, and in the epithelium
and connective tissue of mantle.

Total Seston, Inorganic Seston, and Organic Seston

Sediment resuspension was caused mainly as a consequence of
tidal currents and wind. In this area, maximum values of TS and IS
were found in April. July. December, and January. Higher values
of OS were found in April. December, and January (Fig. 3a).

Inorganic Seston/Organic Seston Ratio

The IS/OS ratio had no clear relation with the reproductive
cycle (Fig. 3a). However, it showed a significant positive correla-
tion with salinity (r = 0.676; P = 0.015; n = 12).

Food index

The FI expresses the quality of the diet available to a filter-
feeding organism. This FI showed maximum values in July, Sep-
tember, and November 1995 (4.99, 5.03, and 4.97%, respectively)
and minimum values in April-May 1995 (2.79 and 2.92%, respec-
tively) and February 1996 (2.78%) (Fig. 3b). It did not show a
clear relation with the reproductive cycle.

110

Luna-Gonzalez et al.

TABLE 1.
Histochemical tests performed on gonad, muscle, and mantle of Argopecten ventricosus.

Substance Tested

Technique

Control

Gonad

Muscle

Mantle

Glycogen

Unsaturated lipids

Acid mucopolysaccharides

Pas-Mall

Oil Red

Al/ian Blue

Rat liver

- 00

++ ro

++ do

None

- oo

++ ro

++do

None

++ ifd

+ ifr

Abbreviations: -, not detected; +. positive reaction; ++, strong positive reaction; do. developing oocytes; ifd, interfollicular connective tissue of
developing stage gonads (male and female area), epithelium and food content of intestinal loop; ifr. interfollicular connective tissue of ripe stage gonads;
mec, mantle epithelium and connective tissue; oo. oogonias; ro, ripe oocytes.

DISCUSSION

The cytological characteristics of the gonad of A. ventricosus in
the Rada del Puerto de Pichilingue, B.C.S. were similar to those
described by Villalejo-Fuerte and Ochoa-Baez ( 1 993) for the same
species in Bahi'a Concepcion. B.C.S.. and for other pectinids. like
Patinopeclen yessoensis (Motavkine and Varaksine 1983) and Pla-
copecten magellanicus (Beninger 1987). Male and female follicles
developed simultaneously and the gametes were spawned at about
the same time.

The gonads of A. ventricosus contained gametes in different
stages of development in all the months during the annual cycle,
although in lesser amount in June when the majority of the speci-
mens were in the undifferentiated stage. Ripe organisms were
present throughout the year, which suggests that this species re-
produces throughout the year. Similarly, the presence of ripe or-
ganisms of A. ventricosus all year has been reported in other lo-
cations of Baja California Sur (Baqueiro et al. 1981, Felix-Pico et
al. 1995).

Although the temperature is an important environmental factor
in the regulation of bivalve reproduction (Sastry 1979). in this
work, neither temperature nor salinity showed a clear relation with
the reproductive cycle of A. ventricosus in the Rada del Puerto de
Pichilingue because partly spawning scallops appear throughout
the annual cycle. Maximum and minimum water temperatures co-
incided with the spawning (histologicaly detected) of August-
September and December, as did the minimum and maximum
values of salinity. The above suggests that the changes in tempera-
ture and salinity may be responsible for triggering spawning, but
did not affect directly the gonadal maturation process.

In this work, the MYI did not show a significant negative
correlation with GSI, but reproductive activity was present year
around. An explanation of this unclear relation of the MYI with Ihc
reproductive activity is that in the Rada del Puerto de Pichilingue
this species uses the available food in the environment more than
inuscle reserves for the gonadal maturation when the food is abun-
dant, and they use the muscle reserves when the food abundance is
poor. A transference of energy from the muscle to the gonad in A.
ventricosus had been suggested by Caceres-Martine/ et al. ( 1990)
and Villalejo-Fuerte and Ceballos-Va/c|ue/ (1996).

Bayne (1976) divided the bivalves into two groups based on
their gametogenic pattern; 1 ) "conservative" species where game-
togenesis occurs from energy stored in the tissue, and 2) "oppor-
timistic" species where gametogenesis occurs when theic \v;is

abundant phytoplankton. In this case. A. ventricosus would be
named both opportunistic and conservative depending on the avail-
able food.

The MYI had a positive correlation with temperature. This may
be the environmental variable that influences the transference of
stored reserves from the adductor muscle to the gonad of A. ven-
tricosus. as happens in A. irradians (Sastry and Blake 197 1 . Barber
and Blake 1981. MacDonald and Bourne 1987). For salinity, a
negative correlation with MYI was observed but the influence of
salinity in the transference of nutrients remained unclear.

Le Pennec and Beninger (1991) observed that through most of
the energy supplied to the developing gametes comes from protein
and glycogen reserves in the adductor muscle, there is also energy
transference from the reabsorption of residual oocytes and from
the transference of nutrients from the intestinal loop to the gonad.
The intestinal loop penetrates into the gonad and has a digestive
function (epithelium with intracellular and extracellular digestion)
and there is a direct transference of the metabolites from the in-

18

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£ 10 ^

6 1
4
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ra

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-•- Total Seston -.- IS/OS Ratio

5,5

5
4.5.

4

35

3

2,5

M

M

JJASOND JF
1995 1996

Figure ,'. Changes in the seston and IS/OS ratio (a), and food index (b)
throughout the annual cycle from the Kada del Puerto del Pichilingue,
B.C.S., Mexico. (Krror bars = SD).

Reproductive Cycle of A. ventr/cosus

111

testinal epithelium to the gonad and therefore to the developing
oocytes. In this work, we found a lot of acid mucopolysaccharides
in the intestinal loop, the mantle, and the perigonadal connective
tissue of the developing gonads (male and female) of A. ventrico-
siis. In the gonads, these carbohydrates can be the result of the
transference from the intestinal loop |Le Pennec and Beninger
1991) or from the mantle (Barber and Blake 1983). In contrast, in
the ripe gonad the acid mucopolysaccharides were few. so we
believe that they were used in the maturation of the gametes.

In the developing and ripe oocytes, we observed a lot of gly-
cogen that probably was the result of the transformation of the acid
mucopolysaccharides. This carbohydrate is converted into triglyc-
erides and is stored in the oocytes to be used as a future energy
source for the larvae (Gabbott 1975). To support this, we found
much unsaturated lipids (oil droplets) in the cytoplasm of devel-
oping and ripe oocytes.

The gross analysis of the seston or the measurement of a single
chemical variable cannot describe fully the nutritive value of
seston. To understand seston as food, it is necessary to determine
its major biochemical constituents (lipid, protein, and carbohy-
drate) (Navarro et al. 1993). These components form the food
material available for scallops and their larvae. TS. IS/OS ratio,
and FI showed no clear relation to the reproductive cycle. It seems
the reproductive cycle was influenced by a combination of the
quantity of food and the muscle reserves.

A. ventricosiis exists in large stocks in the bays of Baja Cali-
fornia Sur (Tripp 1985, Aurioles-Gamboa 1992) but some of these
stocks have been overfished (Chavez-Villalba and Caceres-
Martinez 1992, Caceres-Martinez et al. 1993). This is true in Bahi'a
de La Paz. in which the study area of this work is included. There
is no fishery in this bay now because of the depletion of the A.
venrricosiis population. A management option is the culture of the
species and this idea directed this study. From our results, we can
say that the Rada del Puerto de Pichilingue is not an appropriate
zone for the culture of catarina scallops. This is because the quality
and quantity of food is poor and cannot support commercial pro-
duction. Though reproductive activity was observed throughout
the year as in other locations of Baja California Sur (Baqueiro et
al. 1981, Villalejo-Fuerte and Ochoa-Baez 1993. Felix-Pico et al.
1995), the GSI values in the Rada del Puerto de Pichilingue were
lower (2% less) than those obtained for A. ventricosiis from Bahi'a
Concepcion. B.C.S.. Mexico (Villalejo-Fuerte and Ochoa-Baez
1993).

ACKNOWLEDGMENTS

This study was supported by the UABCS research for the Mas-
ter of Sciences in Aquaculture Program. Special thanks are due to
Javier Cortes Salazar for his technical support during the field
studies. Thanks to Dr. Ellis Glazier (CIBNOR) for editing this
English-language text.

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Journal of Shellfish Research. Vol. 19, No. I. 113-124. 20UU.

BIOECONOMIC ANALYSIS OF A SEA SCALLOP, PLACOPECTEN MAGELLANICUS,
AQUACULTURE PRODUCTION SYSTEM IN NEWFOUNDLAND, CANADA

R. W. PENNEY' AND T. J. MILLS"

' Department of Fisheries and Oceans

P. O. Box 5667

St. John's. Nfld. Canada

AIC 5 XI
'Thimble Bay Farms Ltd.

P. O. Box 381

Botwood, Nfld. Canada

AOH lEO

ABSTRACT We report the results of 2-year pilot scale scallop, Phuopecten nuigelhmicus. culture trials at Charles Arm. in Notre
Dame Bay on the northeast coast of Newfoundland during 1989-1991. We used extrapolations of the growth and survival data from
these trials, as well as records of all capital, labor, and operational costs, to conduct simulation modeling of cash flows associated with
start-up and operation of scallop farms scaled up to commercial size. Our aim was to determine the economic viability of sea scallop
farming in Atlantic Canada using the standard economic evaluation methods of Net Present Value (NPV) and Internal Rate of Return
(IRR), based on production of 55-65 mm (shell height) live, whole, scallops using a suspended pearl net culture system. Two separate
pilot trials were carried out concurrently. In the first, the effect of stocking density and culling on growth and survival were determined
by stocking scallop seed in peari nets at five different starting densities; 50, 75, 100, 150, and 200/net with subsequent culling (thinning)
at two different times during grow-out. In the second trial, the effect of seed grading and net mesh size on growth and survival were
determined by grading seed into two nominal sizes based on shell height, small (< 18 mm) and large (> 18 mm), followed by stocking
in pearl nets of varying mesh size: 4.5, 6.0, and 9,0 mm, at a starting density of 50/net for grow-out. After 2 years of grow-out, size
at age (growth) was significantly related to initial seed stocking density, culling, net mesh size, and seed grading. Survival was
significantly related to initial stocking density, culling, and seed grading but not to net mesh size. The model simulations predict scallop
farming enterprises with vertically integrated culture farms and processing plant operations, and with annual stocking rates of about
1 million or more seed per year, are economically viable in current market and financial conditions. Sen.sitivity analyses indicate farm
business viability is relatively sensitive to changes in sale price received for harvested product, but relatively insensitive to changes
in capital costs, labor, other operational costs, or to mortality. The simulations also predict farm ownership of a processing plant
enhances the economic viability of farming enterprises. These favorable economic projections support the contention that continued
private and government-assisted investment in expansion of a whole scallop culture industry in Atlantic Canada is warranted.

KEY WORDS: Scallop. Placopeclen magellaiiicus. aquaculture, bioeconomics

INTRODUCTION duction of Atlantic sea scallop: ( 1 ) industry dependence on annual

wild seed collection that has proved to be unreliable with wide

The sea scallop. Placopeclen mogellanicus. has been the main- interannual fluctuations in seed settlement (Couturier et al. 1995);

stay of the traditional commercial scallop capture fishery in At- and (2) high production costs for meats that marginalized eco-

lantic Canada for many years. Beginning in the 1960s, consider- nomic viability (Frishman et al. 1980, Gilbert 1987, Gilbert and

able effort has been expended to explore the comtnercial aquacul- LeBlanc 1991, Wildish et al. 1988).

ture potential of the sea scallop (Couturier et al. 1995, Naidu et al. These economic analyses, which focused on "'meat" production
1987) using technology, equipment, and reaiing practices widely as the sole source of farm revenue, cited high meat production
used in the extensive Japanese scallop culture industry (Taguchi costs as the principal obstacle to viability but did not consider the
1977 Aoyama 1989) for the indigenous Japanese scallop. Pali- harvesting and sale of alternative products. However, markets are
nopecten yessoensis. North American markets for scallop products reported outside North America for "meats with roe." whole, live
have traditionally been almost exclusively restricted to the white scallops and various "value-added" products. In Japan, a large-
adductor muscle, or "meats." as they are known in the industry. scale culture industry producing Japanese scallop. Patinopecten
Early efforts to commercialize culture of the giant sea scallop, yessoensis. for sale in a variety of product forms, including whole,
Placopeclen magellaniciis. in Canada's Atlantic provinces were in-shell product has thrived for many years (Ikenoue and Kafuku
based on production of meats intended to compete in these existing 1992). Whole, in-shell queen. Chhiniys operciilaris. and king,
markets. Peclen ma.ximus, scallops are marketed in several European coun-

Development of the culture industry has been slow. Total an- tries (de Franssu 1990, Hardy 1991). but availability typically is

nual Canadian production of cultured scallop has averaged < 100 restricted to markets near fishing ports, because scallops have a

mt since 1993 (Dept. of Fisheries and Oceans Statistics Rept. relatively short shelf life in air (De Franssu 1990). In British Co-

1998). This figure includes both sea scallop. Placopeclen magel- lumbia, live, in-shell, pink scallops, Chlamys nibida. and spiny

lanicus, production from Atlantic Canada as well as production of scallops. C. haslala. < 80 mm in shell height have been supplied

the introduced Japanese scallop, Patinopecten yessoensis. in Brit- in small quantities (< 100 mt per annum) to both domestic and U.S.

ish Columbia on Canada's West Coast. Two factors are considered markets for several years (W. Heath. BC Min. of Fisheries, pers.

to be primarily responsible for the slow increase in cultured pro- comm.). Since 1996, this has been augmented by cultured scallop,

113

114

Penny and Mills

P. xessoensis, production, which has been sold throughout Canada.
the U.S., and Asia. Cultured, in-shell sea scallops are also pro-
duced in small quantities (< 30 mt/annum) from farms in Nova
Scotia and Newfoundland for sale into domestic Canadian mar-
kets.

Beginning in 1992. promotional and market development ini-
tiatives carried out by Thimble Bay Farms Ltd.. have identified a
niche market in Canada and the northeastern United States for a
55-65 mm SH (shell height) sea scallop product, sometimes
termed "princess"" or "•cocktail"" scallops by the seafood industry,
depending upon its intended retail presentation. These are intended
for retail and seafood service industry presentations similar to
those in existing large volume North American markets for soft-
shelled clam (A/ra arenaria). steamers, littleneck. and cherrystone
clams {Mercenaria mercenaria), and oysters (Crassostrea vir-
ginica) (De Franssu 1990). Sale of live, in-shell sea scallops now
account for most of the annual farmed scallop production from the
private company. Thimble Bay Farms Ltd in Newfoundland.

However, efforts to increase market volume have been limited
by unavailability of product attributable in large part to production
bottlenecks caused by unstable seedstock supply. This has limited
total cultured scallop production in Newfoundland to 10-19 mt
annually since 1994 (Dept. of Fisheries and Oceans Statistics Re-
port, 1998). Recent construction of a new scallop hatchery at Bel-
leoram in Newfoundland with an estimated annual production ca-
pacity of 20 million seed (G. Deveau, Ntld. Dept. Fisheries and
Aquaculture. pers. comm.) may resolve the immediate seedstock
supply problem and allow significant expansion in the industry.
This has rekindled industry attention toward production and mar-
keting issues, including whether it is economically advantageous
to invest in market development for whole scallop products.

Harvest and sale of small, in-shell scallops, in particular, may
have a dramatic effect on scallop farm viability. In the United
Kingdom, harvest and sale of small (5-6 cm), in-shell queen scal-
lop, Chlamys opercularis. is considered to be financially advanta-
geous for scallop farmers, because it reduces labor costs associated
with shucking meats and greatly shortens production time (Hardy
1991). This may also be true for sea scallop culture in Atlantic
Canada. Recent consulting studies commissioned by the Provincial
Government of Newfoundland and Labrador seem to support this
contention (Atlaniecon 1992. ARA Consulting Group 199.^). Both
studies developed financial projections suggesting the economic
viability of commercial sea scallop culture might be enhanced, as
compared to meat production, by developing markets for alterna-
tive products, particularly whole scallops < 70 mm in shell height.
The purpose of this paper, is to determine whether continued
private industry and government investment in commercial expan-
sion of this sector is warranted. In this paper, we report the results
of pilot-scale sea scallop culture trials conducted at Thimble Bay
Farms" s leased acreage at Charles Arm in Newfoundland, Canada
during 19X9-1991. These trials sought to determine the biological,
technological, and economic factors associated with producing for
market a whole, in-shell scallop product using a Japanese sus-
pended pearl net culture system. During the pilot trials, scallop
growth and mortality, as well as labor, capital, and operating costs
were recorded during a 2-year production cycle. Data collected
during the pilot trials were used as input into a financial model to
forecast the economic viability of commercial scale farms and,
thus, provide both biological and economic bases for capital in-
vestment decision making for the Atlantic Canadian shellfish cul-
ture induslrv.

This paper has three goals: ( 1 ) to quantify the effects of stock-
ing density, culling during grow-out. initial spat grading, and net
mesh size on scallop growth and survival observed in pilot-scale
culture trials and use these values to define the optimum husbandry
techniques appropriate for future scaled-up commercial opera-
tions: (2) to conduct model simulations forecasting the economic
viability of commercial-scale sea scallop culture farms utilizing
production methodology similar to that used in the pilot trials to
produce a 55-65 mm whole scallop: and (3) to assess the impact
of vertical integration (e.g., farm ownership of a processing plant
along with the culture farm) on projections of economic viability
for scallop farming enterprises.

METHODS

Culture Trials

Pilot-scale culture trials were conducted at Charles Arm. in
Notre Dame Bay on the northeast coast of Newfoundland. This site
is one of two shellfish production areas leased by Thimble Bay
Farms Limited, a private shellfish aquaculture company specializ-
ing in sea scallop and blue mussels. In October, 1989, 50,000
approximately 1 -year-old sea scallop seed, originating from stocks
in Port au Port Bay in western Newfoundland were purchased and
transferred to the Charles Ann site. Scallop seed were stocked into
standard 34-cm square Japanese pearl nets. The pearl nets were
hung using a longline, suspended culture system in vertical arrays
of 10 nets (Fig. 1). Each vertical array was repeated at 0.7-m
intervals along a horizontal subsurface headline suspended at 3-m
water depth and supported by surface floats.

Two separate trials were carried out concurrently. In the first,
scallop seed were stocked at five different starting densities: 50,
75. 100. 150. and 200/pearl net. All nets were 6-mm mesh size.
During the first year of culture in May, 1990, and again, in Sep-
tember, 1990, some of these nets were selected for culling (thin-
ning), while others were left unculled. The nets originally stocked
at 50/net were culled to 25/net, while all others were culled to
50/net. In the second trial, seed were graded into two sizes based
on shell height, nominally referred to as small (< 18 mm) and large
(> 18 mm). The graded seed were stocked in pearl nets of varying
mesh size: 4.5, 6.0. and 9.0 mm. at a starting density of 50/net,
with the exception of the small size grade which, because of their
small size, could not be stocked into the 9-mm nets, because they
readily fell through the mesh. All experimental trials were repli-

Floats

Bottom
Contour

Rock Anchors Pearl Nets

Fisure I. Diajjranimatic cross-sectional representation of the longline
seallop eiillure system used in the pilot trials at Charles Arm, New-
I'oundiand.

BioECONOMic Analysis of P. magellanicus in Newfoundland

115

cated such that each trial had a minimum of six pearl nets (some-
times as many as 10) in each category. In May and September of
both 1990 and 1991 (September. 1990 and 1991 only for the size
grade-mesh size trial), all pearl nets were retrieved and cleaned by
a pressure washer, all scallops were measured for shell height,
counted, and all mortalities were removed.

SAS statistical software (SAS Institute. Inc. 1985) was used for
all statistical analyses of the biological data from the pilot trials.
We used a nested, main effects analysis of covariance model
(ANCOVA. SAS Institute Inc.. 1985) to determine the relation-
ships among stocking density, culling, size at age (shell height).
and survival on each sampling date. Initial starting size of seed-
stock was the covariate to control for initial variation in shell
height among pearl net groups.

Economic Model Parameter Selection

During the culture trials and including the post-trial harvest in
September. 1991, records were kept of all capital and operational
costs, as well as all labor incurred. These records, as well as the
pilot trial growth and survival results, were used as input data to a
spreadsheet-based Lotus'"''^' financial model (Table I ) and extrap-
olated to commercial scale to simulate the startup and operational
costs of commercial-scale farms and to forecast their economic
viability using the standard financial evaluation methods of net
present value (NPV) and internal rate of return (IRR) (Lusztig and
Schwab 1977). All equipment, supply, and labor costs were
sourced from commercial equipment suppliers as of March. 1999
and are quoted in Canadian dollars. Estimates of useful life span of
various equipment were based on practical experience of Thimble
Bay Farms. The purchase price of scallop seed and the sale price
of harvested product are the most recent values quoted for Thimble
Bay Farms. Ltd.

Selection of specific husbandry practices used in the models
can have a major impact on the outcome of the model simulations.
To ensure parameter values selected were as realistic as possible,
we used the results from the stocking density-culling and net
mesh-seed grade trials to select appropriate model input values for
several key parameters. These included net mesh size, time to
harvest for each seedstock cohort, the annual production cycle,
stocking density, survival rate to harvest, and frequency of culling
and handling for net cleaning.

Although a larger mesh size is expected to yield a faster growth
rate, particulariy in the second year of the production cycle, the
6-mm mesh is the largest mesh size capable of accommodating the
smallest of the purchased seedstock (10-15 mm) in year 1. Any
economic advantage attributable to slightly faster growth in 9-mm
mesh nets, as compared to the 6-mm mesh, is outweighed by cost
considerations because of the need to stock nets of two or more
mesh sizes, the utility of which will vary annually, depending on
interannual variations in shell height of the seedstock supply.
Therefore, use of the 6-mm mesh size was assumed in the simu-
lation modeling exercise.

The minimum time to harvest for each seedstock cohort was set
at 15 months. Because the harvesting schedule must be year round,
the annual production cycle from each annual seedstock cohort
was set at January (year 2) to January (year 3) or, in other words,
a 15-27 month production cycle. This production schedule was
determined by analysis of the variability in the size at age data
from the pilot trials.

Assuming no seasonality in the harvesting schedule, we se-

TABLE 1.

Selection of key model parameter values used in the economic
model simulations.

Key Model Parameters

Pearl net (square) specifications:
Cage/mesh size
Stocking density (% of stock

' # per net)

Net clean (# of times per year)

Culling/thinning of stock

Cost/life span (years) of capital equipment;

Pearl nets (bulk order)

Mainline. 365-m coil. 16-mm polypropylene

Anchor and float lines. 365-m coil, 19-mm
polypropylene

Pearl net droplines. 365-m coil. 7-mm polypropylene

Floats. 34 cm

Floats, 200 L

Work boat, 6.8-m aluminum

Outboard motor, 40 hp

Boat eqmt.. star wheel and hydraulics

Vehicle, '/: ton pickup with cap

Processing plant/work shed, 9.3 x 6.2 m

Plant water pumps
Hourly labor rate
Owner/manager's annual salary
Per unit fuel cost (liter, gasoline)
Unit cost of autumn-delivered Spat ( 10-25 mm)
Survival rate to harvest
Time to reach harvest size
Harvest schedule
Product specifications;

Market required product size (shell height)

Ex-plant, per unit scallop sale price
Business and startup fees (Year 1 )
Crop insurance (per million stock)
NPV discount rate (prime + 2%)

34 cm/6 mm
50% @ 25
25% @ 50
25% @ 75
1
None

Sl.80/10
$98.60/8

$233.10/8
$29.00/8
$10.50
$35.00
$9,400/15
$3,695/5
$4,000/5
$23,800/5
$19,300/20
$2,000/5
$8.50
$18,000
$0.60
$0.04
85%
15-27 months
Year Round

55-65 mm

$0.25

$9,205-11.505"

$4,000

8.75%

■' Varies with farm size.

Individual equipment costs were obtained from commercial supply
sources. All other values were obtained from analysis of the pilot trial data
or from Thimble Bay Farms Ltd. records.

lected the following stocking scenario for use in the model simu-
lations: 50% of seedstock would be set at 25 scallops/net; 25% at
50/net: 25% at 75/net. Based on the growth data from the pilot
trials, this stocking scenario should ensure year-round availability
of a 55-65 mm product for harvesting and minimize the likelihood
of scallops exceeding the maximum product size specification be-
fore being harvested. Although the pilot trials had no peari nets
initially stocked at 25/net, we consider the data from the 50/25
stock culled in May 1990 to represent a reasonable estimate of the
probable growth performance of scallops initially stocked at 25/net
for use in the model simulations. However, because scallops
stocked at 25/net would exceed the maximum acceptable market
size in less than 27 months, to ensure year-round availability of
55-65 mm product some seed scallops must be stocked at higher
densities. Analysis of the variability in the size at age data from the
pilot trials indicated unculled peari nets initially stocked at 50 or
75 scallops/net best matched the required market size during the
15-27 month production cycle.

116

Penny and Mills

In the pilot trials, all experimental groups with stocking of
50/net or less achieved survival rates > 85%. Most were > 90%.
Therefore, we considered a survival rate of 85% to be a reasonably
conservative estimate of survival for the model simulations.

Ideally, operational costs are minimized by selection of hus-
bandry practices that allow individual scallops to be handled as
little as possible during the production cycle. Because there are
indications from the pattern of survival data, as well as from other
concurrent farm operations, that excessive handling has a negative
impact on survival, we chose no culling as the preferred production
method for the simulations. This also lowered labor costs. For the
model simulations, it also necessitated optimizing production
solely by varying initial seed stocking density rather than by a
combination of stocking density and culling.

The discount rate for NPV calculations was the small business
cost of borrowing, as of March. 1999, used by the Canadian bank-
ing industry and is calculated as bank prime rate -i- 2%. A 50:50
split between bank loans and owner equity for capital infrastruc-
ture and equipment as well as an operating line of bank credit with
a monthly repayment schedule of 3% of the outstanding balance is
assumed.

Economic Model Simulations

We selected three hypothetical commercial farm sizes, based
on annual seed stocking rates, for the model simulations: 500,000
('/: M), I million (1 M), and 3 million (3 M) seedstock per year.
The half million size model represents a farm size consistent with
a part-time or family operation worked as an income supplement;
whereas, the other two represent possible full-time commercial-
scale farms consistent with the amount of leased acreage currently
utilized by shellfish farms in Newfoundland. Model simulations
assume a year-round market requirement for 55-65 mm SH. live,
whole product that is fully processed in accordance with all ap-
plicable Canadian seafood processing regulations in a farm-owned
federally registered processing plant.

The spreadsheet-based financial model (Lotus"") forecasted
the potential economic viability of each of these three model farm
sizes using the NPV and IRR values. We used a sensitivity analysis
procedure to simulate the effect of variability in specific model
input parameters on the model output. For the sensitivity analyses,
we used an iterative procedure, changing the value of the most im-
portant input variables (as a proportion of cash outflow) individu-
ally by a pre-scl percentage until the NPV at year 10, NPV 10 =0.

To assess the effect of vertical integration (e.g., culture farm
plus a farm-owned processing plant) on over-all economic viabil-
ity of scallop fanning enterprises, we recalculated the model simu-
lations with the capital and operational costs of the processing
plant deleted. This farm model requires assumption of sale of
unprocessed scallops to an ex-farm seafood processor. We used an
iterative process, adjusting the ex-farm price for harvested scallops
in $0,005 intervals to determine the ex-farm price for unprocessed
scallops needed to: ( 1 ) achieve minimal standards of economic
viability (e.g., 0 < NPVIO < $1000: 8.75% < IRR 10 < 9%); and (2)
achieve economic viability projections for farms without the pro-
cessing plant comparable to those for the same size farm with the
processing plant included.

RKSULTS

Peiisily and Culling Trials

At the outset of the culture trial, the mean shell height of all
groups ranged from 19-21 mm (Fig. 2). Beginning with the first

sampling in May. 1990, shell height was significantly related to
stocking density (P < 0.0001). This relationship was maintained
through all sampling periods. The pattern in least-square means
(LSM) among the five initial stocking densities was also signifi-
cant (f < 0.01 or greater) and consistent across all stocking den-
sities (LSM^,, > LSM75 > LSM|„o > LSM,.,,, > LSM.oo)-

Shell height was also significantly related to culling {P <
0.0001 ). The LSMs of culled (thinned) scallop groups were con-
sistently larger in shell height than their unculled counterparts {P
< 0.0001) at the same initial stocking density (LSM^.„„,„,.,,,y,i >
LSM^.^,i..,epc,„ > LSM„„,.„|,^.j). The interaction term of stocking den-
sity X culling date was also significant (P < 0.0001 ) throughout the
sampling period. By the end of the second year of the trial (Sep-
tember 1991) an increase in shell height attributable to culling
(Table 2) was noted at most initial stocking densities. In Fig. 2, the
slope of the lines between adjacent sampling times indicate the
mean growth rate during that interval. Growth rates were highest
during the May to September period, 1990 (first summer season)
and declined considerably thereafter. The highest mean growth
rates, observed in the May 1990 cull group, ranged from 0.142 to
0.176 mm day"', depending upon stocking density, during this
time.

The effects of initial stocking density and culling on survival
were less consistent (Fig. 3). Survival was significantly related to
both initial stocking density and culling date (P < 0.0001 ). How-
ever, the pattern in LSMs was inconsistent among initial stocking

densities (LSM^,, = LSM,

= LSM,,u > LSM75 > LSM,„„).

LSM patterns with respect to culling date was also inconsistent
(LSM,„,|.,,,,c„ > LSM,,.„.,,p«„ = LSM„„,„„,,,) although the May
1990 cull group were consistently larger than the other two groups.
All except the unculled 200/net group had mean survival rates >
80% at the end of the pilot trials. Most exceeded 85%. Overall, the
change in survival attributable to culling was much less pro-
nounced than that for shell height among the experimental groups
(Table 2).

Maximum growth was achieved in the 50/25 cull groups. In
these groups, more than 90% of all scallops were greater than the
minimum acceptable market size by May of Year 2 in the produc-
tion cycle. Back-calculation of the size at age data from May in
Year 2 ( 1991 ) based on the mean monthly growth rate during the
September, 1990 to May, 1991 period projected that 90% of the
50/25 stock culled in May 1990 were probably in excess of the 55
mm minimum market size in January of Year 2 (1991). Thus, the
minimum time to first harvest is approximately 15 months.

Seed Gradin/i and Mesh Size Trials

We used a similar anahtical approach (o determine the rela-
tionships among seed grading and net mesh size with size at age
(shell height) and survival. When graded, the mean shell height of
scallop seedstock in the two nominal size grade categories were
15.3 mm (small grade) and 22.5 mm (large grade). At the end of
Year 2 of the pilot trial in September, 1991, size at age was
significantly related to both mesh size {P < 0.0001 ) and initial size
grade (/-" < 0.03l. The interaction term was not significant {P >
0.05 ). All groups exceeded 45 mm shell height by the end of Year
I and exceeded 60 mm shell height by the end of Year 2 (Fig. 4a).
Increasing mesh size had a positive effect on mean size at age for
both size grades. Howe\er, the mean shell heights of small size
grade groups were sometimes larger at the end of 'tear 2 compared
to large size grade seed in nets of the same mesh size (LSM,,, =
LSM„s > LSM,,, > LSMj ,s = LSM^ „ ).

BioECONOMic Analysis of P. magellanicvs in Newfoundland

117

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91

Figure 2. Mean size at age (shell height) over time of scallops in the stocking density-culling trials. The figure legends follow the convention
"original stocking density / culled density, and date of culling" (e.g., 50/25 May90 means original stocking density = 50, culled density = 25, and
May 1990 was the date of culling). Standard error bars (± 2 SE) are plotted but are obscured by the datapoints.

118

Penny and Mills

TABLE 2.

Percentage i%) change in mean shell height and mean survival at

Year 2 attributable to culling in Spring (May) and Autumn

(September) of Year 1, compared to the unculled stock of the same

initial stocking density. Stocking density values are number of

scallops net^'.

Shell

Height

Surv

ival

Stocking Density

May Cull

Sept. Cull

May Cull

Sept. Cull

50

16.2

14.2

0.5

-2.7

75

2.5

2.6

12.0

5.3

100

20.2

L<i.7

-3.7

-6.7

150

29.4

22.7

0.1

4.4

200

42.5

33.9

24.4

5.7

Survival through Year 2 was not significantly related to net
mesh size (P > 0.05) but was weakly related to initial size grade
(P < 0.04). The small size grade experienced a relatively lower
survival during the trials (Figure 4b) than did the large grade

(LSM^L = LSM4 ,

LSMft, > LSM,

LSMj ,s). Survival

through Year 2 in all groups exceeded 83%, while mean survival
of the large grade exceeded 91%. In September, 1991, the ob-
served mean size at age and mean survival of scallops in the mesh
size-seed grade trials were comparable to those observed in the
stocking density-culling trials.

Effect of Farm Size

The key parameter values used as input to the model simula-
tions are given in Table 1. Net cashflow projections for all three
farm sizes followed similar patterns of an initial cash investment in
business startup (year 0), a further negative net cash outflow in the
first year of operation, followed by a series of positive net cash
inflows in subsequent years, the magnitude of which increased
with increasing farm size (Fig. 5). Net present value (NPV) and
internal rate of return (IRR) values derived from the model simu-
lations indicate both the 1 M and 3 M farms are projected to be
economically viable using a 10-year forecast horizon at present
commercial bank interest rates and market prices (Figure 6). The
smaller ('/: M) farm size is not considered economically viable.
The model simulations predict a trend of increasing NPV and IRR
values with increasing farm size indicating the influence of
"economy of scale" in farm operations.

Annual labor and .seedstock acquisition costs represent > 50%
of the total cash outflow for all farm sizes (Fig. 6). Acquisition of
capital equipment and infrastructure is a relatively smaller propor-
tion of cash outflow when annualized over the 10-year model
simulation cycle. However, much of the cost for capital equipment
and farm infrastructure are concentrated in Year I (processing
plant, work boat, culture equipment, etc.). Labor and debt servic-
ing costs as proportions of total cash outflow over a 10-year cycle
do not vary with farm size. However, acquisition of capital equip-
ment and operational costs both decline proportionally with in-
creasing farm size; whereas, purchase of annual seedstock propor-
tionally increases over a 10- year period. The payback period,
defined as the time to recoup the initial investment assuming op-
erating profits arc retained within the business, is estimated al 4.2
and 3.4 years for the 1 M and 3 M farms, respectively.

Sensitivity Analyses

To determine how robust our viability projections from the
model simulations were, we recalculated the sinuilalions for the

two model farm sizes deemed economically viable with the base
input assumptions (1 M and 3 M farms). We used an iterative
procedure, changing the value of each of the most important input
variables (as a proportion of cash outflow) individually by a preset
percentage until the NPV 10 = 0. Projections of economic viability
for both the 1 M and 3 M farm models are relatively insensitive to
changing value assumptions for most major input variables, in-
cluding capital, operational costs, and mortality (Fig 7). However,
both models are relatively sensitive to changes in sale price. Re-
duction in sale price obtained for harvested product in the order of
20% and 28%, for the 1 and 3 M farms, respectively, reduced the
NPV to zero. This is equivalent to a minimum sale price of $0.20
and $0.18/scallop, respectively.

Effect of Farm-Owned Processing Capacity

Without the processing plant, the '/: M farm is still not con-
sidered to be viable economically (NPV 10 < 0; IRR 10 < 8.75%)
under assumptions of current scallop sale prices. Farms of this size
only become marginally economically viable (e.g., 0 < NPVIO <
$1000; 8.75% < IRRIO < 9%) if the ex-farm sale price for un-
processed scallops exceeds $0.26/scallop. a price that exceeds the
current sale price for processed scallops. For the 1 M and 3 M
farms without processing plants, economic viability becomes mar-
ginal as the ex-farm sale price for unprocessed scallops approach
$0,185 and $0.165/scallop, respectively. To achieve economic vi-
ability projections comparable to those for farms with processing
plants (equivalent NPV or IRR), the sale price for ex-farm unproc-
essed scallops must exceed $0.235/scallop for both the I M and 3
M farms, a difference of only S0.015/scallop for unprocessed ver-
sus processed scallops at current prices. Obtaining such a small
price differential (approximately 6%) for sale of unprocessed scal-
lops to an ex-farm processor may not be realistic, because it would
seem to allow a rather small profit margin for the processor. With
this considered, scallop fanning enterprises with owner-operated
processing capacity are likely more economically attractive than
farms without owner-operated plants.

DISCUSSION

In commercial production systems, growth and survival are the
two major biological rates of importance to cultured seafood grow-
ers. For bivalve mollusks, many factors influence these two vari-
ables. Some are environmental, such as food availability and water
temperature, and others are physiological related to age, size, and
reproductive maturity of the animals themselves (see Shumway
1991 for review). For suspended culture systems, additional stock
husbandry factors must also be included, such as gear depth, type
of gear and mesh size, current velocity, stocking density, and
extent of biofouling (Claereboudt et al. 1994a, Claerboudt et al.
1994 b. Cote et al. 1993, Parsons and Dadswell 1992, Parsons and
Dadswell 1994, Shellfresh Farms Ltd. 1993).

In this paper, we have examined the effect on scallop growth
and survival of the major variables that can be readily manipulated
by scallop farmers, assuming use of a basic pearl net culture sys-
tem styled after the equivalent Japanese industry for the Japanese
scallop. Palinopccten yessoensis. These are selection of stocking
density, gear mesh size, culling (thinning) practices, and seed grad-
ing. In their review of sea scallop culture in Atlantic Canada,
Couturier et al. (1995) considered stocking density the single most
important factor affecting cultured sea scallop growth rates. Al-
Ihough it is tlifficult lo compare growth rates, size at age. or sur-

BioECONOMic Analysis of P. magellanicus in Newfoundland

119

May

Sep
90

DATE

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— ■A— 75«)Sep90

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oo

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S

ay Sop
1 91

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Figure 3. Mean survival over time of scallops in the stocking density-culling trials. The figure legends follow the convention "original stocking
density / culled density and date of culling" (e.g.. 50/25 May90 means original stocking density = 50, culled density = 25, and May 1990 was the
date of culling). Standard error bars are ± 2 SE.

120

Penny and Mills

(a) --i'^
,'■■//

* 4SSniall

- -a - 6/Snnall
--*--45njirge

- ->•- ■ aurge
--.-aLarge

Sep-QO
DATE

096

096

(b)

\ \
\ \
\ \

*N ""v

;

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1

y 092

\ \

\ \
\ \
\ \

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0$4
082
08

- -D - GTSmall

- - * • -45fl^rge

- ->e- - e/Large
-■■-gfl^ge

Sep-90
DATE

Figure 4. Mean size at age (shell height) and mean survival of scallops
in the spat grading-net mesh size trials. The figure legends follow the
convention "net mesh size in millimeters / nominal spat size grade at
time of stocking" (e.g.. 4.5 / small means mesh size 4.5 mm and stocked
spat were small grade). Nominal spat size grades were < 18 mm (small)
and > 18 mm (large). Standard error bars are ± 2 SE but are obscured
by the datapoints in (a).

vival data among different studies, gear, and locations, the growth
and survival rates observed in this study seem consistent with
those reported for sea scallops in suspension culture grown else-
where in Atlantic Canada (Cote et al. 1993, Dadswell and Parsons
1991. Parsons and Dadswell 1992. Parsons and Dadswell 1994.
Wildishelal. 1988).

In our study, size at age and survisal varied with stocking
density, gear mesh si/e. culling practices, and seed grading. Both
size at age and survival tended to decrease with increasing stock-
ing density, a result consistent with previous studies on several
scallop species including the .lapanese scallop. PuUno\H'cU'n yes-
soensis (Yamamoto 1978. Ventilla 1982). the bay scallop. Ar-
gopi'cten inadiaits (Duggaii 1973, Rhodes and Widman 1984),
and the sea scallop. I'Uuopeili'u imifU'lUmkus (Cole et al. 1993).
However. Penney ( 1 99.S ). in a study ol large scallops > 75mm shell
height, did not find a significant relationship between slocking
density and survival. Parsons and Dadswell (1992) also found
survival in sea scallops from New Brunswick to be unrelated lo
stocking density. In the present work, nonrandom handling moi-

Figure 5. Annual net forecasted future cashflows over a 10-year pe-
riod from initial startup for three sizes of sea scallop farms with farm-
owned processing plant included: ": million seed yr"', 1 million seed
yr"', and i million seed yr '. Year I) is the initial capital investment
before startup.

talily, an artifact of our "hatch processing" style sampling proce-
dure, was likely implicated in the inconsistent survival patterns
among the different density groups observed in the pilot trials. This
might explain the anomalous high mortality among the 75/net
groups. Similar instances of nonrandom apparent handling mor-
tality among adjacent groups were noted periodically during the
farm's other commercial operations. We, therefore, consider ex-
cessive or improper handling of gear lo be a more imporlanl factor
influencing survival than stocking density, a finding that would be
consistent with that of other studies (Parsons and Dadswell 1992,
Ventilla 1982, Wildish et al. 1988).

In this work, size at age in pearl net culture was improved by
early culling and by increasing the initial net mesh size. Survival
was also improved by early culling but was unrelated to net mesh
size. Larger sized seed did not maintain their size at age advantage
over small grade seed after 2 years of grow-out. This suggests shell
height variation in I -year-old seed scallops from wild sources is

BioEcoNOMic Analysis of P. magellanicus in Newfoundland
500,000 Seed / Year ^

121

IRR5 = -9.9%
NPV5 = -$78,000

IRR10 = 8.5%
NPV10 = -$1,800

Operations
19%

Capital Equipment
18%

1M Farm

—Sale Price

- - Labor

■ ■ Seedstock

- Other O & M

- — Mortality
—Cost of Peari Nets

-•Total Capital Cost

100 ISO

NPV ( % OOO's)

1 Million Seed / Year

IRR5= 10.2%

IRR10 = 25.8%

NPVS = $9,500

NPV10 = $237,000

Operations

18% ^„g«^^S

rr*?**,,^ Seed

/^fl

::-:-x:x^xN. 2®*

D btS / **iilll^H

"M^^^M^y^

11%

Capital Equipment
16%

3 Million Seed / Year

IRRS = 26.2%
NPV5 = $308,800

IRR10 = 39.4%
NPV10 = $1.12 Million

Operations

15%

Capital Equipment
15%

Figure 6. Mean annual cash outflows by category over a 10-year cycle,
with NPV and IRR values for model simulations of three sizes of farm
operations. (NPVS = NPV calculated over 5 years, etc.). NPV and IRR
calculations were based on cashflows from Figure 5.

0 200 «0 600 800 1000 1200
NPV ( S OOO's)

Figure 7. Sensitivity analysis of the effect of changing value assump-
tions of the major model input variables (reduction in sale price; in-
crease for all cost variables and mortality) on projected NPVIO values
(NPV calculated over 10 years) for the I M and 3 M farm models.
Percentage change in input variables at NPV = 0 indicate the propor-
tional change from the base values for each variable required to re-
duce NPVIO to zero.

likely the result of variation in environmental factors or seed col-
lection husbandry practices rather than within population genetic
variation. However, seed grading before initial stocking may have
commercial value as a means to reduce size at age variation within
individual pearl nets at harvest time.

Selection of production practices for the model simulations was
guided by two general considerations; ( 1) the intended product was
a 55 to 5 mm, whole, live scallop; and (b) harvesting and sale of
product was required year-round with no seasonality in production
volume. Ensuring year-round availability of a 55 to 65 mm product
requires adjustment of farm production practices, including selec-
tion of stocking densities, deciding whether or not to cull and
when, and selecting a harvesting schedule to minimize labor costs,
while still meeting market demands. Variable stocking densities
are required to ensure correctly sized stock are available for har-
vest throughout the year. Economic viability is optimized by early
generation of sales revenue, achieved through timely harvesting.

122

Penny and Mills

However, harvesting should not be so early that within-net shell
height variation is such that a significant proportion of scallops in
harvested nets are under the minimum acceptable market size. This
situation would necessitate the return of large numbers of under-
sized scallops to pearl nets for further on-growing, resulting in
added labor cost.

It would seem unlikely that further minor changes to the basic
pearl net culture system or husbandry practices are capable of
affecting significant improvement in labor costs and, hence, the
outcome of the model simulations. The sensitivity analyses indi-
cate the viability of model farms is relatively insensitive to
changes in capital costs, labor, or other operational costs. Further
significant reductions in labor costs are likely to be achieved only
by substitution to another culture gear entirely or by use of in-
creased mechanization during farm operations. Parsons and Dad-
swell (1994) suggested that, although the initial capital cost of
pearl nets was much lower compared to lantern nets, when han-
dling times and their associated labor costs were factored in, lan-
tern nets and Shibetsu nets gave the lowest over-all cost of pro-
duction. However, this suggestion must be viewed with some cau-
tion. In a previously reported scallop rearing trial in Newfoundland
using scallops > 75 mm shell height, scallops raised in pearl nets
were larger than those raised in lantern nets at comparable stocking
densities (Penney 1995). In the same study, survival was unaf-
fected by gear type.

The reason for the better growth in pearl nets is unknown, but
it may be attributable to differences in water flow (and, hence,
food availability) around and within the two net types. All sus-
pended net culture systems impede water flow, which, in turn,
negatively affects production, a condition that is exacerbated by
increased stocking density, reduced net mesh size, and biofouling
(Claereboudt et al. 1994b. Devaraj and Parsons. 1997. Parsons and
Dadswell 1994). Pearl nets are estimated to reduce water flow by
46-61'^ (Claereboudt et al. 1994b), but no comparative measure-
ments are available for lantern nets. The better growth of scallops
in pearl nets compared to lantern nets (Penney 1995) support se-
lection of the basic pearl net system as the more appropriate net
type compared to lantern nets. In addition, some growers find large
lantern nets clumsy to handle from small boats similar to those
used in our simulations.

Projections of economic viability derived from the model simu-
lations indicate commercial sea scallop farms marketing a whole,
55-65 mm product can be profitable enterprises in Atlantic
Canada. Our favorable projections are in sharp contrast to earlier
economic analyses for culture systems based on production of
adductor meats alone for sale into traditional North American scal-
lop markets (Frishman et al. 1980. Gilbert 1987, Wildish el al.
1988. Gilbert and LeBlanc 1991 ).

These conflicting economic projections for in-shell versus meat
production are likely because of a combination of factors. Rev-
enues from in-shell product sales begin about 15 months after
stocking in our model simulations. In contrast, production of meats
in the .■^0-40 count range (North American scallop markets quote
in number of pieces to make one pound weight) would require an
extra 15 to 20 months of culture (Penney 1995; Penney and Mc-
Kcn/ie. 1996) and would likely generate less ex-farm revenue per
scallop at current North American market prices (L'rner Barry
1999). This protracted culture time woukl also increase capital
costs, because extra pearl nets and other gear are required for each
annual seed cohort, as well as increase labor costs for stock thin-
ning, gear deployment, and in-plant moat shucking.

In Newfoundland, only two companies are currently in com-
mercial production although this will likely increase quickly, be-
cause a total of nine companies and 1 1 culture sites in various
stages of development totaling nearly 400 leased hectares are now
in operation (G. Deveau. Nfld. Department of Fisheries and Aqua-
culture, pers. comm.). Recent annual scallop production by the two
farms currently selling cultured scallops has varied since 1994
from 10-19 mt, the majority of it marketed in whole form. Esti-
mated annual production from a single 1 M farm would be about
17 mt. This is approximately 10-20'^ of all in-shell scallop prod-
ucts currently being sold in Canada from Canadian sources.
Clearly, considerable developmental marketing initiatives would
be required by industry to expand significantly North American
market share for in-shell scallop products sufficient to absorb the
production of a new Atlantic industry composed of several such
farms. Alternatively, the potential for increased development of
other international export markets into such countries as France,
with an existing tradition of acceptance of alternative scallop prod-
ucts (de Franssu 1990) should be determined.

A trend of increasing NPV and IRR values with increasing
farm size indicates significant economies of scale accrue to larger
farms. Despite farm size, annual labor and seedstock acquisition
costs are the largest factors in over-all annual cash outflows. The
cost of labor has been previously recognized as an important com-
ponent of over-all production costs for scallop culture (Atlantecon
1992. Parsons and Dadswell 1994). In our model simulations,
labor is reduced through elimination of the need for culling during
grow-out by selecting appropriate initial stocking densities. This
tends to improve the over-all survival rate as well. Larger sized
farms ( 1 M and 3 M models) are projected to be more profitable
than smaller operations ('/2 M model).

In Atlantic Canada, many shellfish culture farms, particularly
in mussel and oyster culture, have been started as "cottage-style"
ventures operated on a part-time basis as a source of supplemen-
tary family income by persons employed in other industries. If
started by families already employed in the fishing industry, eco-
nomic viability forecasts using NPV or IRR calculations typically
remove from consideration certain capital costs (e.g., cost of boat,
motor, truck, ropes, etc.) that are shared with the fishing enterprise
(e.g.. Ridler 1995). These capital costs are considered to have been
already compensated by the fishing enterprise. The ', : M model is
sufficiently small in scale to be considered this way. If calculated
using these assumptions, the V2 M model is forecasted to be eco-
nomically viable (NPV = $34,000: IRR = 16.59^).

Of particular interest for industry development purposes, is the
effect the owner-operated processing plant has on projected eco-
nomic viability. Vertical integration and increased farm size are
known to have a positive effect of the viability of other shellfish
aquaculture operations (Adams and Pomeroy 1992, Lambregts et
al. 1993). For scallop farming enterprises, irrespectixe of farm
size. NPV- and IRR-bused projections of economic viability
changed only slightly with elimination of the owner-operated pro-
cessing plant as part of the over-all enterprise. This is attributable
to two factors. First, the capital investment in processing capacity
is quite small for processing whole scallops (see Table 1 1. Because
product processing of whole scallops consists of a fairly simple
process of washing and cleaning shells, sorting empty shells, and
packaging, a relatively small building with minimal equipment is
needed. Second, the labor costs for such a simple processing op-
eration are also relatively minor. Processing in-shell scallops

BiOECONOMic Analysis of P. magellanicus in Newfoundland

123

eliminates the need for shucking, which is the most labor-intensive
component of in-plant scallop "meat" processing.

For either the 1 M or 3 M farms, comparable NPV and IRR
values were projected for enterprises with and without processing
capacity at a sale price difference of only $0,015 per scallop for
ex-farm processed scallops versus ex-farm unprocessed scallops. It
seems unlikely that ex-farm processing companies would pay to
the farmer such a small price differential for unprocessed scallops,
because this leaves them a very tight margin for their own capital
and operational processing costs and potential profit. It is far more
likely that processing companies would pay farmers a lower price
for unprocessed scallops, which would have a negative impact on
the economic viability projections for farming enterprises. Thus,
incorporation of an owner-operated processing plant as part of the
business venture would be likely to enhance business viability.
Ex-farm prices for unprocessed scallops in the range of $0.16-
$0.18/scallop would make scallop farming not economically viable
under current conditions regardless of farm size. Nevertheless,
product processing with subsequent direct sale of product to sea-
food buyers and brokers represents a level of business manage-
ment and marketing activity that some prospective farmers may
not choose to pursue. This may be especially true for the Vz M farm
size model that may be a part-time or family operation.

Favorable NPV and IRR projections are not the only criteria
upon which to evaluate the potential for success of any new busi-
ness venture. It should be recognized that many other factors can
and do influence individual business investment decisions that are
not considered in NPV or IRR calculations. Other factors, such as
timing of large cash outflows versus revenues, debt repayment
schedules, other financing arrangements, personal, biological, le-
gal, and regulatory considerations, all may vary on an individual
business and location basis and may also affect the success of any
business venture (Lusztig and Schwab 1977).

Both the NPV and IRR financial forecasting methods used in
our analyses are based on the estimation of future cash flows
generated by an initial capital investment and are commonly used
as decision-making tools by financial analysts to guide investment
in new businesses. Both give explicit consideration of the time
value of money, incorporated through the discounting of cash
flows, which is often related back to the cost of credit (borrowing)
from banks. Accurate forecasting of future cash flows, the basis of
successful NPV and IRR applications, is often a challenge (Lusztig
and Schwab 1977) and cannot anticipate aperiodic potentially cata-
strophic events, such as disease outbreak, major loss of gear be-
cause of ice damage, etc. Despite these indi\ idual situational con-
siderations, favorable general NPV and IRR values such as we
have forecaste from our model simulations indicate the underlying
potential profitability of sea scallop farming in Atlantic Canada.
Our positive NPV and IRR projections for in-shell scallop farms
indicate continued industry and/or government investment to en-
courage commercial expansion in this sector is warranted.

Sensitivity analyses indicate the forecasted profitability is fairiy
robust with respect to anticipated variability in capital and oper-
ating costs, and stock mortality, but is relatively sensitive to fluc-
tuations in sale price for harvested product. This last point must be
closely considered in the start-up of any commercial business ven-
ture of the scale outlined by the model simulations. Greatly in-
creased product availability in the marketplace may exert down-
ward pressure on prices, particularly in the presence of inadequate
marketing efforts. Given the in-shell nature of the product, prices
may not be affected by trends in market prices for traditional

scallop meats, a critical point considering the continuing increase
in Chilean and Chinese cultured meat production as well as past
fluctuations in both price and supply of meats from the North
American fishery (de Franssu 1990). In existing North American
markets, an in-shell sea scallop product would be more likely to
compete (and, hence, to be affected by price fluctuations) with
soft-shelled clam {Mya arenaria). steamers, littleneck. and cher-
rystone clams {Meirenaria mercenaria). and oysters (Cnissostrea
virginica).

We consider further development of a sea scallop farming in-
dustry in Atlantic Canada to be constrained by four factors; (1)
availability of a reliable large-volume annual seedstock supply at
commercially acceptable prices; (2) market development neces-
sary to substantially increase the current, small volume niche-
market status of North American markets for whole, 55-65 mm
products; (3) the reported short shelf life of live scallops (de
Franssu 1990) and; (4) the long-term frequency and severity of
shellfish site closures because of outbreaks of biological toxins.
The first two are inextricably linked. Resolution of the seed supply
problem that has plagued industry expansion for years (Couturier
et al. 1995), possibly by increased hatchery production of seed-
stock, will greatly increase the volume and interannual stability of
available harvested product and, thus, encourage greater market
penetration of the 55-65 mm whole product. Large-scale markets
for whole scallop products will only be developed when produc-
tion volume is sufficient to warrant the required financial invest-
ment for promotional market development. However, live scallops
are reported to have a relatively short ex-farm shelf life as com-
pared to other molluscan shellfish such as clams, oysters, or mus-
sels (de Franssu 1990). Increasing market volume for whole scal-
lop products may require a shift from sale of live product to a
frozen in-shell or other secondarily processed form. This would
have a negative impact on our projections of farm enterprise eco-
nomic viability unless accompanied by commensurate farm-gate
price increases.

Ultimately, the limitation to increased production of whole
scallop products in Atlantic Canada most difficult to mitigate may
be that caused by the distribution, frequency, and prevalence of
biological toxin outbreaks. Scallop species are well known for
their propensity to sequester biological toxins in their mantle, roe,
and hepatopancreas tissue at relatively high levels as compared to
other bivalve species (Shumway and Cembella 1993; Douglas et
al. 1997). Detoxification of affected scallops may be quite slow,
exceeding several months in duration and be quite variable among
individuals (Shumway and Cembella 1993). Frequent and severe
toxin outbreaks may limit expansion of scallop farms to areas
where toxin outbreaks are relatively infrequent and of short dura-
tion. Although this has been the case in Newfoundland, thus far,
continued industry expansion, particularly elsev, here in the Atlan-
tic Canadian provinces may be seriously impeded by toxic event
considerations.

ACKNOWLEDGMENTS

We thank members of the Mills and Jewer families of Bot-
wood, Newfoundland who willingly helped pick, sort, and other-
wise handle scallops during the 2- year pilot trials as well as staff
of Thimble Bay Farms, Ltd. for their patience and help with many
of the labor costing activities. We especially thank Frank Corbett,
DFO Economics Branch, St. John's for much advice and assis-

124

Penny and Mills

tance to set up and i^n the economic analyses. K. S. Naidu and J.
Davis. DFO St. John's provided many useful comments on an
earlier draft. Funding assistance for the pilot trials was provided by

the Department of Fisheries and Oceans. St. John's. Newfoundland
and by the Canada / Newfoundland Inshore Fisheries Development
Aareement.

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Claereboudt. M. R.. J. H. Himmelman & J. Cote. 1994b. Field evaluation
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Cote, J.. J. H. Himmelman. M. Claereboudt & J. C. Bonardelli. 1993.
Influence of density and depth on the growth of juvenile sea scallops
{Placopecten nui.^ellanicus) in suspended culture. Can. J. Fish. Aqiial.
Sci. 50:1857-1869.

Couturier. C. P. Dabinett & M. Lanteigne. 1995. Scallop culture in At-
lantic Canada, pp 297-340. In: Cold Water Aquaculture in Atlantic
Canada, 2nd ed. A. D. Boghen (ed.). Canadian Institute for Research on
Regional Development. Tribune Press Ltd.. Sackville. N.B.

Dadswell. M. J. & G. J. Parsons. 1991. Potential for culture of the giant
scallop Placopecten magellunicus using natural spat, pp 300-307. In:
S. E. Shumway and P. A. Sandifer (eds.). An International Compen-
dium of Scallop Biology and Culture. World Aquaculture Society.
World aquaculture Workshops. No. I.

De Franssu. L. 1990. The world market for bivalves — oyster, mussel,
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Devaraj. M. & G. J. Parsons. 1997. Effect of fouling on current velocities
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Douglas. D. J.. E. R. Kenchington. C. J. Bird. R. Pocklington. B. Bradford
& W. Silvert. 1997. Accumulation of domoic acid by the sea scallop
(Pliuopecien magellunicus) fed cultured cells of toxic Pseudo-nitzschia
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Duggan. W. P. 1973. Growth and survival of the sea scallop, Argopcctcn
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Frishman. Z., A. Noonan. K. S. Naidu & F. M. Cahill. I9S(1. Farming
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Journal of Shellfish Resaiirh. Vol. 19, No. 1, 125-128. 2000.

THE EFFECT OF CYTOCHALASIN B DOSAGE ON THE SURVIVAL AND PLOIDY OF
CRASSOSTREA VIRGINICA (GMELIN) LARVAE

JOHN E. SUPAN,' CHARLES E. WILSON^ AND
STANDISH K. ALLEN, JR'

^Office of Sea Grant Development
Louisiana State University
Baton Rouge, Louisiana 70803
'Coastal Fisheries Institute
Louisiana State University
Baton Rouge, LA 70803
Virginia Institute of Marine Science
College of William & Man-
Gloucester Point, VA 23062

ABSTRACT Survival and ploidy of D-stage oyster larvae (Crassosrrea virginica) were determined following the rearing of embryos
exposed to CB dosages of 0.5 mg/L. 0.25 mg/L. and 0.125 mg/L for 10 minutes, with O.OS'/r DMSO and ambient seawater as controls.
The experiment was replicated three times on the same day with the same procedures and partially stripping the same male oysters;
different females were used for each replicate. CB dosage treatments began when 509c of the eggs reached PBI (24-3 1 min). Embryos
were reared for 48 h at ambient temperature and salinity. Resulting triploid percentages were 13% ± 6.7% (0.125 mgCB/L), 61.8% ±
6.2% (0.25 mgCB/L). and 68.2% ± 14.1% (0.5 mgCB/L). No significant difference {P s 0.05) in mean survival was found between
the three CB treatments. Significant differences in mean survival between the three replicates implies variability because of different
sources of eggs.

KEY WORDS: Crassoslrea virginica. oyster, triploid. cytochalasin B. dosage

INTRODUCTION

Cytochalasin B (CB), a cytokinetic inhibitor, was first used to
produce triploid Crassostrea virginica and Crassoslrea gigas
(Thunberg) over a decade ago (Allen 1986. Stanley et al. 1981).
Optimal treatments; that is. those yielding high proportions of
triploids, have been reported for C. gigas. based on temperature,
dosage, time of application, and duration; namely, 0.5 mgCB/lmL
dimethyl sulfoxide (DMSO)/L of seawater for 20 min at 25 °C,
when 507f of the eggs were at meiosis I (Allen et al. 1989, Down-
ing and Allen 1987). Because C. virginica is less fecund than C.
gigas (Galtsoff 1964), there is more concern for egg survival.
Lower dosages and treatment times of 0.5 mgCB/L for 15 min at
25 "C (Shatkin and Allen 1990) and 0.25 mgCB/L for 10 to 15
min, at 27 to 29 °C (Barber et al. 1992) were suggested to increase
the survival of embryos while maintaining high yields of triploids.

We tested the feasibility of triploid C. virginica production in
Louisiana, based on the premise that higher summertime meat
yields resulting from triploidy could be profitable for the oyster
industry. Triploid induction, using 0.5 mg/L CB. was variable with
commercial size broods (a 4 million eyed larvae). During the first
summer of commercial-scale production, survival of CB-treated
embryos was < 5% compared to s 21% for diploid controls using
stripped gametes. Differences between the salinity at our hatchery
and salinities at sites where broodstock were obtained affected
development time, in particular meiotic synchrony, and have been
identified as major causes of this variation (Supan 1995).

The objective of this study was to investigate the effect of CB

dosage (H,|:p.|,5„„CB ~ M-0.25mgCB ~ M-0.12SmgCB ~ M-OmgCB-

H^:=^) on survival and triploidy induction in C. virginica, and to
determine what component of the variability was attributable to
females, held in identical environments.

METHODS AND MATERIALS

Survival and ploidy of oyster larvae were estimated after ex-
posing embryos to CB dosages of 0.5 mg/L. 0.25 mg/L, and 0.125
mg/L for 10 min. with 0.05% DMSO and ambient seawater as
controls. The experiment was replicated three times on the same
day with the same procedures by partially stripping the same male
oysters; different females were used.

Preparation of Gametes

Gametes were obtained for each replicate in a fashion similar to
the method described by Allen and Bushek (1992). Oysters were
collected from nearshore containers, opened, and their gender was
determined microscopically using gonadal smears. Ripeness was
visually recognized by the presence of prominent genital canals.
Female and male oysters were placed in separate areas to avoid
cross contamination.

Eggs were obtained from three ripe females, randomly chosen
for each replicate. Females were individually dry-stripped (i.e..
without using seawater) to ensure equivalent periods of hydration
(defined as the length of time eggs are exposed to seawater after
stripping) and simultaneous fertilization. The resulting eggs were
pooled and washed of gonadal debris with filtered ( 1 (jim) ambient
(24%c) seawater (FAS) by passing them through a 75 |xm Nytex
screen onto a 15 |xm screen. They were then resuspended for
enumeration and brought to a volume of approximately 8 million
in 1 L FAS. The eggs were allowed to hydrate for 60 min at 28 °C
before fertilization and treatment.

Three male oysters were partially stripped for each replicate by
scraping away only a portion of the gonad into a beaker and then
covering the oyster with plastic wrap to prevent desiccation. Sperm
from the three inales was pooled in a beaker after being washed of
gonadal debris by passage through a 15 |j.m screen.

126

SUPAN ET AL.

100

80

i60

40

20

0

I

Replicate 1

Replicate 2
Replicate 3

>?J-'-'-'?'-'::H

o.'s

0.1'25 025

CBTreatment (mg/L)

mn

control DMSO

Figure 1. Percentage triploidy in C. virginica D-stage oyster larvae after treatment witii cytochalasin B, by replicate.

Fertilization and Treatment

Pooled eggs were fertilized with approximately 10 sperm/egg
and stirred regularly. After fertilization of the 8 million eggs, they
were divided into five treatment beakers each containing 800 niL
of FAS, bringing the eggs per treatment to about 1 .5 M eggs/L.
Eggs from individual beakers were examined microscopically for
polar body formation at appropriate intervals. Treatments began
when approximately SO'/r of the eggs reached PBI (24 to 31 min
among replicates).

Treatments consisted of adding the appropriate aliquot of 1 mg
CB/1 ml DMSO to the beakers of developing eggs to obtain dos-
ages of 0.5 mg/L. 0.25 mg/L. and 0.125 mg/L. Our control con-
sisted of 0.05'/f DMSO (v/v) dissolved in FAS and FAS alone
served as a normal. Treatments lasted for 10 min. Afterward, each
CB-treatment group of embryos was rinsed of CB with FAS over
a 15 \xm screen then placed in separate beakers containing 0.05%

TABLE 1.

Results of analysis of variance (.\N()VAl: Kffect of cytochalasin B

treatment and experimental replication on the percentage Iriploidy

of C. virginica oyster larvae.

Sources of
Variation

DF

F-ratio

I'rob > F

Treatment
Replicate (error)

4y.7()

O.OOOI

()..s2yi

DMSO-FAS solution for 15 min. The embryos from each beaker
were then rinsed of the DMSO solution and put into separate,
labeled culture vessels containing 15 L of FAS for a final culture
density of 15 embryos/L. Culture vessels were aerated and equal
volumes Isochrysis aff. galbana clone CISO added. Embryos were
incubated for 48 h at ambient temperature and salinity until they
reached D-stage. All counts were obtained using triplicate 1 mL
subsamples from each culture vessel. At 48 h, each vessel was
individually drained onto a 40 jjim screen, and subsamples were
placed into 1.5 mL centrifuge tubes and shipped overnight to Rut-
gers University's Haskin Shellfish Research Laboratory for ploidy
determination using fiow cytometry.

TABLE 1.

Results of ANOVA: Post lioc comparisons of mean percentage
triploidy of C. virginica lar>ae b) treatment.

Triploidy*

Treatment

Mean

SD

Comparisons**

0.1 2.S mgCB

0.3594

0.110

A

0,25 mg CB

0.90.52

0.063

B

0.5 mg CB

0.97S.^

0.1.56

B

Ccnirol w/i) DMSO

0.1862

0.009

A

Control w/DMSO

0.0949

0.0S4

A

R- = 0.9616.

'Triploidy = arcsin (\(';'rTripk)iiil (0.01)).
■ * Tukoy's honestly signitlcanl difference (^
SD = Standard deviation.

0.05).

CB Dosage Effects on Larval Ploidy and Survival

127

Data Analyses

Differences among treatment means for survival and percent-
age triploidy were detennined using analysis of variance (SAS
1991). Percentage triploidy was determined as a proportion of
triploid cells among the total number analyzed by the curve-Fitting
program ModFit (Verity Software House. Topsham. ME) (Allen
and Bushek 1992). Survival and percentage triploidy met the as-
sumptions of normality and variance homogeneity after angular
transformation (Dowdy and Wearden 1991). The models used sur-
vival and percentage triploidy as separate dependent variables and
treatments and experimental replicates as independent variables.
Tukey"s Honestly Significant Difference Procedure was u,sed to
test the difference among the treatments and replicates (a = 0.05).

RESULTS

Percentage triploidy and survival were not different between
0.5 mg/L and 0.25 mg/L CB treatments.

Percentage Triploidy

In treatments, mean percent triploidy was 13% + 6.7% for
0.125 mgCB/L. 61.8% ± 6.2% for 0.25 mgCB/L. and 68.2% ±
14.1% for 0.5 mgCB/L. In controls, 1.4% ± 1.3% of the 0.05%
DMSO treatment and 3.4% ± 0.3% of the FAS normal larvae were
triploid. Figure 1 depicts percentage triploidy by treatment and
replicate. Variation seems high among the three replicates; how-
ever, transformed data revealed no significant difference (P <
0.05).

The model (% triploidy = treatments, replicates) defined the
relationship between the treatment effects and percentage triploidy

and explained mo.st of the variability (R~ = 0.9616). Treatment
was highly significant (f < 0.0001), and the replicate effect was
not significant (P = 0.5291) (Table 1). Post hoc comparisons of
mean percentage triploidy found neither significant differences
between the 0.125 mgCB/L and the two controls, nor between the
0.25 mgCB/L and 0.5 nigCB/L dosages (Table 2).

Survival

Figure 2 shows survival by treatment and replicate. On average,
the results demonstrate an inverse relationship between survival
and CB dosage and a lack of effect (slight enhancement) with
DMSO e.xposure. Although there was moderate variability among
the replicates, overall, they all demonstrated the same trends across
treatments.

The model explained a reasonable amount of variation in sur-
vival (R" = 0.7172). Both replicate and treatment were highly
significant {P < 0.0001, Table 3). For treatments, both control and
norinal were the same, and all CB groups were the same (Table 4).
Overall, CB groups had about 20% lower survival than did the
controls.

DISCUSSION

These results support previously reported findings that 0.25
mgCB/L (Barber et al. 1992) and 0.5 mgCB/L (Shatkin and Allen
1990) are appropriate dosages for inducing triploidy in C. vir-
i^inica. However, results are variable depending upon egg or sperm
quality or some other factor (Allen and Bushek 1992).

Treatment Recommendations

We found no statistical difference in percentage triploidy or
survival between the two higher CB dosages. Considering the cost

0.7

0.6

0.5

03

>

0.4

CO

0.1

Replicate 1

Replicate 2

;W;W>W;W;WJ

£ 0.3 -

0.2

0 -■

Replicate 3

I

0.125 0.25

0.5

control DMSO

CB Treatment (mg/L)

Figure. 2. Survival of C. virginica embryos to D-stage larvae after cytochalasin B treatment, by replicate.

128

SUPAN ET AL.

TABLE 3.

Results of ANOVA: Effect of cytochalasin B treatment and
experimental replication on survival of C. virginica larvae.

Sources of
Variation

DF

F-ratio

Prob > F

Treatment
Replicate (error)

11.54
2?. 1 1

0.0001
0.0001

R- = 0.7172

of CB {$10/mg in the U.S.). economics suggest that the lower
effective dosage is preferable, at 28 °C for 10 min. However, with
a range of 54 to 82% triploidy (0.5 mgCB/L) versus 55 to 67%
(0.25 mgCB/L), one is inclined to use the higher dosage. Greater
triploidy might have resulted from a longer (15 min) treatment
time, at the sacrifice of lower survival. For maximum triploid
production, embryos should be exposed to CB for a period of time
long enough to have a high proportion captured at PBI extrusion
but short enough to minimize mortality (Barber et al. 1992). Allen
and Bushek ( 1992) attributed low variance in triploid production to
using meiosis I as a benchmark to begin treatment, claiming to
have effectively removed meiotic rate as a factor. Although the
time of initiation is determined by an appropriate developmental
milestone (i.e., 50% PBI). the duration is fixed and does not ac-
commodate varying meiotic rates. We suggest that appropriate
duration of treatment be addressed by using a developmental
benchmark to cease treatment as it is used for beginning it. Ob-
servation of a subsample of eggs, held at the same temperature but
without treatment, could provide such a cue. Although this cue
must be determined empirically, we suggest 2-5% cleavage might

TABLE 4.

Results of ANOVA: Means and standard deviations (SD) of survival
of C. virginica by treatment with comparisons.

Survival*

Treatment

Mean

SD

Comparisons**

0.125 mg cb

0.5961

0.1204

A

0.25 mg cb

0.5795

0.0860

A

0.5 mb cb

0.4748

0.(»I3

A

Control w/o dmso

0.7.\M

0.2035

B

Control w/dni.so

0.7585

0.2126

B

* Survival = arcsin (V(Normal larvae/embryos)).

** Tukey's honestly significant difference (.'-^ = 0.05).

be appropriate. This benchmark could be used for C. virtfinica or
any bivalve species.

The real solution to improving efficiency of triploid production
is the development and use of tetraploid broodstock. Tetraploid
male oysters produce diploid sperm; when used to fertilize eggs
from diploid females. 100% triploid offspring result (Guo and
Allen 1994).

ACKNOWLEDGMENTS

We are grateful to Jordan Bradford. Lee Hanson, the late Tony
Venterella and his wife. Gayle. and the late Carlo Venterella for
their logistical support during the project, and Mr. Ron Becker for
his guidance and insight. We are also grateful to Wilbert Collins,
Jules Melancon. and Al Sunseri for their donation of broodstock.
Financial support was provided by grants from the Louisiana
Board of Regents [LEQSF (93-96)-RD-B-08| and the Louisiana
Sea Grant College Program [NA89AA-D-SG226].

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Dowdy. S. & S. Wearden. 1991. Statistics for research. John Wiley & Sons
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Galtsoff P. A. 1964. The American oyster. Crassoslrea virginica [Gme-
lin|. Fisheries Bulletin. U.S. Fish and Wildlife Service. 480 pp.

Guo, X. & S. K. Allen Jr. 1994. Viable letraploids in the Pacific oyster
[Crassoslrea gigas Thunberg) produced by inhibiting polar body 1 in
eggs from triploids. Mol. Mar. Biol. Bioteclmol. 3:42-50.

SAS Institute. 1991. SAS system for linear models. 3rd ed. SAS Institute.
Cary. NC. 327 pp.

Shatkin. G. M. & S. K. .Mien. 1990. Recommendations for commercial
production of triploid oysters. / Shellfish Res. 8:449.

Stanley. J. G., S. K. Allen Jr. & H. Hidu. 1981. Polyploidy induced in the
American oyster, Crassoslrea virginica. with cytochalasin B. Aquacul-
lure 23:1-10.

Supan. J. E. 1995. The effects of salinity on the production of triploid
oyster larvae [Crassoslrea virginica Gmelin) in Louisiana. Ph.D. Dis-
sertation, LSU. Baton Rouge, LA.

Jniinuil of Shellfish Research. Vol. 19. No. 1. 129-1.^2. 2000.

DELIVERY OF RIBOFLAVIN TO LARVAL AND ADULT PACIFIC OYSTERS, CRASSOSTREA

GIGAS THUNBERG BY LIPID SPRAY BEADS

C. J. LANGDON.' C. SEGUINEAU," B. PONCE,^ J. MOAL,^
J. F. SAMAIN-

^ Coastal Oregon Marine Experiment Station

Hatfield Marine Science Center and Department of Fisheries and Wildlife

Oregon State University

Newport. Oregon 97365
-IFREMER

Lahoratoire de Physiologie des Invertebres

BP70. 29280 Ploiizane, France

ABSTRACT Lipid spray beads (SB) were prepared containing 13% w/w particulate riboflavin. Beads suspended in seawater lost 73%
riboflavin after 24 h. Release of riboflavin from SB ingested by Pacific oyster (Cmssostrea gigcis) larvae was observed under
epifluorescent light. Riboflavin concentrations in tissues of adult oysters fed on riboflavin-SB were significantly (SNK; P < 0.05)
greater than those of oysters fed on seawater-filled SB. Concentrations of riboflavin in oysters exposed to dissolved riboflavin were
not significantly greater than those of oysters fed on seawater-filled SB, indicating that elevated riboflavin concentrations in oysters
fed on riboflavin-SB were attributable to breakdown of ingested beads rather than uptake of dissolved riboflavin leaked from SB into
the culture medium. SB seem to be a promising means of delivering water-soluble nutrients to bivalve suspension feeders

KEY WORDS: Spray beads, lipid, riboflavin, oyster, larvae. Crassostrea gigas

INTRODUCTION

Little is known of the nutritional requirements of bivalve mol-
lusks despite their obvious importance in aquaculture and natural
ecosystems. The main reason for this lack of knowledge is that
nutritionally satisfactory, defined artificial diets are not available.
The development of microparticulate diets that both retain dietary
ingredients when suspended in seawater and are digestible by bi-
valve mollusks has been a difficult goal to achieve. High surface
area to volume ratios of microparticulate diets together with low
molecular weights of essential nutrients, such as water-soluble
vitamins, results in their rapid loss. For example. Lopez-Alvarado
et al. (1994) reported that > 80% amino acids were lost from
microgel particles after only 2 minutes in aqueous suspension.

To address the problem of rapid loss of water-soluble nutrients
from microparticulate feeds, Langdon and Siegfried (1984) devel-
oped lipid-walled microcapsules for the delivery of water-soluble
vitamins to juvenile oysters [Crassostrea virginica). Later Buchal
and Langdon (1998) and Langdon and Buchal (1998) developed
lipid spray beads (SB) for the delivery of water-soluble nutrients
and therapeutic substances to bivalves. Buchal and Langdon
(1998) found that it was important to soften the walls of lipid-
walled capsules and SB by adding 40% w/w fish oil to the tripal-
mitin walls of the particles in order to make them digestible by
clams {Tapes philippinarwn): however, softening the walls of SB
in this way lowered 24-h retention efficiencies for encapsulated
riboflavin from 97.9 to 85.1% (Buchal and Langdon 1998).

Seguineau et al. (1996) reported that the microalgal species
Isochiysis galbana. Pavlova lutheri. and Skelelonema costatiim
contained high concentrations of riboflavin and thiamine; how-
ever, the concentrations of these two vitamins in scallop (Pecten
ina.ximiis) larvae fed on a mixture of these algal species declined
during growth and development. Seguineau et al. (1996) suggested
that microencapsulated supplements of riboflavin and thiamine
could be used to study the requirements of scallop larvae for these
vitamins.

In this paper, we describe the results of feeding experiments in
which larval and adult oysters ( Crassostrea gigas Thunberg) were
fed on SB containing particulate riboflavin to evaluate the poten-
tial usefulness of SB in delivering low-molecular weight, water-
soluble nutrients to bivalve mollusks.

METHODOLOGY

Spray Beads

Preparation of Spray Beads Containing Riboflavin

Spray beads were prepared containing micronized, particulate
riboflavin (Sigma) based on the method described by Buchal and
Langdon (1998). Briefly, riboflavin crystals were ground to a fine
powder (< 5-(xm particles; McCrone micronizing mill, McCrone
Scientific Ltd). Two grams of ground powder were mixed by soni-
cation at 90 °C with 8 g of a lipid mixture made up with 4.8 g
tripalmitin (Fluka Chemical Co.) and 3.2 g of menhaden oil (light
cold pressed; Zapata Haynie Ltd.). The heated mixture was then
forced under pressure (90 psi) through at atomizing nozzel (SUE-
25B; Spraying Systems Ltd.) supplied with pressurized nitrogen at
10 psi. The beads were collected in a stainless steel cylinder cooled
with liquid nitrogen then stored in the dark at -20 "C until use.

Determination of Encapsulation Efflciency

To determine encapsulation efficiencies. 10 g of SB were first
dissolved in 3 mL chloroform and the vitamin core material ex-
tracted by addition of 3 mL distilled water with shaking. The
aqueous supernatant was removed and the extracfion repeated
three times. Aqueous extractions were combined and the concen-
tration of dissolved riboflavin determined spectrophotometrically
(absorbance at 267 nm).

A subsample of 0.5 mL of chloroform was removed from the
capsule extraction and transferred to a dry. tarred weighing boat.
The chloroform was removed by heating for 24 h at 50 °C. and the
boat was reweighed to determine the weight of extracted lipid.

129

130

Langdon et al.

Encapsulation efficiencies were expressed as the weight of encap-
sulated vitamin (mg) per 100 mg of lipid.

Retention of Riboflavin by SB Suspended in Seawater

Retention of riboflavin by SB was determined by measuring the
proportion of initially encapsulated riboflavin remaining after 24 h
suspension in seawater. To prepare SB for a leakage experiment,
beads were first suspended in 2% polyvinyl alcohol with sonica-
tion. SB were then sieved using a 40-|jim sieve, and beads under 40
p,m were collected on a GF/C filter and rinsed with cold (5 °C)
distilled water. The beads were then washed from the filter with
cold distilled water and collected in a sealed vial and stored at 5 °C
in the dark.

At the start of a leakage experiment. 75 to 1 00 mg of the sieved
(< 40 p,m) SB were suspended in 15 mL seawater (20 °C) by
vigorous shaking. Immediately after suspension (t = 0), 1 mL of
the bead suspension was taken and filtered onto a GF/C filter. The
filtered SB were then washed with 1 mL of chilled (5 °C) seawater.
The filtrate and washings were pooled and stored in the dark at 5
°C for analysis. Riboflavin concentrations were determined as de-
scribed above. The remainder of the SB suspension was placed on
a continuous agitator at 20 to 22 °C. Samples of suspended SB
were taken over a period of 24 h to determine changes in the
retention of riboflavin over time. Retention efficiency (RE) was
defined as

riboflavin retained on the filter

RE= ., „ . T—rr, X 100

riboflavm on inter and m riltrate

Breakdown of SB and Release of Riboflavin by Larvae

Feeding experiments were conducted to determine if oyster
larvae could inge.st and breakdown SB, thereby releasing ribofla-
vin into the digestive system. Broodstock oysters were spawned,
and the resulting larvae were raised on a mixed diet of Isochiysis
gulhaiui (T-ISO) and Chaetocems cakitnins (Breese and Malouf
1975). After 8 days of culture, larvae were sieved onto a 45-p.m
screen, divided into two groups and each resuspended in two liters
of seawater at a density of 1 0 larvae per mL.

Riboflavin-SB at a concentration of 50 SB/p,L were fed to one
group of larvae with gentle aeration to maintain SB in suspension.
After 1 hour, larvae were sieved from suspension using a 45-|jLm
sieve, rinsed with seawater, then resuspended in two liters of fil-
tered seawater and fed on T-ISO alone for 2 hours. The larvae were
then sieved from the culture medium and preserved with 0.59f-
formaldehyde (final concentration made up in seawater, buffered
at pH 8.0 with borax) for microscopic analysis. The second group
of 8-day old larvae were fed on T-ISO alone for 2 hours, then
sampled and preserved for microscopic analysis (as described
above).

Sampled larvae were examined using an epifluorescent micro-
scope (Leica DMRBE: excitation 355-^25 nm. emission 525 nm)
at x400 magnification. Green and yellow fluorescence indicated
the presence of dissolved and particulate riboflax in. respectively,
while red fluorescence indicated the presence of chlorophyll de-
rived from ingested algae.

Breakdown of SB and Uptake of Released Riboflavin by Adults

An experiment was carried out to determine if adult oysters
could breakdown ingested SB and absorb released vitamin into the
hemolymph and tissues, lliree groups of six adult oysters were

separately held in 20 L of seawater and fed for 6 hours on T-ISO
at a concentration of 50 cells/(j.L in combination with one of the
following additions:

1. 20 riboflavin-SB/|jiL (equivalent to a concentration of 1.15
mg riboflavin/L or a vitamin dose of 3.8 mg riboflavin/
oyster):

2. 20 seawater-filled SB/p.L; or

3. dissolved riboflavin at the same concentration provided in 1.
Two grams of riboflavin-SB were suspended in distilled water,

and the suspension was then poured through a 20fjLm mesh sieve.
SB smaller than 20 ixm were collected and filtered onto a GF/C
filter, rinsed, and resuspended in 10 mL of distilled water. Aliquots
of 100 |j,L SB suspension were taken to determine riboflavin and
bead concentrations. Riboflavin concentrations were determined as
described above. SB concentrations were determined using a
Coulter Counter (Model TA2). Seawater and food additions were
replaced every 2 hours over a period of 6 hours. The cultures were
gently aerated to maintain beads in suspension.

After 6 hours, oysters were removed and dissected.
Hemolymph samples were taken with a syringe from both the heart
and the sinus of the adductor muscle. Samples of stomach contents
were removed with a Pasteur pipette inserted through the mouth.
Tissue samples of mantle and adductor muscle were also removed.
The samples were frozen at -20 °C for protein and riboflavin
analysis.

Riboflavin and Protein Analysis

Hemolymph samples were centrifuged, and riboflavin concen-
trations of the supernatant fraction were determined by high-
pressure liquid chromatography (HPLC) (Seguineau et al. 1996).
Mantle and adductor muscle samples were ground in 0.2M HCl
and 0.6N perchloric acid (PCA) and centrifuged. Supematants
were then removed, their volumes adjusted to 2 mL with distilled
water and riboflavin concentrations determined by HPLC
(Seguineau et al. 1996).

Protein concentrations of hemolymph samples were determined
by the method of Bradford (1976), using bovine serum albumen as
a standard. Treatment of adductor muscle and mantle samples with
HCl and PCA for the extraction of riboflavin probably resulted in
the precipitation of most proteins in these samples; therefore,
Bradford assays indicated concentrations of PCA-soluble protein
and peptides in muscle and mantle samples. Riboflavin concentra-
tions were expressed in terms of ng ribofla\ in per mg protein in
tissue samples or per mL of stomach extract.

Statistics

The rate of loss of riboflavin from SB suspended in seawater
was analy/.ed by regression analysis. Riboflavin concentrations in
oyster samples from the three experimental irealments were com-
pared by analysis of variance (ANOVA) (Model 111: Super
ANOVA, Abacus Concepts). Concentrations were log-trans-
formed to ensure homogeneity of variances, as determined by plots
t)f residual values against fitted values. If ANOVA indicated a
significant treatment effect on riboflavin concentration, individual
treatments were compared by Student-Newman-Keuls (SNK)
nuiltiple range test (significance level P < 0.05).

RKSULTS

Encapsulation and Retention Efficiencies

SB were lound to ha\e an encapsulation efficiency of 13'7r w/w
(mg riboflavin per 100 mg lipid I. Leakage experiments indicated

Delivery of Riboflavin to Oysters by Lipid Spray Beads

131

that almost half the encapsulated ribonavin was lost from SB
during the first 3 hours of suspension in seawater, followed by a
more gradual loss over the subsequent 21 hours (Fig. I). Approxi-
mately 27% of the initially encapsulated riboflavin was retained
after 24 hours of suspension, equivalent to 3.3 mg of riboflavin
retained per 100 mg of lipid.

Regression analysis indicated that there was a significant {P =
0.0012) relationship between the log of the fraction of riboflavin
retained and the duration (log time (h)] that SB were suspended in
seawater (Fig. 1 ). The rate of loss of riboflavin could be expressed
in terms of the equation;

Log fraction retained
0.994

0.116 - [0.333 X log time (h)] r" =

Breakdown of SB and Release of Riboflavin by Larvae

Larvae were able to ingest and breakdown SB. liberating en-
capsulated riboflavin into the digestive system. Free riboflavin was

100 -

1 1 1 1 1 1 1 1 1 1

[

-;? 80 -

\

£

\

V

>.

^v

o

^

C 60 -

^.

0

#(.^^

o

^^-^^_^

it

^■^-^^

0)

_ 40 -

^""^■^^,,___^

C

^""^~— ^__^

o

^^^~^^^_^

^H*

c

^^ ^

B 20-

(D

tr.

n -

10 12 14 16 18 20 22 24

Time (h)

0 .2 .4 .6 .8 1

Log Time (h)

Figure 1. Retention of riboflavin by lipid spray beads suspended in
seawater. Top: Change in percentage riboflavin retained by beads over
a 24-h period Bottom: Relationship between log of fraction retained
and log time duration of beads suspended in seawater. Log fraction
retained = -0.116 -[0.333 x log time (h)l; r" = 0.994

Figure. 2. Eight-day-old larvae of the Pacific oyster {Crassostrea gigas)
viewed under epifluorescent light (excitation 355—125 nm, emission 525
nm) at x400 magnification. Average larval shell length = 122 pm. Top:
Larvae fed on ribofiavin-containing lipid spray beads (50 beads/pL)
for 1 hour, followed by a 2- hour period of feeding on T-ISO alone.
Bottom: Larvae fed on Isochrysis sp. (T-ISO) alone for 2 hours.

observed as a diffuse greenish fluorescence in the guts of larvae
fed on SB. and riboflavin crystals present in intact or partially
digested SB were evident as bright yellow points (Fig. 2). The
digestive systems of larvae fed on algae alone fluoresced red be-
cause of the presence of chlorophyll from ingested algae but no
yellow or green fluorescence was evident (Fig. 2).

Breakdown of SB and Uptake of Released Riboflavin by Adult Oysters

ANOVA of log-transformed riboflavin concentrations in oys-
ters fed on riboflavin-SB were significantly greater (SNK; P <
0.05) than those of oysters either fed on seawater-filled SB or
exposed to riboflavin dissolved in seawater (Table 1). The pres-
ence of significantly higher concentrations of riboflavin in the
hemolymph. adductor muscle, and mantle of oysters fed on ribo-
flavin-SB indicated that oysters were able to digest the lipid walls
of SB and absorb released riboflavin. There were no significant
differences in riboflavin concentrations in hemolymph sampled
from either the heart or adductor muscle (ANOVA: P > 0.05).

Concentrations of riboflavin in the tissues of oysters exposed to
dissolved riboflavin were not significantly (SNK; P > 0.05) dif-
ferent from those of oysters fed on seawater-filled SB, indicating
a limited ability of adult oysters to take up dissolved riboflavin
from seawater.

CONCLUSIONS

Feeding experiments indicated that both oyster larvae and
adults were able to breakdown SB and release riboflavin. Free

132

Langdon et al.

TABLE 1.

Concentration of riboflavin in tissues of adult Pacific oysters exposed to either lipid sprav beads (SB) containing 13% riboflavin at a
concentration of 20 SB/pm, seawater-filled SB at a concentration of 20 SB/pL or dissolved riboflavin at the same concentration as that

delivered by riboflavin-SB (1.15 mg/L).

Stomach

Hemolymph

Mantle

Adductor
muscle

Contents

Heart

Muscle

(ng/mg PCA-soluble

(ng/mg PCA-soluble

Treatment

(ng/mL)

(ng/mg protein!

(ng/mg protein)

protein)

protein)

Seawater-filled SB

20 ±4

47 ± 19

21 ±10

198 ±94

177± 115

Dissolved riboflavin

24 ±4

84 ± 53

51 ±32

112 ±22

67 ± 20

Riboflavin-SB

7274 ± 1619

1844±66(J

1 1 65 ± 247

1633 ±573

10636 ±3808

Values are given as means (±1 SE. /;

6).

riboflavin was evident in the stomachs of larvae and elevated
riboflavin concentrations were evident in the tissues of adult oys-
ters after being fed on riboflavin-SB. Adult oysters exposed to
concentrations of dissolved riboflavin, equivalent to those supplied
by encapsulated riboflavin, did no show elevated tissue concen-
trations of riboflavin, indicating that uptake of dissolved riboflavin
lost from SB was not a significant source for adult oysters.

About half of the riboflavin was lost during the preparation of
SB, based on a comparison between the measured encapsulation
efficiency of 13% and a maximum theoretical encapsulation effi-
ciency of 25%. Further losses of riboflavin from SB occurred after
suspending SB in seawater; for example, it can be estimated (based
on Eq. 1) that 39% of encapsulated riboflavin would have been
lost at the end of each 2-hour period of the adult feeding experi-
ment.

In this study, retention of riboflavin by SB suspended in
seawater for 24 h was only 27% compared with 85% reported
by Buchal and Langdon (1998). This difference may have been
attributable to higher encapsulation efficiencies of SB used in
this study, because riboflavin-SB prepared by Buchal and Lang-
don (1998) had an encapsulation efficiency of 4.89^ compared
with an encapsulation efficiency of 13% for SB used in this
study.

The effects of additions of riboflavin-SB on the growth and
survival of oysters needs to be determined in future experiments.
Because of the need to prepare SB with a high proportion (> 60%)
of lipid wall material to ensure encapsulation of the core material,
it is unlikely that SB will be useful in delivering bulk dietary
ingredients, such as protein and carbohydrate, to oysters. However.
SB may be useful in supplementing algal or artificial diets with

water-soluble micronutrients. such as essential ainino acids or wa-
ter-soluble vitamins (Seguineau et al. 1996).

ACKNOWLEDGMENTS

This research was supported by a fellowship from the Organi-
zation for Economic Cooperation and Development (OECD). co-
operative research program: Biological Resource Management for
Sustainable Agricultural Systems. We also thank Jacques Panfili
for his help in taking photographs of larvae fed on SB.

LITERATURE CITED

Bracit'ord. M. M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of prolein-dye
binding. Anal. Biochein. 72:248-254.

Breese. W. P. & R. E. Malouf 1975. Hatchery Manual for the Pacific Oys-
ter. Oregon State University Sea Grant Program. Pub. No. ORESU-H-
75-002. 22 pp.

Buchal. M. A. & C. J. Langdon. 1998. Evaluation of lipid-spray beads for
the delivery of water-soluble materials to a marine suspension-feeder,
the Manila clam Tupcs philippinanim (Deshayes 1853). Acjuacull. Nii-
lii. 4:263-274.

Langdon. C. ,1. & M. A. Buchal. 1998. Comparison of lipid-walled micro-
capsules and lipid spray beads for the delivery of water-soluble, low-
molecular weight materials lo aquatic animals. Aiiiiciciili. Nutri. 4: 275-
284.

Langdon. C. J. & C. A. Siegfried. 1984. Progress in the development of
artificial diets for bivalve filter-feeders. Aqiuiciilture 39:135-153.

Liipez-Alvarado. J., C. J. Langdon. S -I. Teshima & A. Kanazawa. 1994.
Effects of coating and encapsulation of crystalline amino acids on
leaching in larval feeds. Aiiiiaciiliure 122:335-346.

Seguineau. C. A. Laschi-Loquerie. J. Moal & J. F. Samain. 1996. Vitamin
requirements in great scallop larvae. Aqiuiciill. In!. 4:315-324.

Jvuimil of Shellfish Raeciich. Vol. 19. No. I. I3.V138, 2000.

MODELING SEASONAL PROLIFERATION OF THE PARASITE, PERKINSUS MARINUS
(DERMO) IN FIELD POPULATIONS OF THE OYSTER, CRASSOSTREA VIRGINICA

D. J. BROUSSEAU' AND J. A. BAGLIVO^

Department of Biology
Fail fie Id University
Fairfield. Connecticut 06430
'Department of Mathematics
Boston College
Chestnut Hill. Massachusetts 02467

.ABSTRACT A temperature-disease course model was developed to predict the effect of seasonal water temperature changes on
disease progression of Dermo in field populations of Crassostrea virgiiiica. A linear model was used to describe the relationship
between weighted prevalence (disease intensity) and lagged cumulative temperature, where cumulative temperature was used as an
estimate of cumulative harm. The model developed for Long Island Sound showed the strongest relationship between cumulative
temperature and disease intensity when a lag time of 53 days was used. Point and interval estimates for the day(s) of the year when
a mean weighted prevalence of 2 (Mackin Index) is expected at four sites in Long Island Sound are given. This model allows the
grower/manager to predict Dermo intensity in shellfish beds if field water temperature patterns are known. Such information can be
used to select oyster growout beds and determine optimal time to harvest.

KEY WORDS: Perkinsii.'s mariiiiis. Dermo. temperature-disease course model. Long Island Sound

INTRODUCTION

Perkinsus marimis (commonly known as "Dermo"). a proto-
zoan pathogen of uncertain phylogenetic affinities (Siddall el al.
1995) is now well established in Long Island Sound (Brousseau et
al. 1994, Brousseau 1996, Ford 1996, Brousseau et al. 1998) and
has been reported as far north as the Damariscotta River in Maine.
This pathogen is a major cause of oyster inortality in the Gulf of
Mexico and Chesapeake Bay. Its introduction to Long Island
Sound, the third largest producer of commercial oysters in the
U.S.. has prompted efforts to develop management strategies and
husbandry protocols to help control the spread of this disease.

The influence of temperature on the activity of Perkin.sus mari-
mis is well documented. Temperature affects multiplication rates.
virulence (Andrews 1988) and zoosporulation of the parasite (Chu
and Greene 1989), and disease intensity in the host increases with
increasing temperature (Chu and LaPeyre 1993). Temperature is
also believed to be the most important factor affecting the geo-
graphic distribution and seasonal cycle of this pathogen (Andrews
1988, Andrews and Ray 1988. Crosby and Roberts 1990. Soniat
and Gauthier 1989).

Modeling studies also point to the importance of temperature in
the development and maintenance of Perkinsus marinus infections.
Simulations have shown that the timing and duration of long-term
climatic changes are important in determining levels of infection in
diseased (coinfeclion by MSX and Dermo) oyster populations
(Powell et al. 1992); whereas, the results of Hofmann et al. ( 199.5)
suggest that temperature is the primary factor regulating the para-
site in the Gulf of Mexico.

Soniat and Kortright (1998) recently developed a model to
estimate the time to a critical level of Perkinsus marinus in eastern
oysters using a long-term dataset of temperature, salinity, and
parasite infection level. Their model indicates that both tempera-
ture and salinity are important variables in predicting Dermo pro-
gression in areas such as the Terrebonne estuary of Louisiana,
where fluctuations in salinity are high and salinity levels often fall
below 10 ppt. In high-salinity, oyster-producing regions such as

Long Island Sound; however, it is likely that water temperature is
the more important factor in controlling parasite proliferation.

This paper reports the results of a modeling study aimed at
predicting the effect of short-term (seasonal) temperature changes
on disease progression of "Dermo" in oyster populations from
New York and southern New England. The annual cycle of Per-
kinsus marinus infections in oysters from six locations in Con-
necticut, Massachusetts, and New York is presented, and a pre-
dictive temperature -disease course model developed for wild and
commercial oyster beds in Long Island Sound is described. A
discussion of the usefulness of this model to oyster aquaculturists
is also presented.

MATERIALS AND METHODS

Data Collection

Oysters (Crassostrea virginica) were collected twice a month
from six locations in Connecticut. New York, and Massachusetts
from January to December 1997 (Figure 1). Most samples con-
tained 25 oysters; a total of 3,786 animals were studied. Sampling
locations, site descriptions, sample sizes, and ages (juvenile vs.
adult) of oysters sampled are provided in Table 1 . Tissue diagnosis
of Perkinsus iiuuinus was done by culture of rectal and mantle
tissue in fluid thioglycollate medium, as described by Ray ( 1954),
Infections were scored for intensity of disease by use of the mea-
sure originally described by Mackin (1962) as the weighted inci-
dence and later renamed weighted prevalence (Ragone and Bur-
reson 1994). On the Mackin Index, scores of 0.5-1.0 indicate light
infections, scores of 2.0-3.0 indicate moderate infections and
scores of 4.0-5.0 are considered heavy.

Temperatures (°C) were monitored at each site using an Optic
Stowaway"^' Temperature Logger (Onset Computer Corp.). At in-
tertidal sites, the recorder was attached to a stake driven into the
flat, and at subtidal locations, it was suspended over the shellfish
bed along a buoy system anchored to the bottom. As a backup
against loss or failure, teinperatures were also taken by hand sev-
eral times a month. Periodic salinity measurements were taken to

133

134

Brousseau and Baglivo

74'-'W

4h^N

420 N _

NEW YORK

40" N -i

NEW
.JERSEY

Figure 1. Map showing tiie locations of the six study sites used in this study: (A) Oyster Bay, NY, (B) Saugatuck River, Westport, CT, (C) Blacli
Rock Harbor, Bridgeport, CT, (D) Thames River, Waterford, CT, (E) Mystic River, Stonington, CT, and (F) Cotuit, MA.

substantiate earlier reports thiat salinities at tine study sites routinely
run in the 20 to 30 ppt. range (Brousseau 1996, S. Ford pers.
comni). They are shown in Table 2.

Mean Temperature Model

The mean temperature. T(x), for sampling day x can be mod-
eled as a cyclic function

T(x)

A cos[c (X - X|^„,,)|

where p. is mean temperature for the year. A is one-half the range
of mean temperatures (the amplitude). X|„„ is the day with the
lowest mean temperature, and c is the constant needed to make the
period equal to one year (c = 2tt/365.25).

For X between the time of lowest mean temperature and highest

TABLE 1.

Age (adult vs. juvenile) and sampling location, number of samples collected, size of oysters sampled, site description (intertidal vs. subtidal).

and type of oysters sampled (wild vs. cultivated).

Mean

Shell

Shell

Age and

Number of

Length

Length

Sampling Location

.Samples

(mm)

(mm)

Comments

Adult populations:

Black Rock Harbor. Bridgepon, CT

25

38..'i- 133.7

70.4

1. 3

Mystic River, Stonington, CT

23

36.6-130.7

76.2

2.4

Thames River. Waterford. CT

2.^

30.1-122..')

62.1

2,4

Saugatuck River, Wcslport. CT

2.5

.34..3- 112.7

69.4

1.3

Juvenile popukilions ( IW.S/yfi YOY):

Black Rock Harbor. Bridgeport. CT

10

14.8-69.1

34.8

1. 3

Cotuit. MA

24

42.2-104.1

69.6

2.4

Oyster Bay. NY

23

42.S-96.y

65.6

2.4

1 = Interlidal sampling site.

2 = Suhiidal sampling site.

3 = Wild population.

4 = Cultivated population.

Modeling Dermo Progression in Field Oysters

135

TABLE 2.

Salinity measurements taken at tlie six study sites durin!> 1997. In
sample size).

Mean ± SE

Range

Study Site

n

(PPt)

(ppti

Black Rock Harbor. Bridgeport, CT

128

23.1 ±0.2

15,5-31.0

Mystic Ri\er. Stonington, CT

-11

25.1 ±0.5

18.1-28.0

Thames River, Waterford. CT

3

14.9 ±4.9

5.4-21.7

Saugatuck River. Westport. CT

55

22.1 ±0.4

11.5-27.0

Oyster Bay. NY

36

24.8 ±0.2

22.0-26.0

Cotuit. MA

7

26.5 ± 0.3

25.0-27,0

mean temperature, the cumulative temperature. CT(x). is the area
under the temperature curve from time X|^,„ to time x:

CT(x) = JJL (X

J - A sin[c (X

Jl/c.

Separate temperature models were developed for each site
(Black Rock Harbor, n = 309; Cotuit. n = 93: Mystic River, n =
365; Oyster Bay. n = 146; Thames River, n = 365; Westport, n
= 365), Weighted nonlinear least squares analysis was used to fit
the parameters.

Temperature — Disease Course Prediction Model

A linear model was used to describe the relationship between
weighted prevalence y and lagged cumulative temperature for
sampling day x and site s:

y = a + b CT, (X - lag).

In this fomiula. CT, (x - lag) is the area under the mean
temperature curve for site s from the time of lowest mean tem-
perature to time x minus the lag. Cumulative temperature is used
as an estimate of cumulative harm; parasite proliferation is as-
sumed to be a function of ambient water temperature patterns at
each site.

Samples with sampling day on or after the mean low tempera-
ture day for the site formed the working set for the analyses. A
total of 87 samples were used (Bridgeport, n = 23; Mystic River,
n = 20; Thames River, n = 21; Westport, n = 23). The lag was
chosen to maximize the correlation between weighted prevalence
and lagged cumulative temperature. The slope and intercept were
then estimated using linear least squares. The model with best
overall fit was chosen. Bootstrap analysis using 1.000 resamples
was used to estimate the sensitivity of the choice of lag time in the
model (Efron and Tibshirani 1993).

Model Predictions

To make predictions, a weighted prevalence of 2.0 was selected
as the parameter of interest since we considered it a critical stage
in the progression of the disease. Andrews (1988) reported that
some mortalities are likely to occur when the mean intensity for a
population exceeds 1.0; however, severe mortalities (5()-757f) are
not expected until the wp reaches 3.0 (Ray and Chandler 1955,
Mackin 1961. Mackin and Hopkins 1961). Site-specific tempera-
ture models were then used to obtain point estimates for the day
with mean weighted prevalence of 2.0. A bootstrap analysis with
1000 resamples was used to construct 95% confidence intervals for
the day with mean weighted prevalence of 2.0 at each site.

1

0.8
0.6

' — 1 — '

•
•

•

• 1

•

•
1 • ♦

U.4

0.2

0

I

Bpt-A Mys Thm Wpt Bpt-J Cot OB

Figure 2. Side-by-side box plots of disease prevalence in juvenile and
adult oy.sters. Adult populations in Bridgeport (n = 25 samples, median
= 100% infected). Mystic River (n = 23 samples, median = 96% in-
fected), Thames River (n = 23 samples, median = 100% infected), and
Westport (n = 23 samples, median = 100% infected) and juvenile
populations in Bridgeport (n = 10 samples, median = 42% infected),
Cotuit (n = 24 samples, median = 30% infected), and Oyster Bay In =
23 samples, median = 68% infected) are represented.

RESULTS

Descriptive Statistics

Disease prevalence in adult and juvenile oysters from all sites
during 1997 is shown in Figure 2. Median values were between 96
and 100% for adult samples and between 30% and 68%- for juve-
nile samples.

Distributions of weighted prevalences among sites are shown in
Figure 3. The highest median weighted prevalence was found at
the Saugatuck River site, followed by the Thames River and Black
Rock Harbor sites. The median weighted prevalence was highest at
sites where adult oysters were sampled ( 1 .4-2.2); median weighted
prevalence in juvenile oyster samples did not exceed 0.5. Distri-
butions of proportions of oysters in all samples with intensity score
of 2.0 or more on the Mackin scale is given in Figure 4. Median
values for adult samples were between 32 and 60%; median values

4

3

1

A

1

1

|J

'— 1

i

1

1

1 1

' 1

0

■

1

1

OB

Bpt-A lyiys Thm Wpt Bpt-J Cot

Figure 3. Side-by-side box plots of weighted prevalence in juvenile and
adult oysters. Adult populations In Bridgeport (n = 25 samples, median
= 1.76 wp). Mystic River In = 23 samples, median = 1.40 wp). Thames
River In = 23 samples, median = 1.90 wpl, and Westport (n = 25
samples, median = 2.20 wp) and juvenile populations in Bridgeport (n
= 10 samples, median = 0.49 wp), Cotuit (n = 24 samples, median = 0.44
wp), and Oyster Bay In = 23 samples, median = 0.40 wp) are repre-
sented.

136

Brousseau and Baglivo

0.8 •

0.6

Bpt-A Mys Thm Wpt Bpt-J

Figure 4. Side-by-side box plots of proportion with intensity score of
2.0 or more. Adult populations in Bridgeport (n = 25 samples, median
= 40%), Mystic River (n = 23 samples, median = 32% ), Thames River
(n = 23 samples, median = 48%), and Westport (n = 25 samples,
median = 60%) and juvenile populations in Bridgeport (n = 10
samples, median = 13% ): Cotuit (n = 24 samples, median = 12%); and
Oyster Bay (n = 23 samples, median = 4%) are represented.

for juvenile samples were between 4 and W/c The largest pro-
portions were observed at the Mystic River, Saugatuck River, and
Thames River sites.

In adult oysters from Bridgeport, Thames River. Mystic River,
Cotuit and Westport, weighted prevalence values increased dra-
matically during a 50-day period from the beginning of June to the
middle of July. The proportion of individuals with infection inten-
sities of 3.0 or higher also climbed during that time interval. In
oysters from Oyster Bay, the shift from lower to higher weighted
prevalences, and from a low to high proportion of moderate to
severely diseased individuals also occurred over a 50-day period,
but it happened later in the year (Figure 5). This result suggests a
pattern of seasonal parasite proliferation for a population that be-
gins in late spring or early summer and continues over a 7-week
period, before reaching a plateau in mid- to lale summer.

Temperature Models

Temperature model parameter estimates for each site are given
in Table 3. Temperature patterns during 1997 were most similar at
the Black Rock Harbor, Oyster Bay, Thames River and Westport
sites, where mean maximum temperatures were between 21 and 23
°C. At Cotuit. on the other hand, mean temperatures peaked at 26
°C: whereas, in the Mystic River, mean temperatures reached a
maximum of only 19 °C (Figure 6). The percentage of variation
explained by the models ranged from 95 lo 98%. The on.set of
seasonal proliferation of Pcrkiiisiis iiitiriiiiis at the study sites (Fig-
ure 5) coincides with approximate ambient water temperatures of
13 °C at the Mystic River, 16 "C at Bridgeport. Westport and the
Thames River, and 20 "C at Cotuit and Oyster Bay (Figure 6).

Temperature — Disease Course Prediction Model

The prediction inodel with a lag time of 53 days gave the best
over-all fit. explaining 45.1% of the variation in the data. The
niiidel equation

y = 0.9461 1 -t- 0.000899753 CT, (x - 53)

is ba.sed on 50 samples satisfying the condition that the sampling
day minus 53 is between the mean low and mean high temperature
days. Point and interval estimates for the day of the year with mean
weighted prevalence of 2.0 are shown in Table 4.

FMAMJJASOND

Month

Month

Figure 5. Graphs of weighted prevalence over time. Top plot: Bridge-
port adults (n = 25 samples, solid black). Mystic River (n = 23 samples,
solid gray), Thames River (n = 23 samples, dashed black), Westport (n
= 25 samples, dashed gray). Bottom plot: Bridgeport juveniles (n = 10
samples, solid black), Cotuit (n = 24 samples, solid gray). Oyster Bay
(n = 23 samples, dashed black).

For comparison, separate site-based models were developed
and gave similar predictions.

DISCUSSION

Many of the characteristics of the Dermo epizootic in the north-
east are similar to those described for epizootics in other areas.
Disease prevalence is higher in adult oysters than in juveniles.
Infection levels differ among size clas.ses (ages): higher parasite
burdens are found in adult oysters throughout the year. The lower
infection intensities generally reported for juvenile oysters (Ray
1954) are believed to be the result of the relative growth rates of
host and parasite (Mackin 1951. Hofmann et al. 1995). The plateau
of high infection intensity seen in Ihe northeast during the summer

TABLE 3.

Temperature model parameter estimates i\i: mean temperature; X|„„

= time of minimum average temperature: A = amplitude) for each

studv site.

Sampling Site

M

\nv.

A

Black Rock Harbor. Bridgeport.

CT

12.19

i5.\b

10.. sy

Coluil. MA

I.V7()

2,V47

12.49

Myslic River. .Stciniiigloii. (T

1 1 ..^ 1

%).{)!

s.oo

Oyster Bay. NY

11.41)

4(1.14

11.67

Thames River. Waterford. CT

\}.l}

.W..^4

9.36

Saugaluck River. Westport. CT

11..^.^

3S.68

10.49

Modeling Dermo Progression in Field Oysters

137

has been reported for other infected populations as well (Crosby
and Roberts 1990. Soniat 1985). The simulation study by Hofmann
et al. (1995) has suggested that this buffering of infection intensity
at levels of 3 to 4 on the Mackin scale may be attributable to two
factors: (1) a decrease in parasite division rate at high parasite
density; and (2) replacement of oysters that reach a lethal level of
infection with less heavily infected oysters.

The northward spread of Perkinsiis mariiiiis into New England
was not widely anticipated, because it had been viewed as a
"warm-water" pathogen, which required minimal temperatures of
20 °C and extended periods of temperatures above 25 °C to es-
tablish an epizootic (Andrews 1988). Failure to predict the range
expansion that has occurred may be attributable in part to lack of
reliable water temperature data for oyster-growing areas. The most
northerly site in this study. Cotuit, a shallow embayment on Cape
Cod, experienced the highest mean water temperatures with tem-
peratures consistently above 25 °C for over a month (Table 3;
Figure 6), conditions similar to those reported for Delaware Bay
(Ford 1996). The lowest mean water temperatures were recorded at
Mystic, a deep-water site strongly affected by tidal exchange in
and out of Long Island Sound. Site characteristics such as tidal
exposure, water depth, tidal currents, and proximity to rivers or
substantial freshwater inflow can be more important factors in
determining the temperature characteristics of an area than its geo-
graphic location.

Infection levels in oyster populations began climbing when
water temperatures reached 13-16 °C at the Bridgeport. Mystic
River, Thames River, and Westport sites. This finding supports

Temp

TABLE 4.

Point and interval estimates for day of year with mean weighted
prevalence of 2.(1 determined for each study site.

Month

Month

Figure 6. Mean temperature curves. Top plot: Bridgeport (maximum
= 23 °C, solid black). Oyster Bay (maximum = 23 C, solid gray),
Thames River (maximum = 23 'C, dashed black), Westport (maximum
= 22 °C, dashed gray). Bottom plot: Cotuit (maximum = 26 °C, solid
black), Mystic River (maximum = 19 C, solid gray).

Day of Y

ear

Study Site

Point Estimate

Interval Estimate

Bridgeport
Mystic River
Thames River
Westport

237
215
226

208, 237
221, 254
200, 231
211. 241

earlier observations made for the Bridgeport population (Brous-
seau 1996). A later onset of parasite proliferation occurred among
the juvenile oyster populations at Oyster Bay and Cotuit. when
temperatures of 20 °C were reached, but the reason for the differ-
ence in timing is not known. Nonetheless, these results show a
significantly different pattern of infection development from those
reported in oysters from locations further south, where tempera-
tures >20 °C are required for parasite proliferation (Andrews
1988). The reason for these observed differences in the tempera-
ture-time course of the disease are unknown, but possible hypoth-
eses include: ( 1 ) the existence of a low temperature-adapted strain
of the parasite (Bushek and Allen 1996. Dungan and Hamilton
1995) and/or (2) physiological differences in the immune systems
of oysters from different geographic areas.

Soniat (1985) failed to find a correlation between water tem-
perature and prevalence or intensity of Peikiiisiis inaiiniis. but
Crosby and Roberts (1990) found a statistically significant but
weak correlation between water temperature and Dermo intensity.
In a .study that introduced lags into the relationship. Burreson and
Calvo ( 1996) found significant correlation between water tempera-
ture and both prevalence and intensity of Perkinsiis inarinus in the
Chesapeake Bay when lags of 2 to 4 months were used. The
strongest relationship was with a 3-month lag; 46% of the vari-
ability in weighted prevalence and 39% of the variability in preva-
lence was explained.

The model developed for Long Island Sound showed the
strongest relationship between cumulative temperature and Perk-
insus marimts intensity when a lag time of 53 days was used. This
result is similar to previous reports in the literature of significant
correlations between temperature and parasite intensity when tem-
perature was lagged by 60 days or more (Burreson and Ragone-
Calvo 1996). It predicted that the oyster population from the
Thames River would reach critical disease intensity levels (wp =
2) by late July/early August; whereas, similar intensity levels
would not appear in the Mystic River until a month later. The
eventual impact of the disease may depend on the time of the year
when critical disease levels are attained. Very high oyster mortali-
ties were experienced in the Thames River after mid-August 1997
(Janke pers. comm.) but no unusual mortalities were reported in
the Mystic River during the year. The oyster mortality at the
Thames River site may be attributable to high infection levels early
in the season (Fig. 5) and higher mean temperatures during the
year (Table 3). Any mortalities that may have occurred at the
uncultivated sites (Bridgeport and Westport) went largely undocu-
mented.

Water temperature is likely the most important single factor
responsible for the establishment of Perkinsiis marinus in the re-
gion of study, and although not controllable, knowledge of how the
disease responds to differing environmental temperature patterns

138

Brousseau and Baglivo

can be helpful in managing oyster stocks in the face of disease
pressure. Unlike most previous attempts to model the effects of
environmental factors on the development and activity of P. mari-
iiiis epizootics (Powell et al. 1992, Hofmann et al. 1995), this
model has the advantage of being simple to use and having modest
data requirements. It allows the grower to predict disease intensity
in shellfish beds if field temperature patterns are known. The
grower can then use this information in selecting oyster growout
beds and determining optimal harvesting times for his product.

Admittedly, one drawback of using such a simple model for
predicting parasite proliferation in the field is its failure to take into
account additional factors that may affect local patterns of disease
progression such as changing size frequency distributions within
the population, yearly variations in food supply and annual
changes in disease prevalence (Soniat et al. 1998). Also, this model
was developed using only one year of data; it would benefit from
additional tests over a longer time period to substantiate its general

applicability. In spite of these shortcomings; however, the model-
ing approach presented here shows promise, and with further test-
ing could prove to be a useful tool in industry efforts to minimize
the impact of Dermo disease.

ACKNOWLEDGMENTS

We thank S. Ford and R. Smolowitz for providing unpublished
data on Oyster Bay and Cotuit oysters. Our thanks also go to D.
Relyea, F. M. Flower & Sons, and T. Janke, Ram Island Oyster
Co. for providing oysters and valuable conversations during the
course of this study. The following students; K. Cuniff, J. Guedes,
C. Infantolino, C. Lakatos, G. LeCleir, R. Pinsonneault, and A.
Takesy are also appreciated for their help with data collection. The
final version of the manuscript benefited greatly from the com-
ments of reviewers, E. Powell and T. Soniat. This research was
supported by NRAC Grant No. 94-38500-0044.

LITERATURE CITED

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pathogen Perkinsus mariims and its effects on the oyster industry, pp.
47-63. In: W. S. Fisher (ed.). Disease Processes in Marine Bivalve
Molluscs. American Fisheries Society Special Publication, American
Fisheries Society. Bethesda. MD.

Andrews. J. D. & S. M. Ray. 1988. Management strategies to control the
disease caused by Perkinsus marinus. pp. 257-264. In: W. S. Fisher
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Brousseau, 0. J., C. Orsine, M. Rios & W. Zavadoski. 1994. Preliminary
results on Perkinsus prevalence in oyster populations from western
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Brousseau, D.J. 1996. Epizootiology of the parasite, Perkinsus nuiriniis
(Dermo) in intertidal oyster populations from Long Island Sound. /
Shellfish Res. 15;583-.'i87.

Brousseau, D. J.. J. C. Guedes, C. Lakatos, G. LeCleir & R. Pinsonneault.
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258.

Burreson, E. M. & L. M. Ragone-Calvo. 1996. Epizootiology of Perkinsus
marinus disease of oysters in Chesapeake Bay, with emphasis on data
since 1985. / Shellfish Res. 15:17-34.

Bushek, D. & S. K. Allen. 1996. Races of Perkinsus marinus. J. Shellfish
Res. 15:103-107.

Chu, F-L. E. & K. H. Greene. 1989. Effect of temperature and salinity on
the in vitro culture of the oyster pathogen Perkinsus marinus ( Apicom-
plexa: Perkinsea). / Invertebr. Palhol. 53:260-268.

Chu, F-L. E. & J. F. LaPeyre. 1993. Perkinsus marinus susceptibility and
defense-related activities in eastern oysters, Crassostrea virginica tem-
perature effects. Dis. Aquai. Org. 16:223-234.

Crosby, M. P. & C. F. Roberts. 1990, Seasonal infection intensity cycle of
the parasite Perkinsus marinus (and absence of Haplosporidium spp.)
in oysters from a South Carolina sail marsh. /)/.v. .Aquai. Org. 9:149-
155.

Craig, A., E. N. Powell. R. R. Fay & J. M. Brooks. 1989. Distribution of
Perkinsus marinus in Gulf coast oysler populations. Estuaries 12:82-
91.

Dungan, C. F. & R. M. Hamilton. 1995. Use of a tctrazolium-ba.sed cell
proliferation assay to measure effects of in vitro conditions on Perk-
insus marinus (Apicomplexa) proliferation. / Pukaryotir Microbiol.
42:379-388.

Efron, B. & R. J. Tibshirani. 1993. An inlrodiiclion lo the boolslnip. Chap-
man & Hall, Inc., New York.

Ford. S. 1996. Range extension by the oyster parasite Perkinsus marinus
into northeastern United States: response to climate change? J. Shellfish
Res. 15:45-56.

Hofmann, E. E., E. N. Powell. J. M. Klinck & G. Saunders. 1995. Model-
ing diseased oyster populations. I. modeling Perkinsus marinus infec-
tions in oy.sters. J. Shellfish Res. 14:121-151.

Mackin. J. G. 1961. Mortality of oysters. Proc. Natl. Shellfish Assoc. 50:
21-40.

Mackin. J. G. 1962. Oyster disease caused by Denytocystidium marinum
and other microorganisms in Louisiana. Tex. Inst. Mar. Sci. Puhl. 7:
132-229.

Mackin. J. G. & S. H. Hopkins. 1961. Studies on oyster mortality in rela-
tion to natural environments and oil fields in Louisiana. Publ. Inst.
Mar Sci. Univ. Te.x. 7:1-131.

Powell, E. N., J. D. Gauthier, E. A. Wilson, A. Nelson, R. R. Fay & J. M.
Brooks. 1992. Oyster disease and climate change. Are yearly changes
in Perkinsus marinus parasitism in oysters [Crassostrea virginica) con-
trolled by climate cycles in the Gulf of Mexico? P.S.Z.N.I.: Mar. Ecol.
13:243-270.

Ragone, L. M. & E. M. Burreson. 1994. Characterization of overwintering
infections of Perkinsus marinus (Apicomplexa) in Chesapeake Bay
oysters. J. Shellfish Res. 13:123-130.

Ray. S. M. 19.54. Biological studies of Dermocystidiu/n marinum. a tungus
parasite of oysters. Rice Institute, Houston, TX. 1 14 pp.

Ray. S. M. & A. C. Chandler. 1955. Dermocystidium marinum a parasite of
oysters. Exp. Parasitol. 4:172-200.

Siddall. M. E., N. A. Stokes & E. M. Burreson. 1995. Molecular phyloge-
nelic evidence that the phylum Haplosporidia has an alveolate ancestry.
Mol. Biochem. Evol. 12:573-581.

Soniat, T. M. 1985. Changes in levels of infection of oysters by Perkinsus
marinus with special reference to the interaction of temperature and
salinity on parasitism. N. E. Gulf Sci. 7:171-174.

Soniat, T. M. & J. D. Gauthier. 1989. The prevalence and intensity of
Perkinsus marinus from the midnorthern Gulf of Mexico, with com-
inenls on the relationship of the oyster parasite lo temperature and
salinity. Tulane Stud. Zool. Sot. 27:21-27.

Soniat, T. M. & E. V. Kortrighl. 1998. Estimating lime to critical levels of
Perkinsus marinus in eastern oysters, Cras.wstrea virginica. J. Shell-
fish Re.s. 17:1071-1080.

Soniat, T M., E. N. Powell, E. E. Hofmann & J. M. Klinck. 1998. Under-
standing the success and failure of oyster populations: the importance
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1165.

Joiirmil of Shellfish Research. Vol. 19. No. 1. 139-145. 2000.

OSMOTIC TOLERANCE AND VOLUME REGULATION IN IN VITRO CULTURES OF THE

OYSTER PATHOGEN PERKINSUS MARINUS

CAROLINE L. O'FARRELL,'* JEROME F. LA FEYRE,'t
KENNEDY T. PAYNTERr AND EUGENE M. BURRESON'^

^ Departmem of Fisheries Science

Virginia Institute of Marine Science

College of William and Mary

Gloucester Point. Virginia 23062
'Department of Zoology

University of Maiyland

College Park. Maryland 20742

ABSTRACT Growth rate, cell size, osmotic tolerance, and volume regulation were examined in cells of Perkinsus mariiuis cultured
in media of osmolalities ranging from 168 to 737 mOsm (6.5-27.0 ppt). Cells cultured at the low osmolalities of 168 and 256 mOsm
(6.5 and 9.7 ppt) began log phase growth 4 days postsubculture. whereas cells cultured at the higher osmolalities 341. 433. and 737
mOsm (12.7. 16.0, and 27.0 ppt) began log phase growth 2 days postsubculture. During log phase growth, cells from the higher
osmolalities 341, 433. and 737 mOsm had shoner doubling times than cells from the lower osmolalities 168 and 256 mOsm. During
both log and stationary phase growth, the mean cell diameter of cells cultured at 168 mOsm was significantly greater than cells cultured
at 341 and 737 mOsm: the mean diameters of cells cultured at 341 and 737 mOsm did not differ significantly from each other. P.
mariniis cells cultured in various osmolalities were exposed to artificial seawater treatments of 56-672 mOsm (2.5-24.7 ppt). After the
hypoosmotic treatment of 56 mOsm, cells that had been cultured in medium of low osmolality. 168 mOsm. showed only 41% mortality
whereas the cells from the 34 1-. 433-. and 737-mOsm culture groups experienced 100<7f mortality. During the hyperosmotic shock, all
of the groups exhibited mortalities of less than 107r. In P. mariiuis cells cultured in medium of 737 mOsm and then placed in a 50%
dilution, cell diameter increased 13%, which was a volume increase of 44.5%. but cells returned to baseline size (size before osmotic
shock) within 5 minutes. P. marinits cells cultured at low osmolalities can withstand both hypo- and hyperosmotic stress and use
volume-regulatory mechanisms during hypoosmotic stress. Results suggest that transferring infected oysters to low salinity will result
in strains of P. marinus acclimated to low salinity that will be able to withstand periodic events of extremely low salinity.

KEY WORDS: Osmotic tolerance, volume regulation. Perkinsus mariiuis

INTRODUCTION

Perkinsus mariiuis. a parasite of the eastern oyster, Crassostrea
virginica (Gmelin), was first reported in the Gulf of Mexico
(Maekin el al. 1950) but is now observed in C. virginica along the
Atlantic west coast from Maine to Florida and in the Gulf of
Mexico from Florida to Mexico (Andrews and Hewatt 1957,
Maekin 1962, Burreson et al. 1994a). Since the 1950s and espe-
cially since 1986, P. marinus has been a major cause of mortality
in the eastern oyster in the Chesapeake Bay (Burreson and Ragone
Calvo 1996).

The eastern oyster, C. virginica, is an osmoconformer. but the
osmotic tolerances of the parasites Haplosporidium nelsuni (MSX)
and P. marinus living within the oyster are not clearly defined
(Ford and Haskin 1988). Salinity is believed to be an important
environmental factor that regulates the prevalence and intensity of
H. nelsoni and P. mariiuis. These two common oyster parasites,
however, appear to have differing tolerances to hypoosmotic con-
ditions. Ford (1985) reported a reduced prevalence of H. nelsoni in
oysters in salinities lower than 15 ppt. Ford and Haskin (1988)
showed that some killing of H. nelsoni occurred at 15 ppt with
maximum elimination at 9 ppt. suggesting that the pathogen is

*Present address: School of Fisheries, University of Washington, Seattle.
WA 98195.

tPresent address: Department of Veterinary Science. Louisiana State Uni-
versity. Baton Rouge. LA 70803.

ICorresponding author: Eugene M. Burreson. Virginia Institute of Marine
Science, Box 1346, Gloucester Point, VA 23062-1346.

physiologically unable to tolerate low salinities. P. marinus toler-
ates salinities lower than 12 ppt, but the mechanisms that allow
survival in low-salinity environments have not been clearly de-
fined (Ragone and Burreson 1993, Burreson and Ragone Calvo
1996). Studies have shown that low salinity has a retarding effect
on P. marinus developinent (Ray 1954, Maekin 1962, Soniat 1985,
Burreson and Ragone Calvo 1996). In addition, it has been re-
ported that infection intensity of P. marinus is positively correlated
with temperature and salinity (Soniat 1985, Soniat and Gauthier
1989. Crosby and Roberts 1990, Burreson and Ragone Calvo
1996). An in vivo study of oysters infected with P. marinus de-
termined the critical salinity range for pathogenicity to be between
9 and 12 ppt. and that P. marinus was less virulent below 9 ppt
(Ragone and Burreson 1993). Also, the study reported that lower
salinities (6 and 9 ppt) delayed disease development, whereas in-
fections at higher salinities (12 and 20 ppt) increased in intensity
and resulted in higher levels of oyster mortality.

Despite these findings, little is known about the osmotic toler-
ance of P. marinus when faced with hypo- and hyperosmotic
stress. Studies with both free-living and parasitic protozoa have
shown that many protozoa have the ability to adjust their cell
volumes when faced with external osmotic changes (Kaneshiro et
al. 1969, Da Silva and Roitman 1982, Geoffrion and Larochelle
1984, Ahmad and Hellebust 1986, Andre et al. 1988, Cronkite and
Pierce 1989, Hellebust et al. 1989, Darling and Blum 1990, Dar-
ling et al. 1990). Similarly, P. marinus may also utilize physiologi-
cal mechanisms to adjust to its changing osmotic environment.
Only one previous study has been conducted on the osmotic tol-
erance of P. marinus in the absence of host influences (Burreson

139

140

O'Farrell et al.

et al. I994b|. This study reported that cells cultured at 22 ppt and
placed in extreme low-salinity treatments ofO and 3 ppt had higher
than 90% mortality. As a continuation of this work, we investi-
gated the osmotic tolerances and volume-regulatory abilities of P.
maiinus cells, which have been cultured in a range of osmotic
conditions ( 168-737 mOsm or 6.5-27.0 ppt) and exposed to vari-
ous osmotic treatments (56-672 mOsm or 2.5-24.7 ppt).

MATERIALS AND METHODS

In Vitro Cultures of P. marinus

Cultures of P. marinus were maintained in Jeronie La Peyre-
Oyster Disease Research Program- 1 (JL-ODRD-1) medium (La
Peyre et al. 1993) (approximately 737 mOsm or 27.0 ppt) without
bovine serum albumin (BSA) in a humid atmosphere at 28 °C in a
5.0% CO, incubator. Cells from the BSA-free acclimated cultures
were transfened from 737-mOsm culture medium into 168. 256,
341, and 433 mOsm (approximately 6.5, 9.7, 12.7, and 16.0 ppt)
media in a gradual procedure in which cells from 737 mOsm were
placed into 433, 433 into 341 mOsm, etc., with the stepwise trans-
fer occurring every 3 days. For culture maintenance, subculturing
occurred every 2^ wk. Cultures were seeded at a density of 5 x
10* cells per 5 mL of medium for all experiments, and during these
experiments, subculturing occurred every 2 wk. Growth curves for
the groups cultured at 168, 256, 341, 433, and 737 mOsm were
determined by obtaining cell counts with a hemacytometer (Fisher
Scientific) every day for 12 days starting the day after subculture
to determine the time period of log phase growth. The growth rate
study used cells approximately 20 generations (about 1 y ) descend-
ing from the original cultures that were first acclimated to the
different osmolalities. A generation is defined as one subculture.
Cell size experiments used cells that were approximately 25-30
generations descended from the acclimated cultures. The osmotic
tolerance experiments used cultures that were 7-10 generations
descended from the original groups acclimated to the different
osmolalities.

Culture Media

The cell culture medium used for the P. iiniriinis cultures was
the JL-ODRP-1 (La Peyre et al. 1993) without BSA. Media ( 100
mL) equivalent to 168. 256, 341, 433. and 737 mOsm were pre-
pared before each subculture for the different culture groups fol-
lowing methods described by La Peyre et al. ( 1993). In addition to
the reported constituents, the culture medium, depending on the
desired osmolality (168, 256. 341, 433, and 737 mOsm), also
included basal synthetic sea salts (0.3, 0.6, 0.9, 1.2, or 2.2 g), 0.2
g NaHCO,. and KCI (0.0061, 0.0079. 0.0097,0.01 15, or 0.0 177 g)
dissolved in 91.5 mL tissue culture-grade water.

Cell Sizes of Cultured Cells

Cell diameters of the various P. marinus groups in both l<ig and
stationary growth phase were measured by using the NIH Image
Analysis (Version 1.56) Macintosh computer program for particle
size analysis and the MediaGrabber Macintosh program with Ras-
terOps video digitizer board to capture live microscopic images
from an inverted Zeiss light microscope (4()x objective used in all
of the cell size experiments). Cell measurement teclini(.|ues with
image analysis were based on methods described by Weeks and
Richards (1993).

Baseline measurements were initially couducletl to dclermine
whether the groups cullmcd in media with osmolalities ol 168.

341, and 737 mOsm varied in cell size. For log phase growth size
distributions, cells cultured in 168-, 34 1-, and 737-mOsm media
were harvested 6 days after subcultured and transferred to 15-mL
microcentrifuge tubes. Each group of cells was declumped by
repeatedly withdrawing the cells and passing them through a 3-mL
syringe (25G 7/8-inch hypodermic needle). Cells were centrifuged
at 800 g for 15 minutes, the medium decanted, and the cells re-
suspended in 10 mL of isotonic seawater. Seawater solutions (173,
365, and 740 mOsm or 6.7, 13.6, and 27.1 ppt) that were isotonic
to the culture medium of each group consisted of 97.5 mL tissue
culture-grade water, basal synthetic sea salts (0.45, 1.05, or 2.35
g), 0.2 g NaHCO,, KCI (0.0061, 0.0097, or 0.0777 g), and 2.5 mL
HEPES buffer (original concentration = 239.02 mg/mL). After
resuspending the cell pellets in the isotonic artificial seawater so-
lutions, cell solutions were stirred with a vortex mixer (Fisher
Scientific), and a 10-(xL sample was withdrawn from each group
for cell counts using a hemacycometer. Volumes containing 1 x
10°^ cells from each group (168, 341. and 737 mOsm) were cal-
culated, and these cell solutions were added to three different cell
wells (three wells per group) in a cell well plate. From each well
of each of the three groups, three to four images were captured.
The number of cells per image ranged from approximately 40 to 70
cells. Clumped cells that could not be easily distinguished were
excluded. This cell sizing protocol was also followed to measure
cells cultured at 168, 341. and 737 mOsm in stationary phase
growth (2 wk after subculture). Mean cell diameters were calcu-
lated for the culture groups from both log and stationary growth
phase, and the relationship between culture medium osmolality
and cell diameter was examined by a one-way analysis of variance.
Significant differences between the groups cultured at the three
different osmolalities were determined by using the Scheffe post
hoc multiple comparison test.

Osmotic Tolerance

Buffered artificial seawater (ASW) treatment solutions of 56,
135. 222. 305, 386, and 672 mOsm (approximately 2.5, 5.3. 8.5,
1 1.4. 14.4. and 24.7 ppt. respectively) were prepared by dissolving
synthetic basal salts (Sigma Chemical Co.) (0.0. 0.3. 0.6. 0.9, 1.2,
or 2.2 g), 0. 1 1 76 g NaHCO,, KCI (0.0014, 0.0044, 0.006 1 . 0.0078,
0.0097. or 0.0156 g). and 2.5 mL HEPES buffer (original concen-
tration = 239.02 mg/niL) in 97.5 niL of tissue culture-grade water.
After adding these constituents, the mixtures were adjusted to a pH
of 7.5 and then filter sterilized. All of the treatment .solutions, the
BSA-free media for the culture groups, and the isotonic seawater
solutions (used for cell size experiments) were analyzed on a vapor
pressure osmometer (Wescor) to determine osmolalities. Cell den-
sity by hemacytometer and cell viability of the P. marinus cultures
were assessed in each culture group ( 168. 256. 341. 433. and 737
mOsm). To determine cell viability, a lOO-jxL subsample was
placed in a microcentrifuge tube and 10 p-l of 0.05% neutral red
stain added. After 10 min. two 1()-|jl1 aliquots were placed on the
hemacytometer. Both live (stained) and dead (unstained) cells
were counted for at least 200 cells. From each group. 2.0 x 10''
cells were added to sterile 15-mL centrifuge tubes and the volumes
raised to 7 ml, with the treatment ASW at the osmolality equiva-
lent to the medium osmolality. Then, 1 niL of each of these cell
suspensions was centrifuged at 470 i; for 5 min. The supernatant
was decanted and the pellet resuspended in I niL of each of the
ireatment osmolalities (ASW) in a 24-wcll tissue culture plate.
Thus. P. marimis cells cultured in media ol 168, 256. 341, 433. and

Pf.rkinsi!s Marinus Volume Regulation

141

737 mOsm were placed in ASW treatment osmolalities of 56, 135,
222. 305, 386, and 672 mOsm for 24 hours in 24-well microliter
plates at 28 °C in an incubator without CO,. After the 24-hour
incubation. 100 (j.L of neutral red was added and gently mixed
with a pipette tip. Mortality was assessed by counting live and
dead cells in two to three random grid fields with an inverted light
microscope (Zeiss) and a 10 x 10-mm ocular micrometer grid. The
experiment was repeated three times. Logistic regression analyses
with SAS procedure Catmod were utilized to examine the response
of the population (culture group) to the treatment osmolality and to
calculate predicted mortalities (which describe the response of
each population) with 95%- confidence intervals for each of the
culture groups at each treatment osmolality. A logistic regression
model was chosen to represent the binary response of mortality
(live versus dead). In addition, the actual live and dead cell counts
were used for calculating percent mortalities and for an analysis
that compares proportions from independent samples (Fleiss
1981).

Cell Size after Hypoosmotic Shock

Cell diameter changes following a hypoosmotic shock were
measured with the MediaGrabber and NIH Image Analysis sys-
tems. Cells cultured in medium of 737 mOsni were harvested 2-3
wk after subculture, declumped with a 3-inL syringe (25G 7/8-inch
hypodermic needle), and centrifuged at 800 g for 15 min. The
medium was decanted, and isotonic seawater was added to obtain
a volume of 10 mL. Cell density was determined with a hemacy-
tometer, and a volume containing 1x10"^ cells was added to a cell
well. A volume of 173 mOsm ASW was added to the well to result
in a 50'7f dilution of the original seawater solution. Before adding
this calculated volume of the hypoosmotic shock solution, an im-
age was captured to represent time 0. Ten to twenty seconds after
the 50% dilution, a second image was captured as time 1 . Images
were then captured at 1, 3, 5, 7, 10, 12, 15, 20. 30. 45, and 60 min
after dilution. The same cells from the same plane were captured
as images, and thus, the same population experiencing the shock
was represented. These images were analyzed with the NIH Image
Analysis system to determine cell diameters at each time interval.
The experiment was repeated five times. The first experiment used
cells 19 days postsubculture. The second experiment used cells
from a different culture 18 days postsubculture and included time
points of 0 and 10-20 sec, and 1, 3, 5, 10, 15, 20, and 30 min. The
third, fourth, and fifth experiments used cells 20 days postsubcul-
ture and were performed consecutively on the same day with cells
from the same tlask. Experiments 3. 4. and 5 included images
captured at 10-20 sec and 1. 3. 5. 10, 15, 20, and 30 min. Cell
viability was assessed with the vital stain neutral red before the
shock and 30 min after adding the shock solution. The control
experiment used cells 21 days postsubculture and followed the
protocol described above without adding the shock solution; im-
ages were obtained at 0, 1, 3, 5, 10, 15, 20, and 30 min. Cell sizes
after hypoosmotic shock were analyzed with the nonparametric
Kruskall-Wallis test to first examine the effect of each experiment.
To separate out the significant effect of each experiment but still
look at the results of all trials together to examine the overall effect
of the treatment osmolality on cell size, a mean center standard-
ization was used by subtracting the mean cell diameter (total mean
diameter for all time points within each experiment) from each
data point. A second Kruskall-Wallis test was run on the standard-
ized data to examine whether each experiment continued to have a

significant effect on cell diameter. The effect of the experiment
was no longer significant, and the experiments were pooled. A
third Kruskall-Wallis test was used to determine whether time had
a significant effect on cell diameter. Lastly, the Tukey-Kramer
multiple comparison iiosf hoc analysis was implemented to deter-
mine at which time points the mean cell diameters were signifi-
cantly different from each other. An unpaired r-test was used to
determine whether there was a significant difference between the
standardized control diameters and the standardized replicate di-
ameters (experiments pooled) both before the shock and 1 min
after the shock.

RESULTS

Growth Rate

The results of the growth rate study indicated that log phase
growth began approximately 2 days postsubculture for P. marinus
cells cultured in 341, 433, and 737 niOsm (12.7, 16.0, and 27.0
ppt) media. Cultures from the 168 and 256 mOsm (6.5 and 9.7 ppt)
media began log phase growth approximately 4 days postsubcul-
ture (Fig. 1 ). The groups cultured at the higher osmolalities of 341,
433, and 737 mOsm had shorter doubling times compared with the
groups cultured at the low osmolalities of 168 and 256 mOsm. For
the 168-mOsm cells, 35.2 h were required for one doubling and
35.7 h for the 256-mOsm cells. For the higher osmolality cells
from 341, 433, and 737 mOsm, one doubling required 22.8, 25.9,
and 24.4 h. respectively.

Cell Sizes of Cultured Cells

During log phase growth, the mean diameters (± standard error)
of P. marinus cells cultured in media of 168, 341, and 737 mOsm
were 11.8 (±0.191), 9.6 (±0.108), and 9.2 (±0.106) p.m, respec-
tively. The effect of culture medium osmolality on cell diameter

CD
O

1 —

X

a5
O

E

2

uu-

Culture Group (mOsm of media) P

•••■■••■

168 /

50-

/

— G-

256 /

— .^-

341 >°

40-

- X-

433 y^

-D-

737 / /.■*^-~-^

30-

.-/^'■■'

/ J X

//,»-^'

20-

/rtl «'

if

10-

IM / m^T^-'-e ■

/ '^. ■■■-■'' '

0-

_D=Q=d

^^4-'

10 11 12

Days Post-Subculture

Figure L Growth curve of P. marinus cells cultured in media of 168,
256, 341, 433, and 737 mOsm (6.5, 9,7, 12.7, 16.0, and 27.0 ppt).

142

O'Farrell et al.

was statistically significant (P = 0.0001). Cells cultured at 168
niOsm were significantly larger than cells at either 341 (P <
0.0001) or 737 mOsm (P < 0.0001). The differences in diameter
between cells at 341 and 737 mOsni were not significant {P =
0.1565). The mean diameters of stationary phase P. marinus cells
cultured at 168. 341. and 737 mOsm were 8.4 (±0.165), 4.7
(±0.070). and 5.1 (±0.093) |jim, respectively. As observed with
cells from log phase growth, the effect of culture medium osmo-
lality on cell size was statistically significant (P < 0.0001). Cells
cultured at 168 mOsm were significantly larger than cells at either
341 (P < 0.0001 ) or 737 mOsm (P < 0.0001 1, whereas the differ-
ence in cell diameter between the 341- and 737-mOsm groups was
only significant at the 5% level of significance (P = 0.021).

Osmotic Tolerance

Before osmotic shock, the mean viabilities of the P. marinus
cells cultured at 168, 256, 341, 433. and 737 mOsm were 88.2%.
96.2%. 99. 1 %, 99.3%. and 98.8%, respectively. After hypoosmotic
treatments, the percent mortality was lower in groups that were
cultured in low-osmolality media than in groups from higher os-
molalities (Fig. 2). For example, in the extreme hypoosmotic shock
of 56 mOsm (2.5 ppt). mortality was 41% in cells cultured at an
osmolality of 168 mOsm but was 100% in cells that were cultured
at 737 mOsm. Conversely, in the hyperosmotic shock of 672
mOsm (24.7 ppt), groups that had been cultured at low osmolali-
ties as well as high osmolalities all experienced mortalities of less
than 10% (Fig. 2). A logistic regression analysis showed that a
significant relationship existed between treatment osmolality as a
function of mortality [P < 0.001). A comparison of proportions
from independent samples test showed that the mortality response
of the 168-mOsm group was significantly different {P < 0.001)
from the mortality observed for the 737-mOsm culture group at the
56-mOsm treatment. Predicted mortalities determined from a lo-
gistic regression analysis indicated that in low-osmolality treat-

100

90

80-

^ 70-

1 60-
o

^ 50-

I 40-

°- 30-

20-

10-

Culture Group (mOsm of media)
-K- 168

-0-- 256

-h- 341
- • - 433
-B- 737

100 200 300 400 500
Treatment Osmolality (mOsm)

' I '

600

700

Figure 2. PiTccnl miir(:ilily of /'. marinus ci'lis ciilliired in nu-diii of
16S. 256. .141. 4.VV unci 7.17 iiiOsin (6.5, 9.7. 12.7. I6.(). and 27.(1 ppll
and placed in trratinent osnioiaiilii's of 56. 1.15. 222. .1(15, .186, and 672
mOsni (2.5. 5..1. 8.5. 1 1.4, 14.4. and 24.7 ppl) for 24 h. Krror bars =
standard error.

ments. groups cultured at 168 and 256 mOsm have lower mortali-
ties than the groups that had been cultured at 341. 433. and 737
mOsm.

Cell Size after Hypoosmotic Shock

Cell viability was not affected by the 50% dilution; the results
of a viability test indicated a 99% viability before the dilution
(time 0) and 97% viability 30 min after the dilution. From the first
nonparametric analysis, it was difficult to examine the effect of
osmolality on cell size because of variability between experiments
and variability between experimental conditions. After implement-
ing a mean center standardization, however, nonparametric analy-
sis indicated that each experiment did not have a significant effect
on cell diameter (P = 0.8976). Therefore, the results from each
experiment could then be pooled. The overall response to the 50%
hypoosmotic shock was an initial swelling followed by a return to
baseline size (Fig. 3). When placed in the 50% dilution treatment,
P. marinus cells that were cultured at 737 mOsm experienced an
initial swelling between 0 and 30 sec after hypoosmotic shock.
Cells swelled and returned to baseline size within about 5 min. The
mean diameter change during swelling was 0.7 (jim. The initial
mean cell diameter was 5.7 (xm. and thus, the percent diameter
increase during initial swelling was approximately 13%. which
was a 44.5% change in cell volume. The nonparametric analysis on
the pooled, standardized data showed that time had a significant
effect on cell diameter, with a tied P- value of <0.0001. The post
hoc multiple comparison analysis with a P < 0.05 level of signifi-
cance indicated that significant differences existed between the
following time points: 0 and <30 sec. <30 sec and 5 min, <30 sec
and 15 min, <30 sec and 20 min, and <30 sec and 30 min. The
unpaired r-test showed no significant difference between the mean
diameters of the control group and the experimental groups (all
experiments pooled) at time 0 (P = 0.2931), but there was a
significant difference at a significance level of P < 0.05 between
the control group and the experimental groups 1 min after the
shock (P = 0.0022).

DISCUSSION

Continuous cultures of P. marinus can be maintained in low-
osmolality environments (as low as 168 mOsm or 6.5 ppt). Fur-

0 5 10 15 20 25 30

Time (min)

I'lgure .1. Standardised mean cell diameter (pm) of P. marinus cells
cultured al 7.17 niOsm (27.(1 ppl) and placed in 5(l'^f hvpoosmolic
shock (arrow) wilh evperimenis 1-5 pooled and Ihe control experi-
ment. Arrow indicates aclual lime of shock: time represents lime after
shock. Krror bars = standard error.

Perkinsus Mar/nus Volume Regulation

143

therniore. cells maintained in osmolalities ranging from 168 to 737
mOsm (6.5-27.0 ppt) are tolerant of hypo- and hyperosmotic con-
ditions in the treatment range of 222-672 mOsm (8.5-24.7 ppt).
Cells cultured at low osmolalities can also withstand extreme low
osmolalities such as 56 mOsm (2.5 ppt) for at least 24 hours. Thus,
these experiments have shown that cultured cells of P. marinus can
survive both hypo- and hyperosmotic stress. During hypoosmotic
stress, cells increased in diameter, followed quickly by a return to
baseline size (size before osmotic shock), which indicates a vol-
ume-regulatory response. This response helps explain why P.
marinus continues to persist in the Chesapeake Bay despite periods
of low salinity that occur during times of high rainfall and runoff
into the tributaries.

The growth rate study showed that P. inaiiniis cells that were
cultured at osmolalities of 341. 433, and 737 mOsni (12.7, 16.0,
and 27.0 ppt) reached log phase growth before cells cultured at
lower osmolalities of 168 and 256 mOsm (6.5 and 9.7 ppt). In
addition, cells from higher osmolalities had greater rates of mul-
tiplication (shorter doubling time) during log phase than cells cul-
tured in low osmolalities. These results correspond to a study with
trypanosome cultures that showed that media of high osmolality
supported greater multiplication rates than low-osmolality media
(Da Silva and Roitman 1982).

The osmotic tolerance study indicated that P. marinus cells
cultured at low osmolalities experienced reduced mortality when
placed in extreme hypoosmotic conditions when compared with
the groups cultured at higher osmolalities. Because the cells were
already acclimated to the stress of a low-osmolality environment,
they were able to withstand an extreme low osmolality of 56
mOsm better than cells cultured at much higher osmolalities. Ap-
proximately 60% of the 168-mOsm cultured cells survived the
extreme low osmolality of 56 mOsm for at least 24 hours. In this
study, all of the culture groups had low mortalities (<10'7r) after
hyperosmotic stress. Consequently. P. marinus was more tolerant
of hyper- than hypoosmotic shock.

This research showed that the stressor did not seem to be the
magnitude of the shock, but instead the type of shock (hypo- or
hyperosmotic) and the actual osmolality of the challenge treat-
ment. For example, the 737-mOsm cells placed into 222-mOsm
treatment (a difference of 515 mOsm) had much higher mortality
than cells from 168-mOsm placed into 672-mOsm treatment (a
difference of 504 mOsm). Although the magnitude of the shock
was about the same, the hypoosmotic rather than the hyperosmotic
environment was more stressful, as indicated by higher mortality
levels. In addition to the type of stress, the actual osmolality of the
stress affected the level of mortality. For instance, cells acclimated
to 737 mOsm and placed into 386-mOsm treatment (a difference
of 351 mOsm) had much lower mortality «10'7r) than cells from
433 mOsm placed into 56-mOsm treatment (a difference of 377
mOsm), which resulted in 100'7f mortality. Although the magni-
tude of both of the hypoosmotic shocks was similar, mortality was
higher in the treatment with the lowest absolute osmolality, indi-
cating the cells may have a threshold osmolality level needed for
survival.

The results from the osmotic tolerance experiment differ from
the study by Burreson et al. (1994b), which reported much higher
mortality levels in cells acclimated to 737 mOsm and placed in the
same hypoosmotic treatments. The study by Burreson et al.
( 1994b) showed greater than 607^ mortality for cells acclimated to
737 mOsm and placed in treatments of 1 36 mOsm and 2 1 3 mOsm,
whereas this study reports 15^0% mortality in the same low-

osmotic treatments. One difference is that Burreson et al. (1994b)
used P. marinus cells cultured in medium with BSA (known as
JL-ODRP-1 media), whereas this study used cells cultured in
BSA-free medium. However, comparative experiments with cells
acclimated to either medium with BSA or BSA-free medium
showed no significant difference between the effects of the two
types of media on osmotic tolerance. Other factors that may have
contributed to the differences in mortality between this experiment
and the previous one include reported differences in experimental
design such as the age of the cells (numbers of subcultures since
isolation and initiation), growth phase of the cells, and type of
incubator used (CO, or without CO,). For example, the cells in the
previous study were transferred to an incubator without CO, for 1
week before use, whereas cells in our experiment were in an en-
vironment without CO, for only 1 day. The prolonged exposure to
an environment without CO, may have stressed the cells in the
previous study, making them more susceptible to mortality after
osmotic shock. Growth rates are reduced in cultures that have been
transferred to an incubator without CO, when compared with cul-
tures maintained in a 5.09<- CO2 incubator (La Peyre, personal
observation).

Cells cultured at the low osmolality of 168 mOsm were sig-
nificantly larger than cells cultured at the high osmolalities of 341
and 737 mOsm during both log and stationary growth phases. The
cells cultured at the high osmolalities of 341 and 737 mOsin.
however, were not significantly different from each other in size.
The difference in cell size may be due to increased water content
required to match the low osmolality of the dilute external me-
dium. A study with red coelomocytes of the euryhaline polychaete
Gtycera dibranclnata showed cells acclimated to a lower osmo-
lality had a higher "body-wall-tissue water content" and greater
cellular volume than cells acclimated to a higher osmolality (Costa
et al. 1980). An experiment with the amoeba Acanthamoeba cas-
tellanii indicated that the amount of intracellular water increased
when cells were placed in a severe hypoosmotic shock (Geoffrion
and Larochelle 1984). Similarly, the gradual acclimation of the P.
marinus cells from high- to low-osmolality media when develop-
ing low-osmolality cultures may have caused an increase in size as
water initially diffused into the cells, and the cells cultured in the
low osmolality may not have been able to completely return to
baseline size during volume regulation because of the stress of the
prolonged hypoosmotic environment. Cells must maintain certain
levels of metabolites to survive the stress of a low-osmolality
environment. These levels of solutes attract water molecules be-
cause of simple diffusion, and therefore, an increased intracellular
water content results. Studies on the erythrocytes of the bivalve
Noetia ponderosa (Amende and Pierce 1980. Smith and Pierce
1987) and a report on the euryhaline ciliate Paramecium callcinsi
(Cronkite and Pierce 1989) indicated that cells may not always
completely return to baseline (size before osmotic shock) after
volume regulation.

Alternatively, the difference in cell size of the groups cultured
at low versus high osmolalities may be due to a difference in life
stages of the groups that were measured. Cells of P. marinus
divide by schizogony with a cell increasing in size, acquiring a
vacuole, and then releasing several daughter cells (La Peyre and
Faisal 1997). This process could have been occurring with some of
the cells from the low-osmolality cultures during the cell-size ex-
periment, as both small cells and large cells with smaller cells
inside them were observed, whereas the groups measured at higher
osmolalities mainly consisted of small cells. Thus, because the

144

O'Farrell et al.

low- and high-osmolality groups had different growth rates, they
may not have been at the same growth stage when their cell di-
ameters were measured, which could account for the differences in
size between the groups. The cells from higher osmolalities were
not observed as a large parent cell dividing into several smaller
cells, but instead, one cell often appeared to divide into two (data
not shown). P. marinus cells with high growth rates appear to
divide as one small cell dividing into two cells (La Peyre 1996).
The cells at the low osmolality, however, may be larger in size
even before schizogony because of an increased internal water
content. Further studies examining the relationship between me-
dium osmolality. P. marinus growth stage, and cell size would be
useful in understanding the role of osmolality in P. marinus growth
and survival.

During the short-term hypoosmotic stress experiment in this
study, P. nmriniis cells followed a typical cell volume response
that is observed in other organisms by experiencing an initial
swelling and then shrinkage back toward baseline (Costa et al.
1980, Smith and Pierce 1987. Cronkite and Pierce 1989, Darling et
al. 1990). The results indicate that P. marinus cells do not resist
swelling during sudden or extreme external osmolality changes.
The size at the maximum swell was significantly different from the
initial baseline and the acclimated sizes. The erythrocytes of the
clam N. ponderosa exhibited a similar pattern when cells accli-
mated to 935 mOsm were placed in a hypoosmotic shock of 560
mOsm; the cells swelled, thereby increasing their volume by 50%
within 5 min followed by a gradual return toward ba.seline (Smith
and Pierce 1987). Because the cells in this study did swell and
return to baseline size, the results suggest that P. marinus regulates
the intracellular osmotic concentration to regulate cell volume dur-
ing changing external osmolalities. The results reported here along
with other studies by our laboratory (data not shown) and by

Paynter et al. (1997) on intracellular osmolytes (i.e., free amino
acids) used by P. marinus indicate that P. marinus cells utilize
volume-regulatory mechanisms to compensate for osmotic
changes in the external medium. These mechanisms enabled cells
in this study to survive a 50% dilution of the external medium. But
to better describe the specific volume-regulatory mechanisms used
by P. marinus. current studies are focusing on measuring the levels
of intracellular inorganic ions and organic molecules before, dur-
ing, and after osmotic shock to determine their role in volume
regulation.

The results of these experiments help explain why P. marinus
continues to persist in the upper portions of the Chesapeake Bay
tributaries despite periods of low salinities. Periodic increases in
stream flow causing lowered salinities have not greatly affected
the abundance of P. marinus in Chesapeake Bay tributaries (Bur-
reson and Ragone Calvo 1994, Ragone Calvo and Burreson 1995).
The fact that low salinities have not eradicated the pathogen from
these areas may be explained by the results in this osmotic toler-
ance study that indicate P. marinus can use volume-regulatory
mechanisms to adapt to changing external osmolality and become
acclimated to extreme low osmotic conditions. Transferring in-
fected oysters to low salinities may exacerbate the P. marinus
problem by allowing acclimation of the parasites to lower salini-
ties, thereby making them more tolerant of extremely low salini-
ties. As a result, strains of P. marinus that are tolerant of a wide
range of fluctuating salinities, including extremely low-salinity
environments, may develop.

ACKNOWLEDGMENTS

We appreciate the assistance of Lisa Ragone Calvo in experi-
mental design and Robert Diaz in statistics. VIMS contribution
number 2295.

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Da Silva, J. B. T. & I. Roitman. 1982. Effect of temperature and osmolariiy
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Eord, S. E. 1985. Effects of salinity on survival of the MSX parasite Hap-
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Eord. S. E. & H. H. Haskm. I98S. Comparison of /» vitro salinity tolerance
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Journal uf Shellfish Research. Vol. 19. No. 1. 147-151, 2000.

PRODUCTION OF TETRAPLOID PEARL OYSTER (PINCTADA MARTENSU DUNKER) BY
INHIBITING THE FIRST POLAR BODY IN EGGS FROM TRIPLOIDS

MAOXIAN HE, YUEGUANG LIN, QI SHEN, JIANXIN HU, AND
WEIGUO JIANG

South China Sea Institute of Oceanology
The Chinese Academy of Sciences
164 West Xingang Road
Guangzhou. China 510301

ABSTRACT All previou,s attempts to produce viable tetraploid pearl oyster (Pinctudu martcnsii Dunker) by inhibiting the first polar
body and the first mitosis have failed. This study aims to test the possibility of producing viable tetraploids by the intentional process
of crossing triploid females and diploid males following the inhibition of the first polar body. When 0.5mg/L CB was applied to inhibit
the release of the first polar body, about 16.69% of embryos developed as tetraploids, the majority of embryos were aneuploids
(65.48%); about 82% embryos developed as aneuploids in the control group (TDl ). but no tetraploid embryos were found. Ploidy of
embryos in the TDl group mainly fell between 2n and 3n. but ranged from 2n to 5n in the TDCB group. During rearing period, larvae
died heavily. At Day 51 post-fertilization. 2125 spat were harvested, averaging 0.033% of D-larvae cultured. Chromosome analysis
revealed that 1 15 one-year-old pearl oysters consisted of 28.70% diploids (n = 33), 40.87% triploids (n = 47), 1.74% tetraploids (n
= 2) and 28.70% aneuploids (n = 33) with 29, 30, 40, 41. and 43 chromosomes. Comparison of growth showed that aneuploids was
not significantly different from diploids in both shell length and body weight (f > 0.1 ). but significantly smaller than triploids (P <
0.05). This study demonstrated that the production of viable tetraploid pearl oysters with eggs from triploids is possible, and certain
levels of aneuploidy can be tolerated in this species.

KEY WORDS: Tetraploid. aneuploid. triploid. Pinctada marlensii (D.)

INTRODUCTION

Artificial triploid pearl oysters, Pinctada inartensii (D.), have
been successfully obtained (Jiang et al, 1987), Because of their
reduced gonadal development, triploid pearl oysters grow faster
than diploids (Jiang et al. 1993), and pearls cultured in triploids are
significantly bigger than pearls from diploids in pearl size, weight,
and pearl layer (Lin & Jiang 1993). On the other hand, the mor-
tality of triploids isn't different from that of diploids during the
adult stage (Lin et al. 1996). All of which suggest a promising
future for pearl culture by using triploid pearl oysters. Now pilot-
scale testing of pearl culturing in triploids is being conducted in
China. However, the method of inducing triploids by inhibiting
polar bodies rarely produces 100% triploids, and treatment of in-
duction may have deleterious effects on the survival and growth of
induced triploids. If crossing tetraploids and diploids could pro-
duce all-triploids as expected, the use of tetraploids may eliminat
these problems. Tetraploid is commonly induced by inhibiting the
first polar body, the first mitosis division or cell fusion. However,
most of previous attempts to produce viable tetraploids in several
species have failed (Stephens & Downing 1988; Diter & Dufy
1990: Guo et al. 1994; Jiang et al. 1998), which has eluded re-
searchers leading to doubt that tetraploids were inviable in shell-
fish. Tetraploid embryos of pearl oyster were produced with sev-
eral methods, including inhibition of the first polar body, the first
cleavage division with cytochalasin B (CB) or pressure, and cell
fusion with PEG, but none lived to adult age (Jiang et al. 1998).

Although many attempts to induce viable tetraploids in mol-
lusks have failed, there are a few reports of success. For example.
Scarpa et al ( 1993) produced tetraploid mussel {Mytilus gallopro-
vincialis) as an incidental product by inhibiting both the first and
second polar bodies with CB treatment. Out of 29 mussels sampled
at 82 days after fertilization, 5 were tetraploids (17%). Tetraploid
Manila clams. Tapes pliilippinarum (Adams and Reeve), were
found in offspring produced by blocking the first polar body to

induce triploids (Allen et al. 1994). Guo and Allen (1994a) re-
ported that 67% of tetraploid juveniles produced by the inhibition
of the first polar body of eggs from triploid Pacific oysters (Cras-
sostrea gigas Thunberg), and all-triploid Pacific oysters have been
produced by mating tetraploids and diploids (Guo et al. 1996).
These reports renew interests in tetraploid induction in shellfish.
This study aims to induce tetraploidy with pearl oysters by
crossing triploid females and normal diploid males following the
inhibition of the first polar body, and look into the possibility of
this intentional process to induce tetraploid peari oysters.

MATERIALS AND METHODS

Triploid pearl oysters, Pinctada marlensii (D.), used in this
study were produced from 2n x 2n crosses by inhibiting the first
polar body with CB treatment in 1996. Ploidy was confirmed by
chromosome count prior to spawning. Gametes were obtained by
dissecting gonads, and were passed through a 100 \xm screen to
remove the large tissue debris. Fertilization was conducted at 24-
25 °C. Eggs from triploid females (about 7 cm in shell length and
2.5 cm in shell width) were fertilized with sperm from normal
diploid males in 0.6%c ammonia-seawater and treated with 0.5
mg/L CB to block the release of polar body 1 (as TDCB groups).
CB treatment started at 6 min after fertilization and lasted 15 min
After the treatment, eggs were rinsed with 0.1 7f DMSO in seawa-
ter and cultured at a density of about 1/mL in filtered seawater. The
remains of feed and dead larvae were removed at regular intervals
to maintain water quality. The resulting spat were cultured in the
sea. The first treated group (TDCB 1 ) had one female parent;
the other three groups had two triploid females respectively. The
group receiving no CB treatment is as the control (TD), only the
first group had a control (TDl). All groups shared one diploid
male. The experiments were conducted on April. 23, 1998.

To examine the ploidy of embryos, samples of developing zy-
gotes of 2-cell stage were taken, and treated with 0,05% colchicine

147

148

He et al.

TABLE 1.

The ploidy

of embryonic

cells in the treated groups

and the control.

Diploid

Triploid

Tetraploid

Aneuploid

Group

(%)

(%)

(%)

(%)

TDCBl

15.89

11.21

20.56

52.34

TDl

12.00

6.00

0.00

82.00

TDCB2

13.73

8.82

22.55

54.90

TDCB3

5.83

5.85

11.65

76.69

TDCB4

5.00

5.00

1 2.00

78.00

for 15 min, then fixed with Camoy's solution (1;3 glacial acetic
acid and absolute methanol). Fixatives were changed twice. Chro-
mosomes were observed by acetic orcein stain. Briefly, drops of
fixed samples were spread on a slide, stained with 1-2 drops of
orcein stain (2% orcein in 50% acetic acid), and after 15-30 sec,
covered with a cover glass and pressed gently. Slides were exam-
ined with a LEICA DMLS microscope; photographs were taken
with black-and-white film with speed set at 100 ASA. Ploidy of
embryos were determined according to 2n = 28 ± 2, 3n = 42 ±
2, 4n = 56 ± 2, others as aneuploids (normal diploid pearl oyster
has 28 chromosomes). About 100 embryonic cells with good
metaphases were counted for chromosome analysis in each group.
When pearl oysters reached 4 to 6 cm in shell length (on June
6, 1999), 230 pearl oysters were sampled. Each was numbered and
measured for shell length (SL) and whole body weight (BW); a
piece of gill was removed for chromosomal analysis. Gill tissues
were treated with 0.05% colchicine in 50% seawater for Ih. then
treated with 25% seawater ( 1 part seawater/ 3 parts distilled water)
for 30 min, and fixed in a freshly prepared Carney's solution with
three changes of 20 min duration. The tissue was stored overnight
in fixatives in 4 °C. The next day the fixative was replaced by 50%
acetic acid, gill tissue was treated for 10-30 min. then 2-3 drops
of the resulting cell suspension were dropped onto a warmed slide
(40-50 °C) and dried. Slides were stained with 10% Giemsa
(pH6.8) for 40-60 min. Ploidy was determined by examining no
less than five chromosome metaphases with the same chromosome
numbers from gill cells. Individuals with 28. 42. and 56 chromo-
somes were classified as diploid, triploid and tetraploid respec-
tively; any derivation from the euploid chromosome numbers was
classified as aneuploid. Growth comparison between aneuploid
and ployploid was conducted by Student's r-test.

RESULTS

The female parent used in TDCBl had approximately 61.92
million eggs, almost equal to normal diploids of the same size. The
number of eggs obtained from seven triploid females in this study
varied between 2.07 and 61.92 million. The average diameter of

2b

20

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n

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Chromosome numljer
Figure 1. The distribution of chromosome number of embryonic cells
in TDCBl and TDl.

eggs from triploids was 57 jjim, 14% larger than eggs from diploids
(50 (Jim), which corresponded to about 50% increase in cell vol-
ume.

Chromosome examination showed that the ploidy level differed
greatly among groups (Table I ). On average, there were about
16.69 ± 5.68% tetraploid embryos in the treated groups, while
most of embryos were aneuploids, averaging 65.48 ± 13.75%. The
percentage of aneuploids (82.00%) in TDl was higher than that of
TDCBl (52.34%). but the percentage of polyploid (18.00%) was
significantly lower than that of TDCBl (47.667f ). no tetraploids
were found in the control. The distribution of chromosome num-
bers among embryos in TDl and TDCBl is showed in Figure I.
Ploidy of embryos in TDl mainly fell between 2n and 3n. but
chromosome numbers in TDCB I ranged considerably, from 2n to
5n. and there was a peak in 4n.

The survival of the developing eggs in several stages is pre-
sented in Table 2. Due to a heavy mortality in TDCB2 and TDl.
larvae were too few to be sampled for collecting data. The survival
of treated groups varied greatly. At 6 days after fertilization, the
mean survival rate was 24.01% of the total number of D-stage
larvae cultured. At Day 22, only 2.05% of larvae survived. At Day
51,2125 spat of 0.5cm in size were harvested. In TDCB 1,355 spat
were harvested (0.0295% of D-larvae cultured). 737 spat in
TDCB3 (0.0164% of D-larvae cultured). 1033 spat in TDCB4
(0.0531% of D-larvae cultured), the mean harvest rate was
0.033%.

After I -year culture in the sea. shell length had reached 4 to 6
cm. 230 pearl oysters were sampled for size and weight, and ploidy
determination. One hundred and fifteen pearl oysters had good
chrtimosome metaphases and their ploidy was determined, the
ploidy of others could not be determined due to too few
metaphases. Among these 115 samples, two (1.74%) were tetra-
ploids with 56 chromosomes. 33 (28.70%) were diploids, 47
(40.87%) were triploids. and 33 (28.70%) were aneuploids with
29. 30, 40. 41. and 43 chromosomes (Table 3). Reprcsentati\'e

TABLE 2.
The number of D-larvae cultured and the survival at several stages in TDCB groups.

Number of D-larvae
cultured (xlO")

% Surv

val

Group

Day 6 (D-stage)

Day 12

Day L«!

Day 22 (eyed stage)

Day 51 (spat)

TDCBl
TDCB3
TDCB4

1.2(1
4.50
1.95

30.4 1
23.17
18.45

24.78
8.50
9.99

3.31
5.90
6.31

0.96
1.63

3.57

0.0295
0.0164
0.053 1

Production of Tetraploid Pearl Oyster

149

TABLE 3.
Individuals observed and the ploidy of adult pearl oysters from induced groups of triploid females x diploid males

Total

Tetraploid

Diploid

Triploid

Aneuploid

Total

29

3(1 40

41

43

Number
Percentage

115

1.74

33
28.70

47
40.87

33
28.70

10

5 2

13

3

metaphases of ployploids and aneuploids are presented in Figure 2.
Analysis of ;-test showed tliat triploids were significantly big-
ger than diploids (P < 0.03). suppoiling our previous findings
(Jiang et al. 1991). Aneuploids were significantly smaller than
triploids in mean shell length and body weight (P < 0.05). but were
not significantly different from diploids (P > 0. 1) (Table 4) One
tetraploid had the size of 5.38 cm in SL and 22. 2g in BW, one was
5.35 em in SL and 21g in BW. The body weight distribution of 1 15
samples is presented in Figure 3.

DISCUSSION AND CONCLUSIONS

Triploid shellfish are commonly assumed to be sterile due to
their retarded gonadal development. Retarded gonadal develop-
ment and abnormal gametogenesis have been confirmed in
Pinctada martensii (Komaru and Wada 1990; Jiang et al. 1990)
and several other species. It is interesting that some female and
male triploids in mollusks can produce numeral gametes and fer-
tilize with normal diploids, even produce offspring (Allen 1987;
Allen and Downing 1990; Guo 1991; Komaru and Wada 1994; He
et al.l996). In this study, 2-year-old triploid females had between
2.07 and 61.92 million eggs. Reasonable fecundity in triploid fe-
males makes it possible to produce tetraploids through this tech-
nique, but this maybe puts breeders in the unusual position of

*fl

U

5t.

f\-

*v

rAi

i.

HSJ^^J-

Figure 2. Representative metaphases of adult Pinctada martensii (D.I.
A: 2n = 28, B: 2n + 1 = 29, C: 2 n + 2 = 30. D: 3n - 2 = 40, E: 3n - I
= 41, F: 3n = 42, G: 3n + 1 = 43, H: 4n = 56.

needing non-reproductive triploids for commercial culture. How-
ever, the previous research has demonstrated that the daily growth
rate of triploids is obviously greater than that of diploids during the
reproductive period, and there is no significant difference in most
months of the non-reproductive period (Jiang et al. 1991). This
result implies that a small proportion of matured triploids has no
obvious effect on the advantage of faster growth. On the other
hand, retarded gonadal development is not the only reason why
triploids grow faster than diploids.

The TD cross primarily produced aneuploid embryos with
chromosome number between 28 and 42, with an average ploidy of
2.5n (35 chromosomes), which agrees with previous observations
(He et al. 1996). However, no larvae survived through metamor-
phosis in our experiments. An exception is that juvenile Japanese
pearl oysters. Pinctada fticata martensii (a subspecies, Jiang et al.
1993). survived in TD with ploidy of 2n and 3n (Komaru and
Wada 1994). This result differs from the ploidy composition (2n,
3n, and 4n) of Pacific oyster offspring in TD (Guo and Allen
1994b). In TDCB, tetraploid embryos were 16.69%. yet at adult
age, only 1.74% of tetraploids survived. But, 67% tetraploid Pa-
cific oysters were produced by this method (Guo and Allen 1994a).
The percentage of adult aneuploid was 28.70%', smaller than that
of early embryo stage. The ratio of diploid and triploid in adult age
increased to 69.57% from 16.37% in early embryo stage. Spat
harvested were about 0.033% of D-stage larvae cultured. These
data suggested that most of tetraploids and aneuploids died during
rearing or culturing. Guo and Allen ( 1994a) reported that spat of
Pacific oysters were harvested from only one of three replicates,
which were about 0.0738% of the developing eggs. These showed
there was a heavy mortality of larvae produced by crossing triploid
females and diploid males. It is concluded that lower fecundity of
triploids and lower survivorship of larvae may restrict the potential
of this technique for producing viable tetraploids.

Guo ( 1991 ) suggested that the inviability of induced tetraploid
oysters might be due to a cell-number deficiency caused by the
cleavage of eggs of normal volume with a large, tetraploid nucleus.
In oysters and most other mollusks, development is mosaic. Unlike
shellfish, tetraploid fish and amphibians have been obtained; their

TABLE 4.

Comparison of body size and weight between aneuploids and
euploids in Pinctada martensii (D.).

Shell

Chromosome

Individuals

Body

length

Ploidy

numbers

observed

weight (SE)/g

(SE)/cm

Diploid

28

33

23.38(4.75)

5.07 (0.40)

Triploid

42

47

28.01 (7.80)

5.37 (0.65)

Aneuploid

33

20.62(7.83)

4.91 (0.78)

Tetraploid

56

2

21.60(0.851

5.37 (0.02)

150

He et al.

en

D)

O
CO

4

tl^

»-

t

t

26 28 30 32 34363840 42 44464850 52 545658

Chromosome numbers

Figure 3. The body weight distribution of Piiictada martensii (D.) with
different chromosome numbers.

development is not affected by the cell number deficiency prob-
ably because the development is regulative. The problem of cell
number deficiency in tetraploid embryos might be eliminated by
an increase in the egg volume. Eggs from triploids are larger than
eggs of diploids, probably led to a significant reduction in cell
number deficiency and therefor the survival of tetraploids. Pro-
duction of viable tetraploid Pacific oysters (Guo and Allen 1994a)
and pearl oysters in this study supported the cell number defi-
ciency hypothesis. But, why are tetraploids of Mytilus gallopro-
vincialis and Tapes philippiiuintm produced from eggs of diploids
viable, whereas tetraploid Pacific oysters and pearl oysters pro-
duced from diploid eggs are not? Eggs of Mytilus galloprovincialis
have a diameter of about 66-70 [jtm and eggs of Tapes philippi-
nanon are 55-60p.m, larger than the diameter of eggs from Pacific
oysters (47.8 |j.m) and pearl oysters (50 (xm). The remarkable
increase in egg volume may account for it. It may reflect species-
species difference in tolerance to tetraploidy. Although only two
tetraploids were produced in this study, this finding indicated that
tetraploidy can be tolerated in Piiictada martensii (D.), and dem-
onstrated that this method of producing tetraploids is viable.

It is seen that inhibition of the first body release increased the
ratio of polyploid embryos, which maybe result from a variety of
segregation patterns in meiosis (Guo et al. 1992b). Que et al.
(1997) reported that the pattern of chromosome segregation in
meiosis was changed when CB was applied to inhibit the polar
body in eggs from Iriploids. Four types of segregation such as

tripolar segregation, united bipolar segregation, separated segre-
gation and incomplete united bipolar segregation were observed.
Similar patterns of chromosome segregation were found in pearl
oysters (unpublished data). Guo and Allen (1994a) and Que et al
(1997) suggested the production of tetraploids was as a result of
united bipolar segregation. According to this type of segregation,
the united chromosome will undergo equational division, 42 chro-
mosomes are rejected as the first polar body, and 42 chromosomes
remain in the eggs, combining with haploid sperm (14 chromo-
somes) producing tetraploid. In the TD cross, the majority of fer-
tilized eggs went through two meiotic divisions and released two
polar bodies, the extra set of chromosomes segregated randomly.

In addition to ployploid pearl oysters, this process also pro-
duced many aneuploids. This study provided another evidence that
certain aneuploids are viable in shellfish. The viability of aneu-
ploid has been reported in Pacific oysters (Guo and Allen 1994a:
Wang et al. 1999) and Pacific abalone (Haliotis discus liaimi)
(Fujino et al. 1990). The data showed that aneuploid pearl oysters,
as a group, are not significantly different from diploids in shell
length and weight. In contrast, aneuploid Pacific abalone shows no
growth retardation, actually they are bigger than normal diploids
(Fujino et al. 1990); aneuploid Pacific oysters (3n ± n) are larger
than diploids (Wang et al. 1999), but probably because of their
triploidy, not aneuploidy.

It is interesting to note one pearl oyster with 43 (3n -i- I)
chromosomes is the largest in body size and the second largest in
body weight in this study. Guo and Allen (1994a) reported that one
of the aneuploid oysters with 38 (4n - 2) chromosomes is the
largest by whole body weight. These findings suggested that some
aneuploids have the growth advantage and the potential applica-
tion to aquaculture through breeding and selection. Certain aneu-
ploids may also be useful in genetic manipulation. For example,
trisomies and monosomies are of use of the gene transfer or gene
identification. The use of aneuploid has successfully lead to the
transfer of leaf rust resistance from a w ild grass {Aegilops umbel-
lidata) to wheat (Sears 1956).

Further research will focus on the growth and use of aneuploid
pearl oysters, and on how to raise the survival rate of tetraploids.

ACKNOWLEDGMENTS

This study was supported by "863" Project in China (#863-
819-01-03) and Natural Science Foundation of Guangdong. China
(#990315).

LITERATURE CITED

Allen. S. K. .Ir. 1987. Reproductive sterility ol IriploiJ shelltlsh and lish,
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Allen, S. K. Jr. & S. L. Downing. I'M). Performance of triploid Pacific
oyster, Cnissoslreu gigas: Gametogenesis. Cun. J. Fi.sli. Aijiiat. Sci.
47:1213-1222.

Allen. .S. K. Jr.. M. .Shpigel. S. Uning & B. Spencer. 1994. Incidental
production of tetraploid Manila clams. Tiipcs pliilippiihiniin. Ai/iiariil'
lure 128 (1-21:13-19.

Diter. A. & C. Duty. 1990. Polyploidy in the manila clams Riuliiapes
philippinarum. Chemical induction of tetraploid embryos. Aqual. Liv-
ing Resntir. 3:107-112.

Fujino. K., K. Aral, K. Iwadave. T. Yoshida & S. Nakajima. 1990. Induc-
tion of gynogenelic diploid by inhibiting 2nd meiosis in the Pacific
abalone. Hull. Jap. Soc. Sci. Fisheries 5&.\lf<5-\^f>^.

Guo. X. 1991. Studies on tetraploid induclion in tlie Pacific oyster, Cra.v-
scisirea gigas (Thunberg). Ph.D. dissertation, Unixersity of Washing-
ton. Seattle. Washington.

Guo, X. & S. K. Allen, Jr. 1994a. Viable tetraploids in Ihe Pacific oyster
[Crcis.soslreci gigus Thunberg) produced by inhihiling polar body I in
eggs from Iriploids. Mol. Mar. Biol. Bimcchnol. 3( 1 1:42-50.

Guo, X. & S. K. Allen, Jr. 1994b. Reproductive potential and genetics of
Iriploid Pacific oyster, Crassoslrea giga.'i (Thunhergj. Biol. Bull. IS7:
.109-318.

Guo, X., G. Debrosse & S. K. Allen, Jr. 1996. All-triploid Pacific oyster
(Crii.s.^oslreu gigas Thunberg) produced by maling tetraploids and di-
ploids. Aquaculture 142: 1 49- Id I .

Guo, X.. K. Cooper, W. K. Hershberger & K. K. Chew. 1994. Telraplold
induclion with mitosis inhibition and cell lusion in the Pacific oyster,
Cru-mMrea gigas (Thunberg). J. Sliclll'ish Res. 13(11:193-198.

Production of Tetraploid Pearl Oyster

151

Guo, X., W. K. Hershberger. K. Cooper & K. K. Chew. 1992b. Genetic
consequences of blocking polar body 1 with cytochalasin B in fertilized
eggs of the Pacific oyster. Crassostrea gigas: Segregation of chromo-
somes. Biol. Bull. 183:387-393.

He. M.. Y. Lin & W. Jiang. 1996. Studies on the sterility of iriploid pearl
oyster. Pimuida muriensii (D.). Tropic Oceunology 15(2):17-21 (in
Chinese).

Jiang. W., G. Li. G. Xu, Y. Lin & N. Qing. 1993. Growth of the induced
triploid pearl oyster Pinctada mariensii (D.). .\qiiaculture lll:24.'i-
253.

Jiang, W.. G. Li. Y. Lin &. N.Qing. 1987. Induced polyploidization in pearl
oyster, Pinctada martensii (D.). Tropic Oceanology 6(l):37-45 (in
Chinese).

Jiang. W.. G. Li. Y. Lin. G. Xu & N. Qing. 1990. Obser\ation on the gonad
of triploidy in Pinctada martensii (D.). Tropic Oceanology 9(1):24— 30
(in Chinese).

Jiang. W., Y. Lin & M. He. 1998. A study on induction of tetraploid in
pearl oyster. Pinctada martensii (D.). Tropic Oceanology 17(2):45-51
(in Chinese).

Jiang.W., G. Xu. Y. Lin & G. Li. 1991. Comparison of growth between
triploid and diploid Pinctada martensii Dunker. Tropic Oceanology
10(3): 1-7 (in Chinese).

Komaru. A. & K. T. Wada. 1990. Gametogenesis of triploid Japanese peari
oyster, Pinctada fiicata martensii. In: M. Hoshi and O. Yamashita
(Editors). Advances in Invertebrate Reproduction 5. Elsevier, Amster-
dam, pp. 469-474.

Komaru, A. & K. T. Wada. 1994. Meiotic maturation and progeny
of oocytes from triploid Japanese pearl oysters {Pinctada fiicata mar-
tensii) fertilized with spermatozoa from diploids. Aquaculture 120:61-
70.

Lin. Y. & W. Jiang. 1993. A preliminary study on comparison between
triploid and diploid in the cultured pearls of pearl oyster. Tropic Ocean-
ology 12(3):90-94 (in Chinese).

Lin, Y.. M. He & W. Jiang. 1996. An observation on the mortality of
triploidy in Pinctada martensii (D.). Tropic Oceanology 15(2):80-84
(in Chinese).

Que. H.. X. Guo, F. Zhang & S. K. Allen, Jr. 1997. Chromosome segre-
gation in fertilized eggs from triploid oyster, Crassostrea gigas (Thun-
berg), following inhibition of polar body 1. Biol. Bull. 193:14-19.

Scarpa, J., K. T. Wada & A. Komaru. 1993. Induction of tetraploidy in
mussels by suppression of polar body formation. Nippon Suisan Gak-
Aoii/ii 59(1 2):201 7-2023.

Sears. E. R. 1956. The transfer of leaf rust resistance from Aegilops um-
bellulata to wheat. Brookhaven Symp. Biol. 9:1-22.

Stephens. L. B. & S. L. Downing. 1988. Inhibiting first polar body forma-
tion in Crassostrea gigas produces tetraploids. not meiotic triploids. J.
Shellfish Res. 7(3):550-551 (Abstract only).

Wang. Z.. X. Guo, S. K. Allen & R. Wang. 1999. Aneuploid the Pacific
oyster. Crassostrea gigas (Thunberg) as incidental from triploid pro-
duction. .Aquaculture 173:347-357.

Jourmil of Shellfish Reseiiirh. Vol. 19. No. 1. 153-L>7. 2000.

EVALUATION OF FIVE MICROALGAL SPECIES FOR THE GROWTH OF EARLY SPAT OF
THE JAPANESE PEARL OYSTER PINCTADA FUCATA MARTENSII

KATSUYUKI NUMAGUCHI

National Research Institute of Fisheries Science
6-31 Nagai. Yokosuka
Kanagawa 238-0316. Japan

ABSTRACT To estimate the food value of five microalgal species, early spat of the Japanese pearl oyster, Pincuula fucata martensii,
were fed five algal species separately; Pcniovu lutheri. Chaeloceros calchrans. Tetruselmis tetnnhele. Nitzschiii closreriiim. and
Nannochloropsis oculata. The food value of each microalgal species was estimated from the growth rate of hinge length, dry whole
spat, dry shell weight, and dry flesh weight of spat fed each microalgal diet over 19 days in the laboratory. C. calcitrans produced the
best growth of the pearl oyster spat. P. hillwn and T. wtrathele supported moderate growth of the spat. However, growth rate of the
spat fed Nilzschia closlerium was very low and spat fed Nannochloropsis oculata showed negligible growth. These results indicate that
C. calcitrans is an appropriate microalgal diet for rearing pearl oyster spat. P. lutheri and T. tetrathele are also suitable diets for rearing
early spat of pearl oysters.

KEY WORDS: Pearl oyster spat, microalgal diet, growth

INTRODUCTION

Microalgae

Microalgal plankton is the principal food source for bivalves.
There have been many studies on the nutritional value of cultured
microalgae and their promotion of growth in marine bivalves lar-
vae, spat, and juvenile (Ostrea edulis. Enright et al. 1986a. Walne
1963; Cnissostrea virginica, Davis and Gullard 1958; Sciccostrea
commercialis. Nell and O'Connor 1991, O'Connor et al. 1992;
Pinctada fiicata martensii. Wada 1973. Okauchi 1990: Crawo-
doma gigantea. Whyte et al. 1990; Riiditapes philippinanim. Sakai
and Toba 1994),

Pavlova lutlieri (Droop) Green is the inost popular microalgal
species in Japanese bivalve culture and seed production studies
(Scapharca bnniglnonii. Ohhashi and Kawamoto 1980; Pinctada
fucata martensii. Hayashi and Seko 1986; Riiditapes philippi-
nanim. Miyama and Toba 1990. Taba and Miyama 1993; Meretrix
lamarckii, Shitomi and Kodama 1987a, Yanagida and Kodama
1988; Pseudocardiitm sachalinense, Shitomi and Kodama 1987b.
Yanagida et al. 1988).

In a previous study. I reconfirmed that Pavlova lutheri is a
suitable microalga for the growth of early spat of pearl oyster.
Pinctada fucata martensii (Numaguchi 1999). However, there is
little information that evaluates other microalgal species for the
grow th of pearl oyster spat. The aim of this study is to evaluate five
microalgal species as diets for pearl oyster spat.

MATERIALS AND METHODS

Pearl Oyster Spat

Pearl oyster spat used were produced in the Pearl Oyster Seed
Production Center of the Nagasaki Pearl Oyster Fisheries Coop-
erative Association. Spat were obtained approximately 3 months
after fertilization in the hatchery. Average hinge length of the spat
was about 3.5 mm. These spat were reared for 2 weeks in a 30-L
aquarium with water temperature 26-27 °C, salinity 30-32 ppl and
fed an algal diet of Pavlova lutheri.

The algal species used are shown in Table 1 . Algal cultures
were produced axenically in 5-L glass flasks using modified Erd-
Schreiber medium: 100 mg NaNO,, 20 mg Na,HP04 • 12H,0. 50
mgNaSiO,. lOOmg Nitrilotriacetic (NTA). 100 mg Tris (hydroxy-
methyl) aminomethane. 0.4 jjLg Vitamin B,,. 100 p-g Thiamin. I
pig Biotin. 5 mg Clewat 32 (Teikoku Kagaku Ltd. Japan; I g
Clewat 32 contains 3.8 mg Fe. 7.7 mg Mn, 1 .6 mg Zn, 0,07 mg Cu,
6,3 mg Mo. 24.7 mg B. 0.23 mg Co. and some EDTA) in 1-L of
4/5 diluted seawater. The medium was adjusted to pH 7.8 and
sterilized by autoclaving (121 °C, 15 min). All species were batch
cultured at 20 °C with 24-h illumination at an intensity of 5.000
lux.

Because cell size and volume differed for each of these mi-
croalgae, cell size and weight were measured for each species.
Their sizes were measured using a Coulter Counter (Model ZB)
and a Coulter Channelyzer-(Model C-100; Coulter Electronics
Inc. USA). The range and mode of cell diameter for each algal
species was estimated from the histogram of algal cell vol-
ume from Coulter Channelyzer. and the dry weight of each algal
species was determined as follows. Initially, the algal cell con-
centration was determined using a Coulter Counter. A known vol-
ume (20-50 mL) was then filtered though a GF/C glass fiber filter
(Whatman Ltd. England), which was preheated for 2 h at 500 °C
to remove organic substances, to collect the algal cells. The filter
was washed with 0.9% ammonium formate solution to remove
salt and dried at 110 °C for 24 h. The dry cell weight was
then calculated using the algal cell concentration and total
weight of filtered cells. The dry weight of suspended solids in
the seawater used for the experiment was also determined this
way.

Experimental Design

Twenty spat were allocated to each 2-L beaker aquarium with
seawater filtered with 1 ixm cartridge filters. During the experi-

153

154

NUMAGUCHI

TABLE 1.

List of microalgal diets used for the experiment and their cellular characteristics.

Phytoplankton

Cell

Mode

Diameter-

of Cell-

Dry Weight'

'olume'

Range

Diameter

of Cell

(Hm^)

((im)

(Mm)

(pg/Cell)

Haptophyceae

Pavlova liitheri (Droop) Green
Bacillariophyceae

Chaetoceros calcilnms (Paulsen) Takano

Nitzschia closleriiim (HER.) W.Smith
Prasinophyceae

Telraselmis lelralhele (West) Butcher
Eustigmatophyceae

Naimochtoropsis oculala (Droop) Hibberd

57

56

64

335
9

4.5-5.6

4.5-5.6
4.8-7.3

8.0-10.7

2.3-3.5

4.8

4.8
5.0

8.6

2.6

32.5 ± 2.6

70.3 ±4.1
30.9 ± 1.6

251 ± 10

4.9 ±0.1

' Cell volume was measured by Coulter Counter and Coulter Channelyzer.

- Cell diameter range and mode were calculated by the equation of a spherical body from the cell volume histogram measured by the Coulter counter

and Coulter Channelyzer.

' Values are means ± SD (n = 5).

merit, the dry weight of suspended solids in the filtered seawater
was 1.54 ± 0.56 mg/L (n = 4). water temperature was 26-27 °C
and salinity was 30-32 ppt.

Feeding trials, including an unfed control, were carried out over
19 days. Insufficient feeding will give false evaluations of food
value of the microalgae for the growth of pearl oyster spat, so each
feeding diet was supplied in excess in this experiment. Numaguchi
(1999) showed that the optimal feeding concentration of Pavlova
luteri was 2x10^ cells/mL for maximum growth of pearl oyster
spat at 2.6-3.0 mm hinge length. In this experiment, three times
the concentration of P. Iiitlwri (6 x lO'^ cells/mL) was fed to pearl
oyster spat of 3.5 mm hinge length. Other algal concentrations
were calculated from same packed cell volume as one of P. liitheri.
the packed cell volume calculated to product of cell concentrations
and cell volume. The feeding concentration of each algal species
was set as follows; P. liitheri 6 x 10"* cells/mL, Chaetoceros cal-
citransbA x lO'* cells/mL. Tetraselmis tetrathele I x lO"" cells/niL,
Nitzschia closterimn 5.4 x 10'* cells/mL, and Naiiiunhlornpsis
oculata 37.5 x lO'^ cells/mL.

Each algal diet was added to the relevant beaker each morning
at the above concentrations. Seawater in each beaker was changed
every day just before feeding to remove the remaining algae that
might have negatively affected feeding. Over the rearing period.

spat were observed to determine whether they were alive or dead.
Spat attached to the aquarium wall were regarded as alive, and
unattached spat, those with no viscera, or only a shell were re-
garded as dead. Dead spat were counted and removed from the
aquarium.

Spat Growth Measurement

Hinge length of each spat was measured at the beginning and
end of the feeding experiment using a stereoscopic microscope
with a micrometer. Growth rate of spat hinge length per day was
calculated as follows:

Growth rate of hinge length (jj,m/day) = (final average
hinge length - initial average hinge length) / rearing duration

To measure the dry weight of whole spat, the spat shell, and
spat flesh, ten spat were collected randomly from each aquarium at
the beginning and end of the feeding experiment. Each spat was
washed in 0.9'7r ammonium formate solution to remove salts and
was wiped with paper towel. Dry whole spat weight was measured
after spat were dried at 1 10 °C for 24 h on a platinum board. Dry
shell weight was measured after drying the spat on a platinum

TABLK 2.
Growth of hinge length and mortality of pearl oyster spat.

Diet

Hinge Length (pni)

Initial (I) Day)'

Final (19 Day)

Growth

Rate

Mortality

(Uni/Dayl

(%)

281

0

146

10

141

10

66

5

2

10

2

25

Chaetoceros calcilrans
Pavlova liitheri
Telraselmis lelralhele
Nitzschia closteriiim
Nannochloropsis ociiUila
Unfed control

3.405 ± 296-' (n = 20)

3.443 ± 337'' (n = 20)

3.510 ±5l8-'(n = 20)

3.338 ± .37.3" (n = 20)

3.653 ±3.W(n = 20)

3.525 ±.36()-'(n = 20)

8.745 ± 1,285'' (n = 20)
6.217 ± 881" (n = 18)
6.183 ±92r(n = 18)
4.587 ± 962" (n = 19)
3,683 ± 351" (n = 18)
3,.563 ± .346" (n = 15)

Values are means ± SD, values within a column with different superscripts were significantly different (Duncan multiple range test, P < 0.05).

MiCROALGAE AS FOOD FOR PEARL OYSTER SPAT

155

TABLE 3.
Dry weight gain of whole spat, shell, and flesh of pearl oyster spat.

Diet

Whole Spat' (pg)

Shell' (Mgl

Flesh' (pg)

Initial (0 day)

Final (19 days)

Chaeloceros calcitrans
Pavlova liitheri
Tetraselmis tel?'arhele
Nitzschia closterium
Nannochloropsis oculata

Unfed control

1,470 ±533

16.709 ± 6,250'-
9,785 ±4,180''
7,979 ± 3,994''
3,907 ± 1,447"
2,308 ± 674"
1,679 ±410"

1.255 ±452

14.015 ±4.990"
8.280 ± 3,484''
6.852 ±3.185"
3.297 ± 1.202"
2.188 ±548"
1.556 ±389"

215 ±83

2,694 ± 1,302'=

1.505 ±7 IS'

1,127 ±819"'

610 ±275"

120 ± 140"

123 ±53"

' Values are means ± SD (n = 10). values within a column with different superscripts were significantly different (Duncan multiple range test. P < 0.05).

board at 500 °C for 6 h in a muffle furnace to burn away the flesh.
Dry whole and shell weight of each spat was weighed to the
nearest 1 jig using a Micro Balance (Mettler Type M-3; Metiler
Toledo, Switzerland). Dry flesh weight was calculated by subtract-
ing dry shell weight from dry whole weight. Growth rates for the
whole spat, shell, and flesh, in dry weight per day, was calculated
as follows:

Growth rate of weight (jjig/day) = (final average

dry weight - initial average dry weight) / rearing duration

RESULTS

Table 2 shows hinge length of the spat at the beginning and
end of the experiment and growth rate and mortality of the spat
during the experiment. Chaetoceros calcitrans produced the best
growth of the pearl oyster spat in this feeding experiment. Al-
though growth rates of the spat fed Pavlova lutheri and Telra-
selmis tetrathele were about half those fed C. calcitrans. P. lutheri.
and T. tetrathele. both supported good growth rates of pearl oyster
spat. Spat growth rate with Nitzschia closterium was poor.
Moreover, there was almost no growth of pearl oyster spat fed
Nannochloropsis oculata. There was no mortality of the spat
fed C. calcitrans and 5-10% mortality of the spat fed P. lutheri.
T. tetrathele. Nitzschia closterium, and Nannochloropsis oculata.
In contrast, mortality of the unfed control was rather high
(25%).

Weight gain of dry whole spat, dry shell, and dry flesh was
greatest for the spat fed Chaetoceros calcitrans. Weight gain of the
spat fed Pavlova lutheri and Tetraselmis tetrathele was moderate.
Whereas, weight gain of the spat fed Nitzschia closterium was
poor. However, spat fed Nannochloropsis oculata and the unfed
control had very low weight gain (Table 3). Figure 1 shows the
growth rate of dry spat weight, dry shell, and dry flesh of the spat
fed various microalgal diets along with the unfed control. The spat
fed C. calcitrans had the highest growth rate compared to the other
microalgal species. In decreasing order, diets of P. lutheri, T.
tetrathele. and Nitzschia closterium promoted the growth of pearl
oyster spat. The spat fed Nannochloropsis oculata had a negative
growth rate as did the unfed control.

DISCUSSION

Good growth rates of the bivalve are obtained with various
algal cell because of their appropriate cell size for ingestion, their

susceptibility to mechanical or enzymatic digestion by bivalves,
their nutritive and biochemical composition, and their lack of toxic
cell metabolite (Babinchak and Ukeles 1979).

In this experiment, Chaetoceros calcitrans was the superior
microalgal species for maximum growth rate of pearl oyster
spat. Although Pavlova lutheri and Tetraselmis tetrathele were
inferior diets to C. calcitrans, these species supported a moderate
growth rate of pearl oyster spat. These results indicate that C.
calcitrans is an appropriate microalgal diet for rearing pearl oyster
spat; whereas, P. lutheri and T. tetrathele are also suitable diets
for this species. However, Nitzschia closterium was an unfavor-
able diet for the growth of pearl oyster spat. Nannochloropsis
oculata did not promote the growth of pearl oyster spat, sug-
gesting it is an inappropriate diet for rearing pearl oyster spat.
Wada (1973) also showed that Chlorella sp. (now classified
as Nanochloropsis) was a poor diet for pearl oyster larvae.
Walne (1963) indicated that Chlorella stigmattophora. which
has cell wall, is of little value as food for oyster, Ostrea edulis.
larvae. Babinchak and Ukeles (1979) also described that the
cell wall of Chlorella autotropphica was resistant to enzymatic
breakdown by the digestive system of larvae of the oyster, Cras-
sostrea virginica. Nannochloropsis oculata may be similarly
resistant to mechanical or enzymatic digestion by pearl oyster
spat.

The biochemical composition and nutritional components of
microalgae differ between species (Parsons et al. 1961, Epifanio et
al. 1981, Enright et al. 1986b. Whyte 1987). O'Connor et al.
(1992) found that suitable dietary algal species were different for
different growth stages of the same bivalve species. For the pearl
oyster, Wada (1973) indicated that P. lutheri was a more suitable
algal diet than C. calcitrans for larvae; however, for the spat in this
experiment, C. calcitrans was a more suitable diet than P. lutheri.
Fuilhermore, Okauchi (1990) found that Isochrysis gracilis was
suitable algal diet for pearl oyster juveniles. These results suggest
that the nutritional demands of the pearl oyster may change with its
growth stage.

ACKNOWLEDGMENTS

The author expresses gratitude to Dr. T. Horii. National Re-
search Institute of Fisheries Science, for statistical analysis of the
data. This study was supported in part by grants-in-aid from the
Ministry of Agriculture, Forestry, and Fisheries, Japan.

156

NUMAGUCHI

>

a

-o

=1

a
o

(0

bo

51

t

o

>.

(0
■D

0)
+J
(0

o
o

1000

800

600

400

200

800

600

<S 400

200

ItU

120 [ ^[
100 jH

40 ^H

: 1

1

Dry flesh weight

li.

C-cal
—on

P-lut T-tet Ni-cio Na-ocu Unfed

Micro-algal species

Figure I. Variation in growth rate of pearl oyster spat fed various niicroalgal diets. [C-cal] Chaeloceros calcilrans. |P-lutl Pavlova liitheri. |T-tetl
Tctraselmis Ulralltclc, |Ni-iio| \ilzcliia closUrium, |Na-ocu| ^tmnochloropiis oculata.

MiCROALGAE AS FOOD FOR PEARL OYSTER SPAT

157

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for the study of feeding in Crassoslrea viii>iiiic{i veliger larvae. Mar.
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Davis. H. C. & R. R. Guillard. 1958. Relative value often genera of micro-
organisms as foods for oyster and clam larvae. Fish. Bull. 58:203-304.

Enright. C. T.. G. F. Nev\kirk. J. S. Craigie & J. D. Castell. 1986a. Evalu-
ation of phytoplankton as diets for juvenile Oslreci echilis L. J. Exp.
Mar. Biol. Ecol. 96:1-13.

Ennghl. C. T., G. F. Newkirk, J. S. Craigie & J. D. Castell. 1986b. Growth
of juvenile Ostrea ediilis L. fed ChaeWceros gracilis Schutt of varied
chemical composition. / Exp. Mar. Biol. Ecol. 96:15-26.

Epifanio. C. E.. C. C. Valenti & C. L. Turk. 1981. A comparison of Phaeo-
ilacryliim iriconmmm and Thalassiosira pseudonana as foods for the
oyster. Crassoslrea virginica. Acjuacullurc 23:347-353.

Haya.shi. M. & K. Seko. 1986. Practical technique for artificial propagation
of Japanese pearl oyster (Pinclada fucata). Bull. Fish. Res. Inst. Mie.
1:36-68 (in Japanese with English abstract).

Miyama. Y. & M. Toba. 1990. Studies on the seedling production of
short-necked clam Ruditapes philippinarum Adams & Reeve-III food
value of 8 microalga for the lar\a of Manila clam Ruditapes philippi-
narum Adams & Reeve. Bull. Chiba Pref. Fish. E.xp. .Sin. 48:93-96 (in
Japanese with English abstract).

Nell. J. A. & W. A. O'Connor. 1991. The evaluation of fresh algae and
stored algal concentrates as a food source for Sydney rock oyster.
Saccostrea commercialis (Iredale & Roughley), larvae. .Aquacullure
99:277-284.

Numaguchi, K. 1999. Effective feeding concentration of the microalga
Pavlova lutheri for growth of early spat of the pearl oyster Pinctada
fiicata marlensii. J. World Aqua. Soc. 30:290-292.

O'Connor. W. A.. J. A. Nell & J. A. Diemar. 1992. The evaluation of
twelve algal species as food for juvenile Sydney rock oysters Saccos-
trea commercialis llredale & Roughley). Aquacuhure 108:277-283.

Ohhashi. H. & Y. Kawamoto. 1980. Technical development of mass cul-
ture of ark shell. Scapharca broughtonii. Rep. Tech. Develop. Sea
Farming Yamaguchi Naikai Sea Farming Center 6:80-135 (English
translation: in Japanese).

Okauchi. M. 1990. Food value of Isochrysis aff. galbana for the growth of

pearl oyster spat. Nippon Suisan Gakkaishi 56:1343.
Parsons. T. R.. K. Stephens & J. D. H. Strickland. 1961. On the chemical

composition of eleven species of marine phytoplanktons. J. Fish. Res.

Bd. Canada 18:1001-1016; 25:77-87.
Sakai. M. & M. Toba. 1994. Mass culture of Isochrysis aff. galbana V.

food value of mixture of two algal species for the spat of Manila clam

Ruditapes philippinarum. Saibai Ciken 23:1-5 (in Japanese).
Shitomi, S. & M. Kodama. 1987a. Seed production of Asiatic hard clam.

Meretrix lamarckii. Bull. Ibaragi Pref. Fish. Exp. Stn. 61:285-291

(English translation; in Japanese).
Shitomi. S. & M. Kodama. 1987b. Seed production of Sakhalin surf clam.

Pseudocardium sachalinense. Bull. Ibaragi Pref. Fish. Exp. Stn. 61:

292-300 (English translation: in Japanese).
Taba, M. & Y. Miyama. 1993. Gross growth efficiency in juvenile Manila

clam Ruditapes philippinarum fed different levels of Pavlova lutheri.

Bull. Chiba Pref, Fish. E.xp. Stn. 51:29-36 (in Japanese with English

abstract).
Wada, K. T. 1973. Growth of Japanese pearl oyster larvae fed with three

species of microalgae. Bull. Natl. Pearl Res. 1Mb. 17:2075-2083 (Japa-
nese with English summary).
Walne. P. R. 1963. Observations on the food value of seven species of

algae to the larvae of Ostrea edulis. 1 . feeding experiments. J. Mar.

Biol. Ass.. U.K. 43:767-784.
Whyte, J. N. C. 1987. Biochemical composition and energy content of six

species of phytoplankton used in mariculture of bivalves. Aquaculture

60:231-241.
Whyte, J. N. C, N. Bourne & C. A. Hodgson. 1990. Nutritional condition

of rock scallop, Crassadoma gigantea (Gray), larvae fed mixed algal

diets. Aquaculture 86:25—40.
Yanagida. Y. & M. Kodama. 1988. Seed production of Asiatic hard clam.

Meretrix lamarckii. Bull. Ibaragi Pref. Fish. Exp. Stn. 62:338-346

(English translation; in Japanese).
Yanagida. Y.. S. Shitomi & M. Kodama. 1988. Seed production of Sakha-
lin surf clam. Pseudocardium sachalinense. Bull. Ibaragi Pref. Fish.

Exp. Stn. 62:347-357 (English translation; in Japanese).

JoKimil of Shellfish Rfsfurch. Vol. 19. No. 1. 159-166, 2000.

COMBINED EFFECTS OF TEMPERATURE AND ALGAL CONCENTRATION ON SURVIVAL,
GROWTH AND FEEDING PHYSIOLOGY OF PINCTADA MAXIMA (JAMESON) SPAT

DAVID MILLS

Acjiiciciilturt' Co-operative Research Centre

Northern Territory University

Darwin Aqiiacultiire Centre

Department of Primary Industry and Fisheries

Darwin, Northern Territory, Australia

ABSTRACT To determine a suitable culture environment to maximize growth and survival, Pinctmla maxima spat were held at 36
combinations of temperature and algal concentration for 14 days within a flowthrough system. Survival was greatest between 23 °C
and 32 °C. with 35 °C resulting in high mortalities. The optimum temperature range for P. maxima spat found in this study agrees well
with the observed temperatures which limit the natural distribution of P. maxima in Australian waters. Survival of spat was highest
at low algal concentrations. Growth was optimal between 26 °C and 29 °C and at 54 algal cells |j.L"'; however, growth was still
acceptable at algal concentrations as low as 12 cells (jlL"'. The organic content increased with feeding rate and was positively correlated
with specific growth rate. Spat filtration rate declined at high feeding rates, whereas grazing rate increased, with a commensurate
decline in conversion efficiency. It is recommended that P. maxinui spat be maintained within the temperature range of 26 "C to 29
°C and at algal cell densities between 12 and 54 cells |xL"' to maximize spat performance and minimize algal wastage.

KEY WORDS: Pinaada maxima

INTRODUCTION

Following high mortalities of adult silver-lip pearl oysters.
Pinctada maxima (Pass et al. 1987), during the 1970s and early
1980s, there has been a focus on hatchery production for ongrow-
ing and pearl production (Rose et al. 1990). Although there has
been considerable work published on P. maxima spat production
and husbandry, there has been no published investigation into the
role of either temperature or food concentration on spat culture
success.

Temperature is regarded as one of the most potent factors af-
fecting growth and metabolism of marine poikilotherms (Griffiths
and Griffiths 1987) and has been shown to effect many physiologi-
cal processes of bivalves, such as filtration, feeding, respiration,
reproduction, and growth (Bayne et al. 1976).

There is evidence that the Australian distribution of both P.
margaritifera and P. maxima is limited to areas where seawater
temperatures range from 18 °C to 32 °C (Hynd 1955, Pass et al.
1987). High mortalities of up to 80% of wild fished P. maxima in
Western Australia (WA) in the late 1970s and early 1980s were
attributed to reduced disease resistance during periods of low tem-
perature (Pass et al, 1987), This effect may have been enhanced by
the change in temperature (from 19 °C to 26 °C) between the
collection grounds and the farms during transportation.

Rose et al, (1990) investigated the seawater temperatures of the
main Western Australian fishing beds for P. maxima and recorded
bottom temperatures of between 20 °C and 26,8 °C, Surface tem-
peratures showed a larger range ( 19.8-32,3 °C).

There have been several feeding rates used for P. maxima spat,
without any real evidence as to their suitability. Rose ( 1990) rec-
ommended twice-daily feedings of 55-65 cells jjlL"'. whereas
Rose and Baker ( 1994) fed spat a mixed algal diet at 40-285 cells
(xL"' depending on spat size. The algal concentration dynamics in

Current address: Paspaley Pearling Co. P/L. P.O. Box 338, Darwin, NT
0801, Australia,

a batch-fed system will vary with tank size, stocking density, and
feeding frequency.

The aim of this experiment was to quantify the effects of tem-
perature and food availability on the growth, survival, and feeding
of P. maxima and to determine suitable regimes for spat culture.

Materials and Methods

Experimental animals

P. maxima spat were obtained from the Darwin Hatchery Proj-
ect on December 17, 1996. These spat averaged 11 ±0.7 mg with
an initial organic content of 10.9%. Mean initial shell height (dor-
soventral measurement) was 4.3 mm and ranged from 3.3 to
5.2 mm.

System

The system used in this experiment was a modified and scaled-
up version of that described in Mills (1997). There were three
experimental blocks, each consisting of six 100-L temperature-
controlled waterbaths and six elevated 100-L reservoirs. Each res-
ervoir contained an algal suspension at one of the experimental
concentrations and supplied one replicate in each waterbath
through a submersible pump and 4-mm tubing manifold. The flow
rate into each replicate was controlled with 2-L h"' irrigation
drippers. Thus, each waterbath in each block contained one repli-
cate tray at each algal concentration, giving one replicate of each
combination of temperature and food concentration per block and
three replicates of each combination. Different-colored pegs were
used to identify replicates of each algal concentration within a
waterbath. Both the incoming air and algal suspension were pre-
heated to the correct temperature before entering the replicates by
first passing through approximately 4 m of the 4-mm supply lines
coiled within the waterbath. Spat were held individually within
histological cassettes, with 10 spat in each replicate tray.

Trays were supported by the rim in rectangular holes cut into a
32-mm-thick sheet of extruded polystyrene foam, which was
floated within each waterbath and acted as both tray support and

159

160

Mills

insulator. Irrigation drippers and trays were replaced at weekly
intervals to prevent fouling.

Outflow from each replicate was collected from a 4-mm tube
connected to the tray outlet. A 60-|j.m mesh feces retainer pre-
vented contamination of the outflow sample with feces and/or
pseudofeces.

Temperatures and algal concentrations

There were six temperatures used in the experiment: 20, 23. 26.
29. 32. and 35 °C. This temperature range was chosen as it en-
compasses the annual range experienced in Darwin Harbour (23-
32 "O and is similar to that recorded at Broome (Rose et al.l990).
The ambient room temperature was maintained at 20 °C (the mini-
mum temperature attainable in the isothermal room), and all of the
waterbaths at higher temperatures were heated with 300-W glass
immersion heaters. Temperatures of the replicates were checked
twice daily and maintained within ±0.5 °C of the desired experi-
mental temperature. Standard errors of experimental temperatures
were generally ±0.15-0.2 °C.

The initial algal concentrations delivered from the reservoirs
were 10. 20. 40. 80. and 160 cells \}.L'\ with unfed controls
exposed only to filtered seawater. All seawater was filtered to 1
|xm and then passed through a carbon filter to remove possible
contaminants from the intake seawater. which was drawn from a
commercial shipping wharf.

The algal concentration range was chosen to encompass the
optimums found for P. fiicata (Numaguchi 1994a. Krishnan and
Alagarswami 1993) and for P. maxima by Bellanger (1995). and
also the commonly used feeding rates in commercial hatcheries
(80-100 cells |jiL"'). However, the effective algal concentration
surrounding the oyster may be better represented by the concen-
tration in the outflow (Hildreth & Crisp 1976). That the outflow
algal concentration was the same as that within the replicate was
confirmed by comparing the algal concentration in samples taken
from several replicates at 4-h intervals, with the subsequent con-
centration in the outflow. Thus, the results presented are given
relative to the effective (outflow) rather than initial algal concen-
tration. The mean effective outflow concentrations were 0. 6. 12.
23. 54. and 1 10 cells (j.L"'. Algal feeding reservoirs were cleaned
and refilled daily with the appropriate algal suspension.

.Spat were fed an algal diet of equal cell numbers of Tahitian
Isdclirysis sp. (T. Iso) and Chaetoceros miwUeri. These species
have been shown to support good growth and survival of pearl
oyster spat (Taylor et al. 1997. Southgate et al. 1998). Mean algal
cell dry weights were 19 and 20 pg. respectively, and were deter-
mined by the method of Epifanio (1979). Algae was cultured in
20-L carboys using f/2 medium with a 12:12 photoperiod and
harvested at the late exponential stage.

Preliminary trial

A preliminary trial was conducted to determine whether there
was any change in the delivered algal concentration due to cell
damage, growth or sedimentation, or differences in delivery vol-
umes due to differing friction head loss within the system. One
block was run over 24 hours without animals in the system at an
initial algal concentration of 100 cells jjiL"'. Outflow volumes and
initial and final algal concentrations were compared by two-way
ANOVA using a significance level (c») of 0.2.

There were no significant differences in either the xolumes
delivered {P = 0.56) or the oulllow concentration (/' = 0.69).

Initial and final algal concentrations were not significantly differ-
ent (P = 0.78). Subsequent trials showed that the volume of
suspension delivered by a dripper was independent of the number
of drippers on the manifold line at least up to n = 8. This occurs
as the pumps used were not positive displacement, but rather main-
tained a set delivery pressure and possessed a delivery capacity
exceeding that of the combined number of drippers. Thus, the
number or status of drippers on a manifold line had no effect on the
delivery pressure (and hence output) of individual drippers.

Sampling

As it was not logistically possible to weigh and measure all of
the 1080 spat and sample all of the 108 outflows during a single
day. both the startup and sampling procedures were sequenced
over 3 days. A full block could not be sampled on 1 day, as there
was not enough floorspace for all of the outflow collection vessels;
hence, a part of each block was sampled on each day. The se-
quence used was designed to sample one replicate of each treat-
ment combination on each day.

At days 7 and 14, each spat was removed from the histological
cassettes, washed in seawater of the appropriate temperature to
remove adherent feces, and then weighed to the nearest 0.1 mg and
measured to the nearest 0.1 mm (DVH). Outflow volume and
collection duration were recorded, and outflow samples were pre-
served with Lugols iodine for later counting and calculation of
filtration and grazing rates.

Growth was expressed as the daily tissue weight specific
growth rate (SGR) and was calculated according to the following
equation:

SGR = (Ln final tissue weight) - (Ln initial tissue weight)/time
[days|)x 100

The organic content was calculated as:

Organic content (%) = loss on ignition/dry weight x 100

Filtration rate (FR) was calculated by the formula of Bayne et
al. ( 1976) for flowthrough systems:

FRa h"') = a- CO/CO x F

where CI = the initial algal concentration. CO = the final algal
concentration, and F = flow rate (1 h"').

This was converted to a weight-specific filtration rate by the
following equation:

FR (1 h''g-') = FR (1 h"')/tissue weight (g)
Algal grazing rate for each replicate was calculated as:

Grazing rate (Vr ) = C (g)/dry tissue wt (g)

Conversion efficiency was calculated b\ the equation:

Conversion efficiency (Vr) = SGR/giazing rate x organic

content of algae x 100
(modified trom De Sil\a and Anderson 1995)

Statistical analysis

All responses to temperature and algal concentration were ana-
lyzed using a two-way factorial ANOVA model. Although
samples for growth, filtration, and grazing rates were taken at
weekly intervals, because of the plasticity of the spat organic con-
lent the analysis was conducted only on the final values, as these
responses were all calculated relative to spat tissue weight.

Temperature and Algae Effects on P. Maxima

There was very low survival at 35 °C (1.1%). and this tem-
perature was excluded from subsequent analysis because of the
low number of surviving individuals and hence very high selection
pressure on the population.

Any survival percentage data that were not normally distributed
was arcsine transformed before being analyzed (Underwood
1981).

Homogeneity of variances were tested with Cochran's test with
the critical value (CV) calculated as:

CV = largest variance/ S variance

and was compared with a tabulated value with (replicates/
treatment) - 1 and (treatment levels - 1) degrees of freedom.

Normality of response distributions were tested on residuals
(yij-Yi) using the Shapiro-Wilk W test (Zar 1984). If variances
v\ere found to be unequal, or the data had a non-normal distribu-
tion, appropriate transformations were done. Comparison of means
w as only undertaken if the overall ANOVA model was significant,
using the Fisher's protected least significant difference test. Rela-
tionships between measured responses and culture conditions were
examined using regression analysis. A P value <0.05 was consid-
ered significant for all statistical analysis.

Results

Survive/

Both temperature {P < 0.0001) and algal concentration {P =
0.03) affected spat survival, with temperature being a much stron-
ger influence than algal concentration. There was no significant
interaction (P = 0.16). Within the naturally occurring temperature
range for Darwin Harbour (23-32 °C), there was no effect of
temperature on mortality rates, and survival was greater than 90%
(Fig. 1). At 35 °C mortality was almost complete (98.97f ). and at
20 °C survival was significantly lower than at 23 °C. 26 °C. 29 °C,
and 32 °C.

Spat at the lowest algal concentration of 6 cells |xL"' showed
the highest survival, which was significantly higher than those at
23 and 110 cells (aL"' and unfed spat (Fig. 2). It is notable that the
only survival at 35 °C was at the lower algal concentrations (6 and
23 cells |jiL~').

Growth

Tissue SGR responded significantly to both temperature and
algal concentration (P = 0.0008 and P < 0.0001. respectively), but
there was no significant interaction (P = 0.73). Growth increased
with increasing temperature up to 29 °C and then declined from
29 °C-32 °C (Fig. 3). The decrease in growth at 32 °C indicates
that this is approaching the upper temperature limit for the species,
as confirmed by the very low survival at 35 °C. Growth at 29 °C
was more than twice that at 20 °C. and growth at 32 °C was similar
to that at 23 °C. Tissue weights of unfed spat declined, indicating
that there was no significant nutritional value in the filtered sea-
water. In fed spat, growth increased progressively with increasing
algal concentration up to 54 cells |jlL''. after which there was a
slight but nonsignificant decline (Fig. 4). Growth at 54 cells jjiL"'
was approximately 50% greater than that at 6 cells [i.L'\ This
illustrates that P. ma.xima spat are capable of moderate growth
even at very low algal concentrations.

Organic content

Increases in algal concentration were reflected in significant
increases in spat organic content {P = 0.0002), from 9.4% in
unfed spat to >13% at the highest concentrations (Fig. 5). The
organic content of spat cultured at 6 and 12 cells |jiL~' was not
significantly different from the initial value of 10.9%. Temperature
had no significant effect on spat organic content (f = 0.8). nor
was there any significant interaction between temperature and al-
gal concentration (P = 0.8). There was a positive correlation
between the SGR of spat and their organic content (;' = 0.51, P

100

100-

>

120

Temperature (°C)

Figure 1. Survival off. maxima spat after 14 days" culture at various
temperatures. Figures show means ± standard error. Means with simi-
lar subscripts are not significantly different (/* > 0.(15).

Algal concentration (cells ijI' ')

Figure 2. Survival of P. maxima spat after 14 days culture at various
algal concentrations. Figures show means ± standard error. Means
with similar subscripts are not significantly different iP > O.OS).

162

Mills

T3

O
CO

Temperature (°C)

Figure 3. SGR of P. maxima spat at various temperatures. Figures
show means ± standard error. Means with similar subscripts are not
significantly different (P > 0.05).

< 0.0001), with faster-growing spat having a higher organic con-
tent.

Feeding

The two different algal species comprising the diet were
counted separately in outflow samples obtained during the first
week. There was no preferential selection by the spat for either of
the species at any concentration or temperature, and the ratio of T.
Iso to C. miielleri in the outflow was not significantly different

c
B

o
o

o

'c

CO

en

100 120

Algal concentration (cells |J I' ')

Figure 5. Organic content [% of dry weight) of P. maxima spat after
14 days at various algal concentrations. Figures show means ± stan-
dard error. Means with similar subscripts are not significantly differ-
ent (P > 0.05).

from 1 , Henceforth, for the calculation of feeding rates it was
assumed that there was no selection for either species by the spat.
The weight-specific filtration rate increased with moderate in-
creases in algal concentration up to 23 cells (xL"', before declining
significantly at 54 and 1 10 cells |j.L"' (Fig. 6). Filtration rate was
highest at 20 °C (54 L h"'g"') and declined significantly with
increasing temperature to 17.3 L h"'g"' at 32 °C (Fig. 7). This is
an inverse response to that shown in most bivalve studies, in which

(0

5-

2.5

-2.5-

150

Algal concentration (cells |jL ' )

Figure 4. ,S(;R of P. maxima spat after 14 days at various algal con-
centrations. Figures show means ± standard error. Means with similar
subscripts are not signincantly different (/' > (1.05).

125

Algal concentration (cells pi" )

Figure A. Filtration rates of /'. maxima spat at various algal concen-
trations. Figures show means ± standard error. Means with similar
subscripts are not .significantly different (/' > 0.05).

Temperature and Algae Effects on P. Maxima

163

Temperature (°C)

Figure 7. Filtration rate of P. maxima spat at different temperatures.
Figures show means ± standard error. Means with similar subscripts
are not significantly different (P > 0.05).

filtration rate generally increases with increasing temperature up to
a maximum, with a subsequent decline.

The grazing rate (G) increased linearly with increasing algal
concentration from approximately 15% at 6 cells |jlL"' to 136% at
1 10 cells tJ-L"' following the equation:

C% = 0.74 X algal concentration + 18.2 (;-- = 0.96) (Fig. 8)

With the increase in grazing rate, there was a corresponding de-
cline in the gross conversion efficiency from approximately 38%
at 6 cells jxL"' to 5% at 1 10 cells |j,L'' (Fig. 8). As the production
of pseudofeces was not quantified, the gross conversion efficiency
refers to growth from algae grazed, rather than ingested. The loga-

o

c

o

0)
it:
(D

c
g
(/)

>

c
o
O

O

-| 1 1 I 1 r

0 20 40 60 80 100 120

Algal concentration (cells \i ''^)

Figure 8. Grazing rate and conversion efficiency of P. maxima spat at
different algal concentrations. Figures show means ± standard error.
Means with similar subscripts are not significantly different iP > 0.05).

rithmic decline in conversion efficiency iCE) can be described by
the equation:

CE = [128.4 X algal concentration (cells [jlL'' )1-"^-"

As the grazing rate increased greatly in response to increasing
algal concentration without a commensurate increase in growth
rate, it seems likely that the majority of the algae grazed were
rejected as pseudofeces. Neither the grazing rate nor conversion
efficiency was affected by temperature, nor was there any signifi-
cant interaction of the two factors.

Discussion

Survival

The pattern of survival exhibited in this experiment is consis-
tent with the observations of Pass et al. ( 1 987 ), who concluded that
the natural distribution of P. maxima was limited to areas with a
seawater temperature range of 18-32 °C. Although it was not
possible to examine the effects of temperatures below 20 °C. it is
apparent from the significantly lower survival at 20 °C that the spat
were approaching their lower tolerance limit. The very low sur-
vival at 35 °C indicates that this is above the upper tolerance limit.
as foreshadowed by the reduced growth at 32 °C. The reduction in
survival at 20 °C. and the very low survival at 35 °C. reflects the
results of Doroudi et al. (1999) for P. margaritifera larvae, in
which there was no development at either 20 °C or 35 °C. The
optimum range for P. margaritifera larvae was found to be be-
tween 26 °C and 29 °C, which is slightly narrower than that found
for P. maxima spat in the present study (23-29 °C). Numaguchi
and Tanaka (1986) investigated the effects of temperatures from
7.5-35 °C on P. fucata and concluded that the lower and upper
tolerance limits were 15 °C and 32 °C, respectively, with the
optimum range being from 17.5 °C to 28 °C.

Temperatures on both the natural pearl oyster beds and pearl
farms in WA would occasionally be high or low enough to be
deleterious to P. maxima spat, as they generally range from 20 °C
to 32 °C (Rose et al. 1990). In the Northern Territory (NT), sum-
mer inshore water temperatures are generally 31-32 °C from De-
cember to April (Padovan 1997) and may be approaching stressful
temperatures. Wada (1953) observed that the temperature of the
main deepwater commercial oyster grounds in the N.T. was the
same throughout the water column and averaged 29 °C during the
summer. The lower temperatures offshore may be more conducive
to growth and reproduction than warmer inshore waters.

As surface seawater temperatures reach higher levels than bot-
tom waters, pearl oysters hung from longlines (typically 1-3 m
deep) may experience temperatures greater than 32 °C. especially
in sites farther to the north such as the Kimberly region in northern
WA. and the NT, and in calm sheltered bays. These temperatures
may be at or near the tolerance limit of the species, and although
there does not seem to be any direct mortality associated with
them, there could be significant sublethal effects such as reduced
growth, reproductive output, pearl quality, and resistance to stress-
ors such as cleaning and handling. This may account for the lower-
quality spawnings and gametes produced by oysters from farm
longlines compared with those from the offshore fishing grounds
(Rose et al. 1990), although this may be partly attributable to the
frequent cleaning and handling of farm oysters.

High water temperatures may be more critical in NT hatcheries,
as during the summer air temperatures commonly reach 34 T. and
this may be reflected in the temperature of the rearing tanks. Cur-

164

Mills

rently. the industry addresses this by shading of the seawater sup-
ply and rearing tanks; however, temperatures may still reach
stressful levels. Stressed larvae and spat may be more susceptible
to disease and suboptimal water quality. Algal cultures used to
feed larvae and spat are grown at temperate water temperatures
(20-24 °C). and problems may occur as a result of the abrupt
increase in temperature experienced by the algae when it is trans-
ferred from the algal culture system to the spat culture tanks.
Minaur (1969) noted that P. lutheri became moribund at tempera-
tures above 30 °C and attributed this as a major problem in at-
tempts to rear P. maxima larvae and spat.

Numaguchi (1994b) considered that an increase in the mortality
rates of farm-held P. fucata was due to su.stained elevated tem-
peratures of greater than 28 °C. This may have been related to
temperature stress combined with reduced food intake and higher
metabolic costs, as the same author demonstrated that filtration
rate declines dramatically at temperatures above 28 °C (Numagu-
chi 1994c). whereas catabolic losses increase at higher tempera-
tures (Numaguchi 1995). There appears to be a similar process in
P. maxima. Assuming that the organic content of the spat at day 7
was the same as that at the beginning of the experiment, then at
35 °C the mean filtration rate over all algal concentrations at 35 °C
was 9.6 L h~'g"' compared with >30 L h"'g"' at temperatures
from 20-32 °C. Unfed .spat showed greater tissue weight loss with
increasing temperature. This strongly suggests that if the experi-
ment had been extended, there would have been large mortalities
in unfed spat, particularly at the higher temperatures. The combi-
nation of these two factors indicate that at 35 °C there is reduced
feeding and increased metabolic costs, leading to negative growth
and increased mortality.

Algal concentration had a small but significant effect on spat
mortality, with the survival rates at 6 cells jxL"' significantly
greater than those of unfed spat and those at 23 and 1 1 0 cells p,L" ' .
The only surviving spat at 35 "C were unfed, and at the lower fed
algal concentration. This is probably a result of the stimulatory
response in filtration rate at higher algal concentrations (Fig. 6).
resulting in an increase in metabolic rate and energetic costs, and
con.sequently a more rapid loss of body tissue and death.

Grawlh

As previously found by Mills ( 1997), there was no significant
relationship between the initial weight of spat and the subsequent
SGR within the spat si/e range used (/' = -0.11. P = 0.08).

Increasing growth with increasing temperature up to an asymp-
totic point, followed by a rapid decline, is a common pattern for
bivalves (Bayne et al. 1976). A similar pattern was shown by a
temperate strain. P. fucata (Numaguchi and Tunaka 1986), ex-
po.sed to temperatures ranging from 7.5 °C to 35 °C.

The relationship between preasymptote temperalure and
growth of P. maxima spat can be described by the second-order
polynomial equation:

Growth (SGR 'A day ' ) = -19.85 -I- 1.6 temperature - 0.027
tempeialure' (/~ = 0.98)

From this equation, the calculated temperature of zero growth
is 17.7 °C. which agrees well with the esliniale of the lower tem-
perature limiting the distribution of /'. iiia\uua by Pass et al.
(1987) of 18 "C.

Growth relative to algal concentration showed a pattern similar
to that obtained by Numaguchi ( 1994a) for P. fucata spat, in which
growth increased rapidly up to a concentration of 20 cells p.L ',

with no advantage of further increases in concentration. In this
study, growth increased rapidly up to 12 cells (j.L"', with further
increases in concentration producing slightly higher growth. This
lower threshold value for P. maxima may reflect the very high
filtration rates attainable in this species. Yukihira et al. (1998b)
calculated that the algal concentration for maximum scope for
growth (SFG) of adult P. maxima was 20-30 cells |jiL"'. Above
this concentration the calculated SFG declined and was negative
above 90 cells \xL'. The decline was primarily due to a large
reduction in the absorbed energy as a result of a decrease in ab-
sorption efficiency. Similarly, the SFG of P. maxima spat calcu-
lated by Bellanger (1995) predicted that growth would decline at
T. Iso concentrations greater than 17 cells |xL~'. Although the
results of the present study indicate that low algal concentrations
may still promote good growth, there is no evidence that higher
algal concentrations are detrimental. Bellanger (1995) could not
separate pseudofeces from true feces, and consequently the ab-
sorption efficiency was underestimated. It is possible that spat may
have different energetic characteristics than adults. Alternatively,
there may be an acclimation to higher algal concentrations over
time, which cannot be compensated for in short-term studies.

Preasymptotic growth at various algal concentrations can be
described by the equation:

Growth (SGR^f day"') = 2.921 +0.05 algal concentration

(cells p.L"') - 3.795"'* algal concentration (cells (xL~')~

(r- = 0.93)

This equation predicts a maintenance ration (where SGR = 0)
of 1.45 cells |j.L~'. This value is substantially lower than that of
Bellanger ( 1995), where the SFG was calculated to be 0 at 7.6 cells
|i.L~'. Given that growth was still quite high at 6 cells (xL~' in this
experiment, the estimate obtained in this study would appear to be
a more accurate estimate of the maintenance concentration. Yuki-
hira el al. (1998a) calculated that the SFGs for P. maxima and P.
margaritifera were very high even when exposed to very low algal
concentrations (5 cells |jiL"'). Hayashi and Seko (1986) monitored
chlorophyll a levels and growth of P. fucata on pearl farms in
Japan and concluded that maintenance requirements were met by
algal concentrations that result in chlorophyll a levels of 3 (ig L~',
whereas levels of 4-5 p.g L"' were required for good growth and
reproductive development. This was equivalent to 6 and 10 cells
(xL"' of P. lutheri. respectively. P. fucata appears to be adapted to
more eutrophic conditions than P. nuixima. as chlorophyll a levels
in Darwin Harbour are generally from 0.5 to 3 ixg L~' (Radovan
1997) and similarly low levels occur in the main fishing grounds
off Broome (Rose et al. 1990). Mean chlorophyll a le\els recorded
at the Broome fishing grounds were from 0.3 to 0.9 |j.g L ' (Rose
et al. 1990), equivalent to approximately 0.(j-1.8 cells jxL"'. Thus,
the calculated maintenance ration in this experiment agrees well
with observed food levels in the field. The ability to thrive in
conditions of very low food concentrations is due to the ability to
process very large volumes of water (\'ukihira et al. 1998a). As
growth rates increased by 50% from 6 to 54 cells jiL"', it may be
that growth of oysters in the field is commonly food limited.

Organic coiiltnt

Organic content is rarely determined in bi\al\e studies, and
there are few references to pearl oyster spat. Given that spat or-
ganic content increased with both algal concentration and SGR,
and that SGR increased comnicnsuralcly with algal concentration,
it is possible that the increase in organic content is related to the
SGR rather than the algal concentration per se. This would agree

Temperature and Algae Effects on P. Maxima

165

with the results of Taylor et al. (1997), who found that the organic
content of P. wu.xiimi spat increased with higher SGR despite a
reduced weight-specific ration fed to the fastest-growing spat.

Feeding

A limitation of the experimental method utilizing histological
cassettes is that it is impossible to collect biodeposits: thus, the
estimation of ingestion, absorption, and conversion efficiencies
cannot be carried out. The filtration rates obtained in this experi-
ment are very high compared with those of the previous experi-
ment and other published filtration rates. This is probably due to
the small size of the spat used, as the weight-specific filtration rate
generally declines with increasing size according to the equation:

FR (L h"') = aW'^ (Bayne et al. 1976).

Thompson and Bay ne ( 1 972 ) found that the weight exponent for
mussels less than 1 g dry weight was higher than that of larger
mussels. Thus, the very high filtration rates found in this study
may reflect the very small spat used. Yukihira et al. (1998a) dem-
onstrated that smaller P. inaxiiiia and P. maigantifera spat had a
considerably higher filtration rate than larger animals. Using the
equation developed by Yukihira et al. (1998a). CR = 10.73
W^^'^, the predicted filtration rate for the mean final spat ash-free
dry weight used in this trial (0.0023 g) would be 0.115 L h^'
compared with a measured value of 0.09 L h"' .

Yukihira et al. (1998a) found that the filtration rates obtained
for P. maxima and P. margaritifera were among the highest re-
corded for any bivalve species. A similar result was found for P.
margaririfera by Pouvreau et al. (1999).

Reduction in filtration rate is a common response to increasing
algal concentration (Bayne et al. 1976). The trends found in this
study are similar to the findings of Bellanger ( 1 995 ), in which algal
concentrations above 17 cells |jiL"' resulted in a decrease in the
weight-specific filtration rate. In both cases, filtration rates initially
increased with moderate increases in algal concentration and then
declined at higher algal concentrations.

Reduction in filtration rate with increasing temperature is con-
trary to results from other studies on pearl oy.sters. Numaguchi
(1994c) found that the filtration rate of 2-year-old P. fucata (5.7-
6.1 cm shell height) increased with increasing temperature up to
the tolerance limit before sharply declining. A similar pattern was
shown for P. fucata spat (Numaguchi 1994a). The unusual results
in this study are probably an artifact of the differences in size of
the spat at the various temperatures, and a high rate exponent.
Mean final dry tissue weight at 20 °C was 7 ± 1 .5 mg and increased

commensurately with temperature up to 21 ± 1.7 mg at 32 °C.
Filtration rates of the largest spat (17 L h^'g"' at 32 °C) ap-
proached those obtained by Mills (1997) of 7.3 L h 'g"' and those
of Bellanger (1995) (11.9 L h"'g-'). To eliminate any potential
effects of different-sized spat, a short-term experiment would have
to be conducted with similar-sized spat at all temperatures.

The lack of a temperature effect on grazing rate may also be an
artifact of the variations in spat size at the different experimental
temperatures, as the increase in filtration rate by smaller spat
would have masked the increase in grazing rate with higher tem-
peratures.

The increase in grazing rate with increasing algal concentration
reflects the relatively low corresponding decrease in filtration rate.
As growth did not increase proportionally, the extra algae grazed
at higher algal concentrations was probably rejected as pseudofe-
ces. This was consistent with observations that pseudofeces were
produced at initial algal concentrations above 20 cells |a.L~'. A
similar observation was made by Bellanger (1995) at algal con-
centrations greater than 22 cells |xL"'. This increased rejection as
algal concentration increases is reflected in the decrease in con-
version efficiency from approximately 37% at 6 and 12 cells (xL~'
to approximately 5% at 54 and 1 10 cells |jlL"'.

Both the grazing and growth rates in this study are substantially
higher than those recorded by Mills ( 1997), suggesting that growth
is heavily dependent on food intake. This is consistent with the
higher growth at higher algal concentrations.

In the present study the growth rate at 6 cells |xL"' was still
quite high, although Bellanger (1995) predicted it to be negative.
It is likely that the metabolic costs in that study were overesti-
mated, leading to erroneous conclusions as to the predicted growth
at various algal concentrations.

On the basis of the results of this study. P. maxima spat should
be maintained at temperatures between 26 °C and 29 °C, and algal
concentrations between 12 and 54 cells |jlL^'. Within these culture
parameters, spat growth and survival will be optimal, and the
efficient utilization of algal cultures will be maximized.

ACKNOWLEDGMENTS

This research was funded by the Co-operative Research Centre
for Aquaculture and supported by the Darwin Aquaculture Centre
of the Department of Primary Industry and Fisheries. Northern
Territory. The author is grateful to the staff of the Pearl Oyster
Propagators and the Darwin Hatchery Project, who supplied the
spat and microalgae.

REFERENCES

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121-159. In: B. L. Bayne (ed.). Marine Mussels: Their Ecology and
Physiology. Cambridge University Press. Cambridge. UK.

Bellanger. J. 1995. Effects of food density on the feeding physiology and
resultant scope for growth for the juvenile pearl oysters, Pinctada
maxima (Jameson) and P. all^ina (Lamark). Honours Thesis, James
Cook University, Townsville, Queensland. Australia.

De Silva. S. S. & T. A. Anderson. 1995. Fish Nutrition in Aquaculture.
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Doroudi. M. S.. P. C. Southgate & R. J. Mayer. 1999. The combined effects
of temperature and salinity on embryos and larvae of the black-lip pearl
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Epifanio. C. E. 1979. Growth in bivalve molluscs: nutritional effects of two
or more species of algae in diets fed to the American oyster. Crasso-

strea virginica (Gmelini and the hard clam Mcrceiuiria mcrcinariail..).

Aquaculture 18:1-12.
Griffiths. C. L. & Griffiths. R. J. 1987. Bivalvia. pp. 1-88. In: Animal

Energetics, vol. 2. Bivalvia through Reptilia. Academic Press. London.

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Hayashi, M. & K. Seko. 1986. Practical technique for anificial propagation

of Japanese pearl oyster. Bull. Fisli. Res. Inst. 1:39-68.
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Mar. Biol. As.roc. UK 56: 1 1 1-120.
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Mills. D. 1997. Evaluation of histological cassettes as holding containers
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pearl oyster, Pinciada fiicala martensii (Dunker, 1850) in Ohmura Bay.
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Numaguchi, K. 1994c. Fine particles of the suspended solids in the pearl
farm. Fish. Eng. 30:181-184.

Numaguchi, K. 1995. Effects of water temperature on catabolic losses of
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Numaguchi. K. & Y. Tanaka. 1986. Effects of salinity on mortality and
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Western Australian silverlip pearl oyster, Pinctada ma.xima (Jameson)
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son). Asian Fish. Sci. 10:1-8.

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Yukihira. H.. D. W. Klumpp & J. S. Lucas. 1998b. Comparative effects of
microalgal species and food concentration on suspension feeding and
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Journal of Shellfish Rcscanh. Vol. \9. No. 1, 167-174. 2000.

INFLUENCE ON UPTAKE, DISTRIBUTION AND ELIMINATION OF SALMONELLA
TYPHIMURIUM IN THE BLUE MUSSEL, MYTILUS EDULIS, BY THE CELL SURFACE

PROPERTIES OF THE BACTERIA

BODIL HERNROTH,' ANNHILD LARSSON,^ AND LARS EDEBO^

' The Royal Swedish Academy of Sciences

Kristineherg Marine Research Station

SE-450 34 Fiskebdckskil, Sweden
'Department of Radiation Physics

Sahlgrenska University Hospital

SE-413 45 Goteborg. Sweden

Department of Clinical Bacteriology

Sahlgrenska UniversityHospital

SE-413 46 Goteborg. Sweden

ABSTRACT This study was carried out to investigate whether the cell surface charge of Salmonella typhinutrium could influence the
kinetics of uptake, distribution, and elimination in the blue mussel. Mytiliis edulis. The bacteria (1 |j.m) were labeled with '"'Tc"' in the
presence of stannous fluoride. Two different concentrations of stannous fluoride were used to produce differences in the cell surface
charges of the bacteria. A set of mussels in the investigation were also given "'Sn-labeled microspheres (15 p,m) together with bacteria
to compare the impact between particle size and cell surface properties on the distribution kinetics. The distribution of radiolabeled
particles in the mussel was followed and analyzed with a computer-aided gamma camera that can detect two isotopes simultaneously.
Finally, the mussels were dissected and the radioactivity in the fractions was measured with a well-shielded Nal(Tl) detector. The
reduced cell surface charge of S. typhimitrium enhanced the preingeslive selection on the gills or labial palps as well as the postingestive
selection in the digestive glands in such a way that it became similar to the microspheres, despite the size differences. The uptake of
the bacteria labeled in the presence of less stannous fluoride was significantly lower. However, the subsequent absorption of these
bacteria in the digestive gland was greater, because the recovery of radioactiv ity outside the digestive tract was higher than for the more
manipulated bacteria and the inicrospheres. Likewise, the elimination of the more manipulated bacteria was similar to that of the
microspheres and significantly higher than that of the less affected bacteria. It is concluded that the cell surface properties of bacteria,
possibly the charge, influence the uptake, distribution, and elimination in M. edulis and that this factor could have the same influence
as size on the uptake capacity.

KEY WORDS: Mytilus edulis. bivalves, molluscs. Salmonella typhimurium. gamma camera, ""'Tc'^-labeled bacteria, surface, uptake,
preingestive selection, postingestive selection

INTRODUCTION microalgal metabolites have been proved to influence mussel feed-

Because of their efficient filter-feeding mechanism, bivalves '"§ ^^^avior (Ward & Targett 1989). This indicates that the lamel-

are capable of accumulating large numbers of microorganisms '''^''^"'-"'^ t"^^'^« have some ability of premgestive selection, pre-

from the surrounding water. ^""^^'''>' °" '^e gills or the labial palps, which is not only related

Problems related to microbes in terms of pathogenic bacteria '° ^'^^ t."' ^'^^ '° o*er particle characteristics. In addition, the

and viruses in bivalves can be a major deterrent when developing possibility of postingestive selection in the digestive tract has been

a sustainable plan for utilization of coastal resources. The plank- suggested (Shumway et al. 1985, Smith & MacDonald 1997).

tonic bacterium Vibrio parahaemolyticus, as well as bacteria as- Digestion in invertebrates includes extracellular and intracellular

sociated with fecal pollution, such as Salmonella. Shigella, and digestion processes. The extracellular digestion is a fast process

Closlndiwn spp.. have caused numerous outbreaks of gastroen- "lat dominates during intestinal digestion. It results in low absorp-

teritis in connection with consumption of seafood (Matches and tion efficiency and pooriy digested "intestinar" feces. The intra-

Abeyta 1983, Rodrick and Schneider 1991. Wilson and Moore cellular digestion is a slow process in the diverticular folds of the

1996). In addition, viruses, such as the small, naked viruses (e.g. stomach. The epithelial cells of the folds phagocytose and digest

Calici and Norwalk viruses), hepatitis A. and enterovirus, are con- small particles with high efficiency resulting in good absorption

sidered as health hazards in utilizing bivalves for food (Sinder- and well-digested "glandular feces" (Morton 1973. Decho &

mann 1990. Enriques et al. 1992. Cliver 1997). To improve risk Luoma 1991). The hemocytes of the bivalves are also functioning

assessment and develop satisfactory control methods in respect to in nutrient digestion and transport as well as in internal defense

public health, the basic knowledge about the regulating mecha- (Cheng 1984). They contain numerous lysosomes capable of re-

nisms for uptake, distribution, and elimination of microbes in bi- leasing hydrolytic enzymes and reactive oxygen radicals (Winston

valves has to be improved. et al. 1991). Birkbeck and McHenery ( 1982) showed that bacteria

A study on filtration capacity of particles in Mytilus edulis such as Micrococcus roseus and Staphylococcus aureus, resistant

(Mohlenberg and Riisgard 1978) showed a marked decline in the to the lysozyme of M. edulis, were rejected intact, whereas Es-

uptake of particles smaller than 7 jxm. which fell to 20% at 1 [xm. cherichia coli. M. luteus and Bacillus cereiis. which were sensitive

Also, it has been shown by Allison et al. (1998) that particles to lysozyme, were killed after ingestion. Rogener & Uhlenbruck

(3^0 jj-m) enriched in metals were rejected in M. edulis, and (1984) found that invertebrates recognized and bound so-called

167

168

Hernroth et al.

heterophilic antigens or ubiquitous chemical structures sucli as
lipopolysaccharides and zymosan, wiiicli are often present on the
surface of microorganisms. This indicates that recognition of cell
surface characteristics of the prey might be a regulating mecha-
nism for selection in the digestive gland.

We hypothesize that not only size but also cell surface prop-
erties of particles might influence the uptake and the subsequent
distribution and elimination of microorganisms in ^4. ediilis. To
test this, a gamma camera technique was used for in vivo recording
of blue mussels with respect to uptake and elimination of radio-
labeled Salmonella lyphinnirium (ca. 1 p,m) and microspheres ( 15
|xm). After the gamma camera experiment, the radioactivity in
dissected fractions of the mussel tissue was measured with well-
shielded Nal(TI) detector to follow the distribution into different
organs. The labeling procedure for S. nphimiiriiim has been de-
scribed in the previous study by Hernroth et al. (2000). Bacteria
with different cell surface charges were obtained by using different
concentrations of stannous fluoride.

MATERIALS AND METHODS

Bacterial Strain and Growth Conditions

S. typhinnuium .^95 MR 10. a nonvirulent. chemotype-Rd mu-
tant (Edebo et al. 1980) was grown for 16 h in glucose broth
(Lindberg et al. 1970) (pH 7.0-7.2) at 37 °C on a rotary shaker
(200 rpm). The bacterial suspension was washed three times by
centrifugation (2000 rpm. 10 min. 4 °C) in 3 mL 0.9% NaCI to
remove the culture medium. The pellet was resuspended in 2 mL
0.9% NaCl (2.5 x 10'^' mL"').

Radiolabeling and Chemical Modification of Bacteria

Stannous fluoride (SnF-,) was used to reduce "'"'Tc"' to facilitate
the labeling of the isotope (Perin et al. 1997). It also binds to
protein structures intracellularly as well as at the cell surface
(Rhodes 1991 ). It has been shown in a previous study by Hernroth
et al. (2000) that stannous fluoride can chemically modify the cell
surface charge of 5. typhimiiiiiim. The bacteria showed differences
in electrophoretic mobility when 80 jjLg SnF, and 800 |j.g SnF,.
respectively, were u.sed in the labeling procedure. The mobility for
bacteria treated with 80 [x.% SnF, (5.4 x 10~" m" x V"'s"') was not
significantly different from untreated 5. typliimurium (4.7 x 10 "
m- X V"'s~'), whereas the mobility for bacteria treated with 800
SnF, was significantly reduced (2.3 x 10"'^ nr x V^'s"'). The
same treatment to label bacteria with different cell surface prop-
erties was used for this study.

One milliliter of bacteria suspension (2.5 x 10" mL"') was
mixed with 2 mL of 37 "C 0.9% NaCl containing 80 or 800 fjLg
SnF, and then incubated with approximately 50 MBq ""Tc"'-
pertechnetate for 20 min at 37 "C on a rotary shaker (200 rpm).
After incubation, the bacteria were cenlrifuged and washed three
times. Ascorbic acid (0.25 mg niL ') was added to the washing
solution to prevent reoxidation of the reduced ""Tc"'. The bacteria
were resuspended in 1 mL 0.9% NaCl.

To control the cell size and shape of S. lypliinuoiiini and ob-
serve possible formation of aggregates, the batches of labeled bac-
teria were inspected in a microscope (12.5 x lOOx magnificalion)
before feeding took place. As references, unlabeled .V. lypliimiirinni
bacteria were used.

V(a/)i/i/v of '"'•"Tc-lxiheled Bacteria

The viability of the bacteria was chccketl using a lluorescence
assay (LIVE/DEAD «((( Light 1^1 Bacterial Viabililv Kit. Molecular

Probes. The Netherlands). Triplicates of ''''Tc'":80 and '"*Tc"':800
were compared with unlabeled bacteria from the same culture,
using epifluorescence microscopy (Zeiss Axioscop, Exciterfilter
BP450-490. Dichroic reflector 510, and Barrier filter LPS 159,
Zeiss, Germany). The suspension of bacteria was diluted to 10^ x
mL"' in sterile filtered (Schleicher & Schuell, Keene, NH, FP
030/3) seawater (32 PSU) and incubated with the fluorescence
probe for 15 tnin at 12 °C. The intact plasma membranes of live
bacteria give green fluorescence, whereas compromised mem-
branes of dead ones give red fluorescence (Haugland 1996). The
viable bacteria were calculated as part of 100 cells.

Mussel Experiment

The experiment was carried out during April and May 1998.
Blue mussels, M. edulis, were collected at 1 m depth in the Aby
Fjord, on the west coast of Sweden (tidal amplitude 20 cm). The
salinity, when sampling, was 28 PSU, and the temperature was
6 °C. Mean shell length of the 32 mussels was 7.6 ± 0.8 cm, shell
width was 3.4 ± 0.7 cm, and flesh wet weight was 1 1 .6 ± 3.5 g. The
mus.sels were cleaned from epiphytes and stored in running sea-
water (32 PSU and 8 °C). Two days before the experiment started,
mussels were placed individually in hanging baskets in filtered
(Millipore, 0.3 jjim) seawater (32 PSU, 1 2 °C) for adaptation to the
experimental .setup, in which it was necessary to mix the water by
magnetic stirring. The water (eight mussels in 15 L) was ex-
changed daily. It was continuously oxygenated, and the mussels
were fed the nanoflagellate Isochrysis ffulhana. One hour before
the start of the experiment, each basket with one mussel was
transferred to a beaker with 700 mL filtered (Millipore, 0.3 m) and
oxygenated seawater (32 PSU, 12 °C). The beaker was placed on
the magnetic stirrer in front of the gamma camera. Ten minutes
before addition of bacteria, the mussel was given 1 niL of a sus-
pension of 10" niL"' of/. gLilbana as a trigger for filtration, and the
activity was visually confirmed. Thereafter, the radiolabeled mi-
crospheres and/or bacteria were added to the beaker. The final
concentration of bacteria was approximately 3 10" niL"', and ap-
proximately 32.4 MBq of '"Tc"' activity was added to the water.
The concentration of the microspheres was approximately 1.5 10*
niL '. and the activity of "''Sn was approximately 1.1 MBq. All
values used in this study have been corrected for the half-life of the
isotopes.

Sixteen mussels were given 5. typhinnuium labeled in the pres-
ence of 80 |jLg SnF, (designated '"Tc'":80). and 16 mussels were
given S. typhimurimn labeled in the presence of 800 (xg SnF,
(designated ''''Tc"':8()0). Within each group of mussels. 50% were
given "\Sn-labeled microspheres (NEN TRAC microspheres, Du
Pont) together with the bacteria. The nondegradable microspheres
were made from styrene-divinyl ben/ene resins and were uniform
in size (15 (xm) (designated "'Sn:ms).

Each mussel was exposed to radiolabeled microbes/
microspheres for 5 hours. During this time the radioactivity was
continuously recorded by the gamma camera. Then the mussel was
carefully rinsed and repositioned in front o{ the camera, but now in
clean seawater for recording of elimination of radioacti\ ity. Fi-
nally, the mussel was dissected and the radioactivity of the organs
and tissue tractions was measured using a well-shielded Nal(Tl)
detector ( 15 cm in diameter; Nuclear Enterprises, UK) in a low-
activity laboratory. The dissected fractions were the fimbriae part
of the mantle, one pair of gills, one pair of palps, pericardial gland
including the pericardium, gonail. one pair of kidneys, digestive

Influences of 5. Typhimurum in M. Eduus

169

gland, terminal part of intestine, crystalline style, mantle, posterior
adductor muscle, foot, anterior adductor muscle, and retractor
muscle. The digestive gland was transected to distinguish the an-
terior part, including the esophagus, stomach, and diverticular
folds (designated the stomach) from the posterior part, including
the direct and recurrent intestine and the blind sac (designated the
liver). The terminal intestine (designated the gut) was dissected
separately. The dissected part of the kidneys was one of the lon-
gitudinal canals that lie on either side of the body at the root of the
gills and the closest connected tissue (designated the renal). As the
transit time of the radioactive particles in the mussel tissue was
unknown, the dissection was done with different time lags, and
the.se were randomized among the mussels. Meanwhile, the mus-
sels were stored as under the pre-experimental conditions, with a
daily exchange of water but without any food supply. The time
lags were 5. 20, 28, and 54 hours for the mussels fed on ''''Tc"':80
(n = 4 in each group). The mussels fed on ''''Tc'":800 were dis-
sected after 5 (/i = 4). 20 {n = 4). 28 (/i = 2). 54 (n = 2). and
68 (II = 4) hours. It should be pointed out that these differences in
numbers of mussel dissected at different times were not planned
but were a result of the time-consuming dissection.

Gamma Camera Technique

The gamma camera technique (MAXI II General Electric.
Hermes Sy.stem NuD, Nuclear Diagnostic. Hilgersten. Sweden)
was used. The camera continuously visualizes the distribution of
radioactivity in the mussel. Furthermore, by outlining the regions
of interest (ROIs) on the screen, the radioactivity in the region was
quantified and listed and displayed as curves of activity versus
time. The ROIs chosen in this study were the images of the stom-
ach and gut regions. The parameters were calculated from the
stomach region as follows:

Uptake = the fraction (% ) of the initial amount of the given
radioactivity that was accumulated when the maximum value in
the ROI was reached. The initial radioactivity in the beaker rep-
resented the given activity. The maximum value was normalized
to the initial radioactivity to avoid differences due to variations in
the given activity, differences due to the distance between the
mussel and the camera, and differences in the geometry of the
mussels.

Elimination = the reduction (%) from the maximum value
measured in the ROI. The reduction was determined after 20 hours
of measurement. These values were normalized to the maximum
value in the ROI to avoid differences in the uptake capacity.

Statistical Analysis

The influence on the uptake and the elimination of the bacteria
due to the chemical treatments (80 or 800 p.g SnF,) and to the
presence or absence of "'Sn:ms was analyzed using two-way
analysis of variance (ANOVA) and a Tukey test to allow multiple
comparisons (Zar 1995). The variance of the uptake and elimina-
tion of '"'Tc"':80. ''^Tc'^:800. and "''Sn:ms was analyzed using
one-way ANOVA and a Tukey test (Zar 1995). To obtain inde-
pendent measurements for the ANOVA analysis, the '™Tc'":80 and
'*"Tc'":800 groups included mussels fed exclusively on bacteria,
and the ' '^Sn:ms group included the same numbers of individuals
randomized from the mussels fed simultaneously on bacteria and
microspheres.

Because of the differences between ''"Tc"':80 and ''"Tc"':800 in
time lags before dissection, some of the mussels were excluded to

equalize the groups when analyzing the distribution of the mi-
crobes in the mussel tissue. The excluded mussels were the four
fed on ''^^Tc"':800 dissected after 68 h and two mussels randomly
chosen among those fed on "''Tc"':80 dissected after 28 and 54 h.
respectively. The Mann-Whitney rank sum test (Sokal & Rohlf
1995) was used to compare variances in content of radioactivity in
the different fractions. Pearson product moment correlation
(Snedecor & Cochran 1989) was used to investigate whether an
increased amount of radioactivity in mussel tissue outside the di-
gestive tract was related to a decrease in the digestive gland. In all
of the statistical analyses, Sigma Stat version 2.0 (Jandel Scientific
Software. San Rafael. CA) was used.

RESULTS

Viability of """"Tc-Labeled Bacteria

The viability of the unlabeled bacteria was 96.4 ± 1.4%. The
viability of '"'Tc'":80 was 95.9 ± 1.7%. and for ''''Tc'";800 it was
89.6 ± 4.2%. The microscopic inspections showed the same size
and shape of the bacteria compared with the unlabeled bacteria,
and no aggregates were observed.

Uptake and Elimination of Radiolabeled Bacteria and Microspheres in
M. edulis

When mussels were given S. typliiumriuin labeled with a small
amount of stannous fluoride ('^''Tc"':80; Fig. I A), as well as ra-
dioactive microspheres ("''Sn:ms; Fig. IB), the uptake of bacteria
in the stomach was slow and small and in the gut it was neariy
inconspicuous, whereas the microspheres rapidly accumulated in
the stomach and later in the gut. The uptake when '''^Tc"':80 was
tested alone (Fig. IC) was similar to that of the bacteria in the
mixture (Fig. I A). Mussels given bacteria labeled with more
stannous fluoride ('"'Tc'":800: Fig. 2A) as well as "'Sn:ms (Fig.
2B) showed similar uptake kinetics for the two kinds of particles.
A similar pattern appeared for '^"Tc"':800 alone (Fig. 2C).

Two-way ANOVA confirmed that the presence of the ' '""Snims
affected neither the uptake nor the elimination of ^''Tc"':80 and
''''Tc'":800, but the difference due to the amount of stannous fluo-
ride used for the labeling of the bacteria was significant (Table 1 ).
The uptake (Fig. 3) and elimination (Fig. 4) varied on an individual
basis. Still, the statistical analysis showed that the variances in the
processing of ''"Tc'":80 compared with '"Tc"':800 and "'Sn:ms
were significant. The uptake of "'Snims and of ''''Tc'";800 was
significantly higher than that of '^''Tc"':80 [one-way ANOVA. F =
32.4; degrees of freedom (df) between subjects = 1; P < 0.001,
post hoc Tukey test]. Similarly, the elimination of ' '-'Snrms and of
'''^Tc'":800 was significantly higher compared with that of ''''Tc'":
80 (one-way ANOVA. F = 20.8, df between subjects = 5; P <
0.001. post hoc Tukey test).

The Distribution of Radioactivity in M. edulis

The radioactivity in the different organs and tissues showed
great differences between individuals. Despite this, the differences
between the three different groups (""Tc'":80. ""Tc'":800. and
"■^Sn:ms) were pronounced. Most of the activity of """Snims in
the digestive tract (Fig. 5) was recovered in the stomach, in the
liver, and in the gut. The activities of ''''Tc"':80 and '"'Tc'^rSOO
were even higher in the stomach and liver. "'^'Tc'":80 could hardly
be detected in the gut. The gills contained significantly more

170

Hernroth et al.

Fig. 1A: ^'Tc'":80

Fig. IB

1200

0,6 1
0,4

Fig. 1C;

^'Tc'iSO

0,2

_^

0

mmmA^mJii

600
Time (min)

1200

Figure 1. Chart lines showing the radioactivity measured in the ROIs (stomach and gut) in a mussel which was simultaneously given 5.
typhimuriiim labeled with ""Tc"' in the presence of 80 ng SnF, ((A) '"'Tc"':801 and microspheres labeled with '"Sn ((B) "'Sn:ms). (C) Shows a
mussel which was given ""^Tc^iSO only. The amount of radioactivity is normalized to the initial amount given to the mussel.

"''Tc'^iSO than ■''Tc"':800 (ANOVA, df = 15. P = 0.028). There
were low values of ''*Tc"\ and ' ''^Snims were almost undetectable
in the mussel tissue outside the digestive tract. When analyzing
fractions with detectable activity, the gonad, posterior adductor
muscle, mantle, and renal showed significantly (Mann-Whitney
rank sum test) higher activities of ""Tc'";80 than of ''"Tc"';800
(Table 2). The correlation analysis (Table 3) showed that when the
amount of '^''Tc'":80 in the digestive gland decreased, the amount

in gonad, adductor muscle, mantle, and renal increased. This was
not the case when comparing the corresponding values for '^''Tc'":
800.

The Transit Time of "^Tc" in M. edulis

The number of mussels dissected at 5. 20, 28, and 54 h were too
few for any statistical ANOVA of transit time, but the general

0,6

> > 0.4

0.2

Fig. 2A: ^'Tc'^:800

1^

600
Time (mm)

1200

Fig. 2B: "^Sn:ms

99T„m.,

Fig 2C: '^Tc"':800

600
Time (mm)

1200

Figure 2. C'harl liius shoHlng (he nididaclivlty nuiisurcd In (he KOIs (stomach and gut) in a mussel which \\as simultaneously given S.
lyphimurium laheled with ''''iv'" In the presence of 8()() jig SnF, ((A) ''''rc"':8(l(() and microspheres labeled with '".Sn ((H) "'Sn:ms). (C) Shows
a mussel which was given ''''Tc"':8U0 only. The amount of radioactivity is normalized to the initial amount given to the mussel.

Influences of 5. Typhimurum in M. Edulis

171

TABLE 1.

Two-way ANOVA table, comparing the influence on the uptake and
the elimination of S. typhimuriuin in M. edulis due to the chemical

treatments of the bacteria (""Tc^iSO or ""Tc'-'iSOOl and to the

absence or presence of the microspheres in the medium (mono- or

multiple medium).

df

SS

Source of variation for uptake

Chemical treatment ('"'Tc"':80
or ''''Tc"':800)

Mono- or multiple medium

Residual

Total
Source of variation for elimination

Chemical treatment ('"'Tc"':80
or ''^Tc'":800)

Mono- or multiple medium

Residual

Total

6080

^^9 ~> j9***

1
28
31

0.0166 0.0001 (NS)
4341
1053

6698

1021
(20)21 8348
(23) 24 16.384

1

16.848***
2.568 (NS)

*** P < 0.001.

NS, not significant: P > 0.5.

df, degrees of freedom; SS, sum of squares.

patterns based on the mean values in Figure 6 gave some indica-
tions. In the posterior adductor muscle and the mantle, there was a
reduction of radioactivity from '''Tc":80 and '''^Tc"\800 after 28 h.
The most rapid accumulation and the highest values were mea-
sured in the gonads of the mussels fed '*'^Tc"^80. The amount of
'^Tc^iSO did not decrease in the gonad and the renal during 54 h.
The mussels fed on '''Tc'":800 did not show any reduction in the
renal after 68 h (Fig. 6). In the digestive tract, there was also
detectable activity after 54 h. In the digestive gland, there was 0.45
± 0.26 MBq x g'' for '"'Tc"':80 and 0.57 ± 0.03 MBq x g"' for
''^Tc'":800. In the gills, there was 0.16 ± 0.06 MBq x g"' from
'^^Tc"\80 but not detectable values from '"'Tc'":800.

80

60

> 40 ■
0)

3

o

t

f

o

+4-

t

^

o

o

^

99tc"

99-,

113c

':80 ^"Tc'^iSOO ' '-^Stiims

Figure 3. The uptake of radioactivity in the stomach ( % of the given
amount) when its maximum activity was measured, in 16 mussels fed
on S. typhimurium labeled with "Tc"' in the presence of 80 and 800 ng
SnF,, respectively ('"""Tc:80 and '"'"'Tc:800) and the microspheres la-
beled with "'Sn ("'Snims). Box plots display the median of the data,
the lower and upper quartiles. and the lowest and highest values ob-
served.

^^Tc'^:80 ^^Tc'^:800 ''"'^Snims

Figure 4. The elimination of radioactivity ( % of the maximum value in
stomach) determined 20 h after the experiment started, in 12 mussels
fed on S. typhimurium labeled with ''''Tc"' in the presence of 80 and 800
Mg SnF,, respectively ("""'TcrSO and """'Tc:800) and the microspheres
labeled with "'Sn (""'Sn:ms). Box plots display the median of the data,
the lower and upper quartiles, and the lowest and highest values ob-
served.

DISCUSSION

S. typhimuriuin 395 M RIO is an Rd-tnutant deficient of the
0-antigenic polysaccharide side chain and with a pronounced nega-
tive surface charge (Edebo et al. 1980). Hernroth et al. (2000)
described the chemical manipulation of S. typhimurium, using
stannous fluoride. The electrophoretic mobility toward a cathode
was significantly reduced for ''''Tc"':800 when compared with
99jj,m.gQ gjjj untreated bacteria. In this study we have found dif-
ferences in the mussel processing of the differently manipulated
bacteria.

The preingestive selection of particles is expected to take place
on the gills or on the labial palps. The structure of the gills is
known to divert particles due to size (Riisgard et al. 1996), and it

0,45

0,4
0,35

0,3
0,25

0,2
0,15

0,1

0,05

0

■ 99mTc80

[]99mTc800

B113Sn:ms

i

it

I

Gills

Palps

Stomach

Liver

Gut

Figure 5. Distribution of radioactivity of S. typhimurium [+ standard
deviation) labeled with '"''Tc'" in the presence of 80 and 800 (jg SnFj,
respectively (''"""Tc:80 and ''''"'Tc:800) and microspheres labeled with
"-'Sn ("'Snims), in the digestive tract Igills, labial palps, posterior
part of the digestive gland (stomach) and the anterior part (liver) and
terminal intestine (gut)]. The columns are based on the mean values
(per g tissue) from 12 mussels dissected within 54 h.

172

Hernroth et al.

TABLE 2.

Mann-Whitney rank sum test: comparison of median values

(''■'Tc"' X mg"') in posterior adductor muscle, mantle, gonad, and

renal from mussels fed on S. typhimurium. labeled in the presence of

80 and 800 pg of SnF,, respectively.

Group

Median

25%

75%

P < 0.05

Adductor: 80
Adductor: 800
Mantle:80
Mantle:800
Gonad:80
Gonad:800
Renal:80
Renal: 800

12
12
12
12
12
12
12

0.99
0.29
4.67
1.58
1.5
0.63
42.5
17.4

0.47
0.15
1.53
0.37
0.85
0.12
27.5
5.. 36

2.35
0.55
6.23
2.61
3.22
1.00

59.5

30. 1

Yes
Yes

Yes
Yes

has been suggested that potentially nutritive particles will be se-
lected relative to inert particles on the labial palps (Hylleberg &
Gallucci 1975. Newell & Jordan 1983. Bayne et al. 1993). The
uptake of '''■^Tc"':800 and """Snims was similar and much faster
than that of '''*Tc"':80. showing that the size alone did not deter-
mine the uptake, because the "^''Tc"'-labeled bacteria were much
smaller (approximately 1 \Lm) than the microspheres ( 15 (j.m). The
higher uptake capacity of ''''Tc"':800 was correlated to a decrease
of the net negative cell surface charge, indicating that negative
charge might antagonize uptake. This study showed discrimination
in ingestion of the less manipulated bacteria, and significantly
more bacteria of this kind were "trapped"" on the gills. A proper
explanation to this requires further studies, and we suggest that it
should include electrostatic repulsion and also hemocytic attach-
ment or engulfment of bacteria on the gills.

The elimination of the bacteria was also affected by the chemi-
cal modification of the cell surface. The elimination was signifi-
cantly lower for ''''Tc'":80 than that for '^Tc'":800 which again was
similar to that of "'Sn:ms. The less-modified bacteria were to a
high degree accumulated in the stomach part of the digestive
gland, but were hardly present in the gut. According to Birkbeck
and MacHenery (1982). this indicates a postingestive selection
based on phagocytic activity. These authors showed, in their study
of M. cihilis. that the processing of bacteria after phagocytic uptake
in the hepatopancreas digested the bacteria into polymers that were

TABLE 3.

Pearson Product Moment Correlati4)n tahle from dissected fractions

of the mussel tissue. The relationship hetween the contents of
radioactivity (over time, as described in Materials and Methods) in

the digestive gland and the gonad, posterior adductor muscle,

mantle, and renal in mussels in = 12) fed on S. lyphiiniiritim labeled

in the presence of 80 and 800 pg SnF, (marked as :80 and

:800), respectively.

(:onad:80

Adductor:80

Mantle:80

Renal:80

Digestive

-0.696**

-0.898***

-0.846***

-0.825***

gland:8()

(;onad:800

Adductor:800

Mantle:800

Kenal:80U

Digeslive

-0.3.56 iNS)

-0.395 (NS)

-0.095 (NS)

-0.244 (NS)

gland:8()()

** P<0.0\.

*** p < ().()() I.

NS, not significant; P > 0.05.

>, 0,8

I 0,6

o

<? 0,4

^^ 0,2

I Gon 80

5 20 28 54 68

Time (h)

0,15

o

CD

0,05

20

28
Time (h)

54

68

Figure. 6. Distribution of radioactivity of .V. typliiiiiiiriiiiii labeled >\ith
''"Tc"' in the presence of 80 and 800 pg .SnF,, respectivelv (""Tc"':80
and '"'Tc"':800) in posterior adductor muscle (Adduc), mantle (Mant),
gonad (Gon), and renal (Ren) (NB: different scales). The columns are
based on the mean values (per g tissue) from mussels dissected after 5,
20, 28, 54, and 68 h.

transferred to other sites in the mussel, whereas most of the
lysozyme-resistant bacteria were rejected as fecal production. It
has prc\ iouslv been shown that M. cdiilis can lyse bacteria extra-
cellularly (Pricur 1981 ). but the slow processing and the preferen-
tial absorption of ""Tc"':80 compared with ""Tc'":800 that evi-
dently occurred in our study indicated a predominance of phago-
cytosis and intracellular digestion of the less-manipulated bacteria.
Absorption of radioactivity from ""Tc"':80 was supported by the
relationship between the decrease of radioactivity in the digestive

Influences of S. Typhimurum in M. Epulis

173

gland and the appearance in organs and tissue outside the digestive
tract. This was not found for '*'*Tc"':800. These bacteria were less
absorbed and were more directly transferred into the intestine. The
faster elimination with lower absorption efficiency shown for
'"Tc'^'iSOO and ' ''Sn:ms indicated extracellular digestion.

Radioactivity was still detectable in the digestive tract of the
mussels dissected after 54 hours. This observation was made in a
closed system in which the water was exchanged only on a daily
basis and should not be compared with the depuration study of
Martinez-Manzanares et al. (1992). They showed a rather rapid
elimination of Salmonella spp. after purification in running sea-
water. However. Plusquellec et al. (1994) managed to detect Sal-
monella spp. in air-stored mussels. 20 days after contamination.
Minet et al. (1995) found culturable cells of 5. typhinniriiim in the
gut 1 week after contamination. The possibility for extracellular
survival of 5. typhimurium in the digestive tract, including the
gills, as indicated by the presence of radioactivity after 54 hours,
needs further investigation. Extracellular survival can thus be a
cause for shellfish-borne gastrointestinal infections and should be
included in risk assessment. Likewise, we found '^"''"Tc distributed
in mussel tissue outside the digestive tract, such as gonads, kidney,
mantle, and adductor muscle, 1-2 days after exposure to the bac-
teria. This might be caused by degraded bacteria but might also be
an effect of resistance to phagocytosis and killing.

In the previous study by Hemroth et al. (2000), the stability of
the isotope bindings to S. typhimurium in seawater was not sig-
nificantly different for '"Tc'^iSO and ''"^^''^SOO. Fragile binding
could increase the amount of hydrolysed, reduced technetium or
free pertechnetate, but these radiochemical impurities did not in-
fluence the uptake capacity of ''''Tc™ in the mussel. The possibility

of diffusion of released '^''Tc'" into the mussel tissue could, as
pointed out. interact with the measurement of the distribution of
radioactivity in the mussel tissue. However, as the binding stability
of '"Tc"' was comparable for ""Tc'":80 and '"Tc'":800, this could
not explain the differences in the distribution of these microbes
shown in this study.

The viability estimated with the fluorescent probe of the la-
beled 5. typhimurium was initially good (96% for '^''Tc'^:80 and
90% for '''^Tc"':800), and the microscopic inspections confirmed
intact cell size and shape and no aggregates. Thus, it was consid-
ered that the same numbers of viable "''Tc'^'iSO and ^''Tc'":800
were given to the mussels. The differences in the uptake between
''"Tc"':80 and '"'Tc"':800 occurred directly from start, indicating
that viability was not the discriminating factor for uptake.

This investigation has shown that the uptake, distribution, and
elimination of microbes by the blue mussel are strongly influenced
by the cell surface characteristics of the microbe. This factor might
be at least as important as particle size. We suggest that recogni-
tion for phagocytic uptake might play an important role in the
processing of microbes.

ACKNOWLEDGMENTS

We thank Prof. Magne Alpsten for providing us with the
gamma camera facilities; Britta Ahlstrom, MD, for valuable advice
concerning the culturing of bacteria: and Ann-Sofi Rehnstam-
Holm, PhD, for discussing the manuscript. This research was sup-
ported in part by the Adlerberth Foundation and by the Sustainable
Coastal Zone Management within the Foundation for Strategic
Environmental Research.

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Iowa State University Press, Ames.

Ward. J. E. & N. M. Targett. 1989. Influence on marine microalgal me-
tabolites on the feeding behaviour of the blue mussel Mytilus edulis.
Mar. Biol. 101:313-321.

Wilson. I. G. & J. E. Moore. 1996. Presence of Salmonella spp. and
Campylobacter spp. in shellfish. Epidemiol. Infect. 116:147-153.

Winston. G., M. N. Moore. I. Straatsburg & M. A. Kirchin. 1991. De-
creased stability of digestive gland lysosomes from the common mussel
Mytlits edulis L. by in vitro generation of oxygen-free radicals. Arch.
Environ. Contam. To.xicol. 21:401-408.

Zar, J. H. 1995. Biostatistical Analysis, 3rd ed. Prentice-Hall Inc.. Upper
Saddle River. NJ.

Joimuil of Shellfish Research. Vol. 19. No. I. I7.'i-181, :()0().

ON THE POSSIBILITY OF USING RADIOACTIVE LABELING AND GAMMA CAMERA
TECHNIQUE TO STUDY SALMONELLA TYPHIMURIUM IN THE BLUE MUSSEL,

MYTILUS EDULIS

BODIL HERNROTH,' ANNHILD LARSSON,^ MAGNE ALPSTEN,^
AND LARS EDEBO^

' The Royal Swedish Academy of Sciences

Kristineberg Marine Research Station

SE-450 34 Fiskebdckskil. Sweden
'Department of Radiation Physics

Sahlgrenska University Hospital

SE-413 45 Goteborg, Sweden
^Department of Clinical Bacteriology

Sahlgrenska UniversityHospital

SE-413 46 Goteborg. Sweden

ABSTRACT This paper presents a radiolabeling method for Salmonella typhimiiriiim to be used for in vivo studies on the kinetics
of uptake in blue mussels with a gamma camera technique. S. lyphimurium bacteria were labeled with technetium-99m (''^Tc"') at
conditions preserving the viability in seawater and the cell surface properties of the bacteria. Stannous fluoride (SnF,) was used to
facilitate the binding of '^''Tc'" to the bacteria. The toxicity of SnF, could inhibit the growth of bacteria, and it can also bind
extracellularly and reduce the negative cell surface charge of the bacteria. Additionally. SnF, can cause radiochemical by-products such
as Tc-stannous colloids, which might interact with the image analysis. To optimize the labeling, two different concentrations of SnF,
used in the labeling process were evaluated. Neither the efficiency nor the stability of the binding of '*''Tc'" to the bacteria increased
significantly, when the amount of SnF, was increased 10-fold. Both treatments of bacteria reduced the viable counts, whereas the
viability assessed microscopically with fluorescent probe was affected only little. However, after incubation in seawater, the viability
was reduced for cells treated with the highest concentration of SnF,. Still, approximately 60% remained viable. Presence of radioac-
tivity, not bound to bacteria, was investigated by centrifugation in Percoll. Less than 4% of probable Tc-stannous colloids were found,
and they were therefore not considered to be disturbing the imaging analysis. The net negative surface charge of the bacteria, examined
by measuring the electrophoretic mobility, was significantly reduced when the concentration of SnF, increased, but was still negative.
Radioactive particles, formed by mixing '^''Tc"' and SnF, in the absence of bacteria, were rapidly taken up by mussels in a way similar
to that of the more heavily labeled bacteria. When less SnF, was used for labeling of the bacteria, different uptake and processing
kinetics were seen. Thus, to keep the natural conditions, the concentration of the labeling compounds have to be minimized. The study
showed that it is possible to balance the labeling method and get a valuable tool for following the uptake and fate of 5. lypliinmrium
in blue mussels.

KEY WORDS: Gamma camera, radiolabeling, 99m-technetium, "'''"'Tc, Salmonella typhimurinm. bacteria, Mylilus ediilis, bivalves,
molluscs, uptake, elimination, enteric infections

INTRODUCTION fluoride (SnF,) has been used to reduce "^Tc"' from +VII to

-i-IV, which facilitates its binding (Rhodes 1991, Perin et al, 1997).

The blue mussel, Mytihis ediilis. is in nature exposed to a g^p^ j^ ^^^^ knovjn for its bactericidal effect (Tsao et al. 1982.

mixture of particles and is able to accumulate high numbers of Caufield et al. 1987. Oosterwaal et al. 1989, Oosterwaal et al.

microbes from the suiTOunding water. The potential for the mussel 1991 ), and therefore the amount used in the labeling process

to become a carrier of food-borne diseases is therefore significant, ^i-if, to be selected with care. SnF, acts as an inhibitor of glyco-

and several repons have pointed out the molluscs as commonly lytj^- enzymes, as it binds to SH groups. The metal ions interact

incriminated in outbreaks of enteric infections (West et al. 1985, ^jth both Gram-positive and Gram-negative bacteria, and the

Martinez-Manzanares et al. 1992, Wittman and Flick 1995, Ripa- antimicrobial effect depends on the concentration of free ions

belli et al. 1999). Depuration studies on bivalves have shown great as well as the chemistry of the ions in the specific system (Scheie

individual variations between mussels (Heffernan and Cabelli 1994). In addition, the concentration of SnF, must be used

1971, Plusquellec et al. 1994) and vaiiations due to bacteria spe- with care, as it might influence the cell surface properties of

cies (Plusquellec et al. 1998). This stresses the necessity of studies the bacteria. Olsson and Oldham (1978) have proved that the

on the uptake and elimination of microorganisms in individual binding of metal ions to bacteria alters their cell surface charge

bivalves to establish satisfying monitoring programs and to im- and adherence ability. With the amount of stannous fluoride

prove risk assessment with respect to public health. used by Plotkowski et al. (1987) in the '-'"'Tc'" labeling of the

Gamma-emitting radionuclides in bacteria can be used for in Pseudomonas aeruginosa, the electrophoretic mobility was not

vivo imaging distribution. Technetiuni-99m ("'^Tc"') isotope has changed, but Perin et al. (1997) showed that the '^'Tc"' labeling of

been used as a radiotracer when studying phagocytosis of viable S. ahortnsovis demands a higher concentration of SnF,. As it has

bacteria in vertebrates (Plotkowski et al. 1987. Bernardo-Fiiha been shown that particle processing in M. edidis can be effected

et al. 1991, Perin et al. 1997). In the coupling process, stannous when the particles are enriched in metals (Wang et al. 1995, Al-

175

176

Hernroth et al.

lison et al. 1998), the concentration of stannous in the labeling
process is critical.

An indirect labeling method has been used to follow the dis-
tribution of leukocytes in humans (Mock and English 1987,
Puncher and Blower 1995). In these assays, leukocytes were la-
beled intracellularly by phagocytosis of colloidal compounds of
'''Tc"' and SnF, (Tc-stannous colloid). The.se studies also demon-
strated that active compounds might have occurred as nonspecific,
cell surface-bound labeling with low affinity and soluble radiocol-
loids. Radiochemical by-products might also occur in the direct
labeling of bacteria. As a side reaction, the reduced technetium can
bind to more low-affinity binding sites or together with SnF-, form
Tc-stannous colloids. There is a possibility that released pertech-
netate or Tc-stannous colloids might be ingested by the mussel and
confuse the image analyses of the bacteria. Because of the com-
plications described, the labeling method has to be optimized to
avoid decreased viability of the bacteria and radiochemical impu-
rities. Furthermore, alteration of the surface properties by the la-
beling process has to be taken into consideration, as this could
affect the processing of the bacteria in M. ediilis.

Salmonella can appear in the marine environment because of
fecal contamination (Prazeres Rodrigues et al. 1989, Papa-
petropoulou & Moschopoulos 1996, Wilson & Moore 1996) and is
of great interest in terms of shellfi.sh safety. The aim of this study
was to investigate and evaluate the ""Tc"'-labeled Salmonella n-
phimithum as a tool to study its uptake and fate in M. edulis. S.
typhimurium 395 MR 10 was chosen because it is nonvirulent and
known to be well accessible to phygocytosis, killing, and degra-
dation in mammalian systems (Edebo et al. 1980). Considering the
evaluation of the method used in the direct labeling of 5. abor-
Uisovis (Perin et al. 1997), we compared the effect of two different
concentrations of SnFj on (I) the viability of labeled 5. typhimii-
riiim in seawater. (2) the labeling efficiency of the bacteria and the
stability of the label in seawater, (3) the formation of Tc-stannous
colloids during the labeling process, (4) the cell surface charge of
the labeled bacteria, and (5) interaction of the labeled bacteria with
M. edulis.

MATERIALS AND METHODS

Bacterial strain and growth conditions

S. lyphimiirium 395 MR 10 (chemotype Rd. deficient of O-
antigenic poly.saccharide side chain and most core sugars of the
lipopolysaccharide) has been described by Edebo et al. (1980). A
single bacterial colony was harvested from a nutrient agar plate
(beef extract, Oxoid 10 g; peptone, Oxoid 10 g; NaCl 8 g; glucose
7.5 g: and agar 1.47r) and cultured in glucose broth, pH 7.0-7.2
(Lindberg et al. 1970) at 37 C tin a rotary shaker (200 rpm) for 16
h. The bacteria were washed three times by centrifugation (2000
rpm, 10 min, 4 °C) in ().9'/f NaCl and resuspended in 2 niL ().9"r
NaCI (2.5 X 10" mL '). With these culturing conditions, the bac-
teria are considered to reach the prestationary phase.

Kadiolnheling of bacteria

One millililei- of the bacterial suspension (2.5 x 10" mL"') was
incubated with approximately 50 MBt| ""Tc"'-perlechnetate and 2
mL of 37 "C ().9'/r NaCl containing 80 and SOO (xg .SnF,, respec-
tively, to cause reduction of '"'Tc'". After incubation for 20 min at
37 °C on a rotary shaker (200 rpm I. the bacteria were centrifugcd
and washed three limes. Ascorbic aciil (0.25 mu x niL ') was

added to the NaCl to prevent reoxidation of the isotope (Rhodes
1991). The bacteria were resuspended in 1 mL 0.9% NaCl.

Bacterial viability

For estimation of the effect of the labeling procedure on the
viability, bacteria treated with ""Tc'^-pertechnetate as well as with
80 (n = 6) or 800 (n = 6) p,g SnF,, respectively, were compared
with control bacteria incubated in 2 mL 0.97c NaCl (n = 6) only.
The suspensions of bacteria were serially diluted in phosphate-
buffered saline (NaCl 0.15 M, sodium phosphate 0.01 M, pH 7.2
7.4), spread onto nutrient agar plates using a spiral plating system
(Spiral System Inc.. Cincinnati, OH), incubated at 37 °C for 24-48
h. and the colonies counted and the colony-forming units per mL
(CFUs X mL"') calculated.

The viability of the bacteria was also investigated using the
LIVE/DEAD® SflcLight™ Bacterial Viability Kit (Molecular
Probes, The Netherlands). Live bacteria appear with green fluo-
rescence (SYTO 9), whereas the red fluorescence of membrane-
impermeant propidium iodide dominates membrane-compromised
bacteria (Haugland 1996). The bacterial suspensions were diluted
(5 X 10'^' X mL"') in sterile filtered (Schleicher & Schuell. Keene,
NH; FP 030/3) seawater (33.69 PSU, 6 °C), incubated on a rotary
shaker (200 rpm) with the fluorescent probe for 15 and 180 min,
observed in an epifluorescence microscope (Zeiss Axioscop, ex-
citation filter BP450-490, dichroic reflector 510. barrier filter
LP5I59, Zeiss, Germany), and the fraction of viable cells calcu-
lated. After all labeling processes, the bacterial suspensions were
observed in a light microscope ( 12.5 x 100 times magnification) to
check possible effects on shape and size and aggregate formation.

Efficiency and .stability of the ''''Tc'" labeling

After the labeling process, the radioactivity of ''''Tc'"-labeled
bacteria was measured using a well-shielded Nal(Tl) detector (15
cm[diameter|; Nuclear Enterprises, UK) in a low-activity labora-
tory. The labeling efficiency was expressed as percentage of the
initial activity bound.

The stability of the binding was tested by incubation of three
batches of bacteria (final concentration 5 x 10'' x mL"' ), labeled in
the presence of 80 and 800 (xg SnF, respectively, in filtered (Mil-
lipore 0.3 fjim) seawater (33.69 PSU, 6 °C). Triplicate samples
were taken within 3 min and then after 15. 30, 60, and 180 min.
Particles >0.2 (jitTi were separated from the water using sterile
filters (Schleicher & Schuell: FP 030/3), and the filtered volume
was collected in vials and the fractions were measured in the
well-shielded Nal(TI) detector. The bounded part was expressed as
the particulate fractitin of the total radioactivity.

Radiochemical by-products

Possible formation of Tc-stannous colloids in the labeling so-
lution was investigated by separation in Percoll (n = 3), with a
density of 1.12 g x mL"'. One milliliter of the labeled bacteria was
layered on the Percoll, and the tubes were centrifuged for 20 min
at 2000 rpm. Free '"'Tc"'-pertechnetate and two colloidal suspen-
sions were used as references. The colloidal suspensions were
formed when ""Tc"' was incubated in the presence of 80 and 800
(j-g SnF,, respectively, without bacteria (in this paper called Tc-h80
and Tc-(-800). After centrifugation, the tubes were placed in front
ol the gamma camera and the separated parts were measured and
calculated as a percentage of the total radioactivity.

Stl'dyinc. S. Typhimurivm in M. Edlius

177

Cell microelectrophoresis

The cell surface charge of the bacteria, labeled in the presence
of 80 and 800 \x.g SnF,, respectively, was investigated using mi-
croelectrophoresis (Mark II. Rank Brothers Ltd., Cambridge, En-
gland). The electrophoretic mobility (m~~ x V"'s~') of the chemi-
cally treated bacteria was compared with that of untreated bacteria.
The bacteria were diluted in 5 niM KCl. and the time needed to
pass a 180-|xm grid in the electric field (90 V; distance between
electrodes. 64.6 cm) was measured 10 times. The variances be-
tween the treatments were analyzed using one-way analysis of
\ariance on ranks (Student-Newman-Keuls method) (Sokal &
Rolph 1969).

Uptake by M. ediilis of radiolabeled bacteria and possible by-products

Mussels were kept in circulating seawater of approximately 33
PSU at 6 °C and fed the nanoflagellate Isochrysis galbana before
the e.xperiment started. The mean length of the mussels was 7.1 ±
0.5 cm. and the mean wet tlesh weight was 9.1 ± 3.0 g. Two sets
of two mussels each were used to study the uptake of 5. ryplwmi-
riiim labeled in the presence of 80 jjig SnF^ (S:80) and in the
presence of 800 (xg SnF, (S:800). As control mussels, two were
given the colloidal suspension (without bacteria) incubated with
'"Tc"' and 80 p.g SnF. (Tc-l-80), two mussels were given the sus-
pension incubated with '"'Tc'" and 800 |jLg SnF, (Tc-f800), and two
mussels were given a suspension with free ""Tc"'-pertechnetate
(free Tc).

Single mussels were positioned in front of the gamma camera,
and hung above the bottom in beakers containing approximately
700 mL of filtered (Millipore 0.3 jjLm) seawater (33.7 PSU). The
water was kept at 6 °C, well mixed with a stirrer, and oxygenated
during the experiment. Labeled bacteria or reference solutions
were added to the beaker. The final concentration when gi\en the
bacteria was approximately 5 x lO*" mL"'. The distribution of
radioactivity was visualized for 5 h using a conventional, com-
puter-aided gamma camera technique (MAXI II, General Electric:
Hermes-system NuD. Nuclear Diagnostic. Hiigersten Sweden) as
shown in Figure 1 . By outlining the region of interest (ROI) of the
image, the amount of radioactivity in the chosen region was mea-
sured. The ROI chosen for this study was the area where the
radioactivity was accumulated after passing the gills, identified as
the stomach. The uptake was estimated as the accumulated fraction
of the given amount of radioactivity (%), measured when the maxi-
mum value in the ROI was reached and the uptake rate was cal-
culated (7f min"'). The values were normalized to the initial
amount of radioactivity to eliminate differences in the given
amount of activity and geometric properties, such as mussel size
and shape and the distance between the mussel and the camera.
The radioactivity was corrected for the half-life of the isotope
(6h).

RESULTS

Viability of the '"""Tc-labeled bacteria

Compared with the unlabeled bacteria, the viable counts on
agar plates were significantly reduced for both S:80 and S:8()0.
When analyzing the unlabeled bacteria. 187 ± 29 x 10^ CFU were
found. The CPUs for S:80 and S:800 were 33 ± 16 x 10^ and 10
± 2 X 10'. respectively, corresponding to a reduction of 82 and
9.'i9K compared with the control. The fluorescence assay showed
(Fig. 2) that after incubation for 15 min in seawater, the viable

B

D

•

D

•

D

%

0

Figure 1. Gamma camera imaj^e of a mussel after beinji given bacteria
labeled with ''''Tc"' in the presence of 8(M) (ig SnF,. (A) Concentrated
along the gills of the mussel. (B) Accumulated in the stomach region.
(C) Directed to the gut. (D) Transported as fecal content through the
gut.

fraction of S:80 (88 ± 6%) was similar to that of the unlabeled
bacteria (94 ± 2%). The corresponding value for S:800 was 81 ±
%%. After incubation for 180 min in seawater. the viable fractions
of the unlabeled bacteria and S:80 remained unchanged, being 97.3
± 0.6% for the unlabeled bacteria and 96.9 ± 2.2% for S:80,
whereas for S:800 it was significantly reduced (59.2 ± 4.5%). The
microscopic inspections showed that the cell size (approximately 1
p.m) and shape were not altered for S:80 and S:800. and no ag-
gregates were observed.

Efficiency and stability of the ''"'Tc'" labeling

The efficiency of the labeling of S:80 (77 ± 7%) and of S:800
(70 ± 14%) was not significantly different. The amount of '^'^Tc'"
released during the first hour in seawater was approximately 37%
for S:80 and 30% for S:800. During the following hour, the bound
i«j^m jfgygj more stable (Fig. 3).

Radiochemical by-products

After centrifugation in Percoll (Fig. 4). free Tc stayed on the
top layer (96 ± 17r), as did the bacteria. S:80 (95 ± 2%). and S;800

120

D15 min
■ 180 min

Control S:80 S:800

Figure 2. Viable cells (% of the total number of cells) (+SD. n=6),
estimated by fluorescence assay, of S. typhimurium labeled in the pres-
ence of 80 (S:8(l) and 800 (S:800) \i% SnF,, respectively, and unlabeled
S. typhimurium (Control). The viability was estimated after 15 and 180
min of incubation in seawater.

178

Hernroth et al.

^ 100

>

ni

e'
o

H

50

150

200

100
Time (min)

Figure 3. The particulate fraction ( % ) of the total amount of ^'^Tc"*
(+SD, n=9l from S. typhimurium labeled in the presence of 80 (S:80)
and 800 (S:800) fig SnF,. respectively, measured during 180 min of
incubation in seawater.

(92 ± 1 %), and less than 4% were found at the bottom. In the tubes
with colloids formed during the incubation of '^''Tc'" with ShF,
(without bacteria), more were found at the bottom. The bottom
fraction increased with the amount of SnF,. showing 48 ± 17% of
the radioactivity in Tc+80 and 71 ± 22'7o in Tc+800.

Cell microelectrophoresis

One-way analysis of variance on ranks (Kruskal-Wallis)
showed that the electrophoretic mobility for S. lyphimuriuin was
affected by the treatments of the bacteria (Table 1 ). There was a
statistically significant reduction in electrophoretic mobility for
S:800 compared with S:80 and untreated bacteria. The median
value for S:800 was 2.3 x 10"" m" x V's"'. For S;80 it was 5.4
X 10"'' m" X V~'s"' and for untreated bacteria 4.7 x 10"'' m" x

Uptake by M. edulis of radiolabeled bacteria and possible by-products

Preliminary studies on the uptake of labeled bacteria by mus-
.sels showed that initially the radioactivity accumulated in the gill
area and subsequently in the stomach and gut region (Fig. I).
Figure 5 displays the curves from the measurements of radioac-
tivity in the stomach from the two mussels given bacteria (S;80
and S:800, respectively). The accumulation of S:80 in the stomach
region was 1 1 ± 1.4% of the given amount of radioactivity and the
process was slow (0.04 ± 0.01% min"') and nondynamic. Of the
given activity of S:800. 32.7 ± 0.28% was measured in the stom-

■ top
n bottom

Tc+80 Tc+800 S:80 S:800 free Tc

Figure 4. The mean percentage of the total amount of "Tc"" activity
(n=3) accumulated in the top and the bottom layers of the test tube
after centrifugation in Percoll. The columns show '"Tc"' incubated
only with 80 (Tc+80) and 800 (Tc+800) m8 SnF,, S. typhimurium la-
beled with ''■'Tc™ in the presence of 80 (S:80) and 800 (S:800) fig SnF,,
and free ''''Tc'"-pertechnetate (free Tc).

ach. The accumulation was faster (0.36 ± 0.20% min"'). and the
reduction came in pulses.

Figure 6 displays the curves from the measurements of radio-
activity in the stomach from the two mussels fed on the colloidal
suspensions (Tc-i-80 and Tc-f800. respectively). The radioactivity
from Tc4-80 was 21.2 ± 3.6%. and the uptake rate was 0. 14 ±
0.01% min"'. For Tc-l-800. the uptake was 31.1 ± 2.2% and the
process was faster (0.31 ± 0.02% min"'). The dynamic movements
of the radioactivity in the stomach were similar between these
mussels. The radioactivity from the two mussels given free Tc was
below the limit of detection.

DISCUSSION

S. rypliiinuriiini 395 MR 10, used in this study, is a deep rough
(chemotype Rd) mutant (Edebo et al. 1980). its surface is more
hydrophobic and negati\ely charged than in most other Salmo-
nella, and it forms a homogenous single-cell suspension in water.
Labeling with '""Tc"^ using the high amount of SnF^ (S:800) re-
duced the net negative charge of the bacteria as studied by use of
electrophoresis. When less SnF, (S:80) was used, no effect on
charge was discerned. Wang et al. ( 1995) and Allison et al. (1998)
have suggested that the cell surface properties of particles will
intluence the preingestive selection on the labial palps. Our results
are in accordance with these suggestions. The reduced net negative
charge of .S:8()() was probably a consequence of accumulation of

TABLE 1,

One-way ANOVA on ranks (Kruskal-Wallis) comparing the electrophoretic mobility (nr x V"'s"'l for S. typhimurium, treated with 80 and
800 ng SnFj, respectively (S:80, S:800), and untreated .S. typhimurium (control I (post hoc Student-New man-Keuls).

Group

n

Median

25%

75%

Control

1(1

4.7 X 10""

4.1 X I0-'

5.1 X 10-'

S:80

10

.'i.4x 10"'

4.7 X 10-"

t.f, X 10""

S:8()0

10

2..1X 10-"

2.1 X nr"

2.5 X lO""

Comparison

DitTerence of Ranks

P

q

P < 0.05

S:800 vs. S:80

l.'i6

.1

.■S.6

Yes

S:80 vs. control

24

2

1.2S

No

Control vs. .S:8()()

1,12

2

7.06

Yes

Studying 5. Typhimvrivm in M. Edulis

179

0,5

o ~

c «

>= 0,25
5

S:80

60

120 180

Time (min)

240

300

0,5 -1

o ->

£■= 0,25 -
> 5

o

'■5

Tc+80

60

120 180

Time (min)

240

300

Stomach
Gut

Stomach
Gut

0,5 y

S:800

Tc+800

180

240

300

Time (min)

Figure 5. Curves showing the "Tc"' activity in the stomach and gut
(observation time 5 h) of mussels fed S. typhimurium, labeled in the
presence of 80 (S:80» and 800 (S:800) (ig SnF,.

positively charged metal complexes at its surface. These com-
plexes might work as ligands for binding to mussel receptors or
mainly operate by reducing the electrostatic repulsion between the
bacterial particles and the recipient mussel surface. The differences
in the cell surface properties between S:80 and S:800 might be a
possible explanation for the differences shown in uptake and ki-
netic handling of the bacteria in the mussels.

Previous studies by Mayhew and Brown (1981) and Tseng and
Wolff (1991) showed that SnF, inhibits the growth of the bacteria.
This was also the case in our study. The viability in terms of viable
counts was significantly reduced for S:80 and still more so for
S:800. Bacteria in the prestationary phase w,ere used for the label-
ing experiment, but log phase might have been a better condition
for preserving the viability. However, the suppressed growth on
agar did not correspond to the viability estimated by use of a
fluorescent compound, probing the integrity of the barrier of the
cell membrane, indicating that the labeling process may impair
growth and division without conspicuously disturbing the cell
membrane barrier. S:80 was better maintained during the incuba-
tion in seawater. but initially the viability of S:80 and S:800 was
similar. Our evaluation is that the differences shown for the uptake
should not be explained by differences in viability, since this phe-
nomenon appeared directly from start when the viability of S:80
and S;800 was still comparable. The similarity between S:80 and
S:800 in cell membrane integrity, size, and shape made us judge
them as equal prey when given to the mussels.

>
•a

0,25

60 120 180 240 300

Time (min)

Figure 6. Curves showing the '^Tc" activity in the stomach and gut of
mussels (observation time 5 h) given a suspension of by-products
(probable "Tc^-stannous colloids) formed during incubation with the
isotope and 80 ^g SnF^ (Tc+80) and the isotope and 800 (Tc+800) ^g
SnF,.

According to Ross et al. (1984). the size of Tc-stannous col-
loids is approximately 1.5 |j.m. which is close to that of 5. typh-
imurium. When giving the mussels the suspension with complexes
formed between '''^'Tc'" and SnF,. without involvement of biologi-
cal material, radioactivity was also accumulated in the mussels, in
a way very similar to that of the more heavily labeled bacteria.
These results indicate that the metal complexes on the surface of
bacteria play a mediating role in the uptake process and that by-
products formed when labeling the bacteria can influence the im-
aging analysis. The fraction of activity not bound to the bacteria
was not greater than the fraction of by-products found when ana-
lyzing free pertechnetate, which indicates that it might include free
or hydrolyzed pertechnetate and not only colloids. However, these
fractions of "probable colloids" produced less than 4% of the total
amount of radioactivity, and the influence on image analysis was
considered to be of minor importance for the purpose of this study.

The amount of free or hydrolyzed pertechnetate in the bacterial
suspension could not be established. The labeling efficiency was
not significantly different comparing S:80 and S:800. The mean
efficiency was 73.3%, and there is no evidence that the excess of
'*'*Tc" was separated from the bacteria through the washing steps.
In addition, a released fraction of radioactivity from the bacteria
suspension appeared during the incubation in .seawater. However,
this study showed that even though the mussels were offered only

180

Hernroth et al.

free pertechnetate (free Tc), the uptake was not detectable and did
not affect the measurements. The possibility of passive diffusion of
free pertechnetate cannot be excluded and needs further investi-
gation.

The appearance of soluble '^''Tc"' was not significantly greater
for S;800 than for S:SO after 180 min of incubation, even though
the viability was more reduced. Thus, only a limited proportion of
the bacteria were lysed. or lysed bacteria did not release the ra-
diotracer. The feeding activity of the mussels can be stimulated
both for paniculate or nonparticulate food (Thompson and Bayne
1972). Cell leakage due to lysed bacteria could elicit a chemosen-
sory response, which might explain the preferential uptake of S;
800. However, the intact state of cell membranes and similar up-
take of Tc-stannous colloids argue against such an effect. Aggre-
gation of the bacteria would also affect the ingestion, but as no
aggregates were found by the microscopic inspection, this expla-
nation is rejected.

In summary, this study showed that there seems to be a higher
uptake capacity and a more dynamic processing of the bacteria in

the digestive gland due to the amount of SnF, used in the labeling
process. The disturbance of the processing stresses the importance
to keep the bacteria in a state as natural as possible. Although
stannous fluoride is a toxic component to bacteria, it can be used
as a reducing agent in the labeling process to produce a stable
gamma-emitting bacterial tracer. However, the concentration used
for this purpose has to be taken in consideration when studying
uptake of viable bacteria in mussels. Bacteria labeled with gamma-
emitting radionuclides, such as '^''Tc'"-pertechnetate. have the po-
tential to be used in numerous applications of bivalve research.

ACKNOWLEDGMENTS

We thank Assoc. Prof. Staffan Wall, Department of Physical
Chemistry, Goteborg University, for helping us with the determi-
nation of the electrophoretic mobility of the bacteria. This study
was funded through grants provided by Adlerberth Foundation
and the Sustainable Coastal Zone Management (SUCOZOMA)
project of the Foundation for Strategic Environmental Research
(MISTRA).

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Journal of Shellfish Research. Vol. 19. No. I. I8.V186. 2000.

ISOLATION AND CHARACTERISATION OF A cDNA ENCODING AN ACTIN PROTEIN FROM

THE MUSSEL, MYTILUS GALLOPROVINCIALIS

GUILLAUME MIXTA, PHILIPPE ROCH, AND
JEAN-PAUL CADORET*

Defense et Resistance chez les Invertebres Marins
(DRIM) IFREMER-CNRS- Universite
de Montpellier 11 - Case courrier 80-2
Place Eugene Bataillon F-34095
Montpellier Cedex 05. France

ABSTRACT A full-length complementary DNA encoding an actin was isolated from a Mytilus galloprovincialis hemocyte library.
This actin displays a typical 376 amino acid open reading frame. Northern blotting indicated that the expression of the actin gene is
particularly abundant in muscular tissues. This actin cDNA will be useful as a potential genetic marker as a standard for expression
level in genetic regulation studies and will allow screening for the whole gene as well as its upstream regulation sequences.

KEY WORDS: Actin. Mytilus galloprovinciaUs. mollusk

INTRODUCTION

Actins are highly conserved contractile proteins ubiquitous in
all eukaryotic cells. In muscle cells it is important in myofibrillar
contraction, and in non-muscular cells these proteins play a role in
diverse functions such as motility, phagocytosis, chromosome
movements, and transport of niacromolecules within the cells
(Kom 1978). Muscle-specific actins can be distinguished from
cytoplasmic actin in vertebrates by their primary sequences
(Vandekerckhove and Weber 1978). For example, the amino acid
Val 10 is characteristic of cytoplasmic actin. while Val 17 is typi-
cal of muscular actin. Wesseling proposed 3 boxes in the N-
terminal region as diagnostic for the family to which an actin
belongs (Wesseling et al. 1988). In invertebrates, actins also have
both muscular and non-muscular functions, but these two classes
are not readily distinguished on the basis of amino acid sequence.
Indeed, invertebrate muscular forms of actins are closer to P-cy-
toplasmic pattern of vertebrate. In practice, rigorous analysis of
tissue expression is necessary in order to distinguish between the
different forms. Actin genes are very abundant and constitutively
expressed. As such they have been subject to numerous studies
also among invertebrates (Gomez-Chiarri et al. 1994; Horard et al.
1994; Lardans et al. 1997; Cadoret et al. 1999).

The bivalves rely on an innate immune defence based on both
cellular and humoral components which interplay to eliminate po-
tentially infectious microorganisms. One such innate immune
mechanism is the production of antimicrobial peptides which have
been recently identified in mussels of the genus Mytilus sp. (Hu-
bert et al. 1996; Charlet et al. 1996; Mitta et al. 1999a.b). A deeper
knowledge of this defence systein would allow the establishment
of health controls to detect bivalve immunodeficiency, the selec-
tion for disease resistance with a coupling of immunology and
genetics or by referring to classical genetics, the characterisation of
immune genes could be exploited in genetic quantitative selection.
Finally, genetic transformation constitutes another promising strat-
egy to obtain resistant strains by various modifications systems. As
part of this strategy, the identification of constitutive genes like the
actin. that provide tools in the study of regulation mechanism of

*Cortespondence to; jean.paul.cadoret@ifremer.t'r

the identified peptides was undertaken. We isolated a full-length
actin cDNA and carried out initial inapping of its expression by
Northern blot experiments. This is a first step toward the identi-
fication of promoter regions as well as the sequencing of the whole
gene.

MATERIAL AND METHODS

Animals and Hemolymph Collection

Adult mussels (Mytilus galloprovincialis) were obtained from a
commercial shellfish farm (Palavas, France, Gulf of Lion) during
winter. The hemolymph of 20 mussels (approximately 0.5 mL/
animal) was extracted via a 23G needle plus syringe, directly into
an equal volume of the anti-aggregant buffer. Modified Alsever
Solution (MAS. Bachere et al. 1988). and immediately centrifuged
at 800 g for 15 min at 4 °C. The cell pellet was air-dried and stored
at -80 °C until required.

Actin-Specific and Ribosomal ISS-Specific DNA Probes and Screening
of cDNA Library

Poly (A)* RNA from adult mussel hemocytes were used to con-
struct a cDNA library in the ZAP Express Vector (Stratagene. La
Jolla). Reverse transcription and polymerase chain reaction (PCR)
were used to prepare a DNA probe corresponding to hemocytic
actin. Three jxg of total RNA (see below for RNA isolation) were
submitted to reverse transcription using the Ready-to-Go You-
prime first strand beads kit (Pharmacia). One-fifth of the reaction
was directly used as a template for PCR with two primers designed
from a consensus actin sequence by M. E. Unger and G. Roesijadi
(1993) for the oyster Crassostrea virginica. and renamed Avil
(5'TAA TCC ACA TCT OCT GGA AGG TGG 3') and Avi2
(5'TCA CCA ACT GGG ATG ACA TGG 3'). PCR was per-
formed in 50 (iL with 40 cycles consisting of 1 min at 94 °C, 1 min
at 60 °C and 1 min at 72 °C with 1 .5 mM MgCL and 1 (JiM
primers.

The resulting 846 base pair fragment corresponding to an actin
cDNA fragment was cloned using the pCR-Script Amp SK (-I-)
Cloning Kit (Stratagene, La Jolla). The plasmid containing the
actin cDNA fragment was called pBSAct.846. The pBSAct.846
insert was labeled with |'-P] by random priming using the Ready-

183

184

MiTTA ET AL.

ARNm

First strand cDNA

846 bp actin cDNA
fragment

■ AAAAA(A)n
TTTTTT(T),,

Reverse transcription

Avi2

<

Avil

■ mTT(T)i

Polymerase Cham Reaction

(specific amplification of the actin cDNA fragment)

Cloning, [ "P] labelling
and cDNA library screening

Actin cDNA

Complete
aclin cDNA

• AAAAA(A),
TTTTTT{T),

5 ' RACE-PCR

AAAAA(A),

Figure I. Complete characterization of Mytilus galloprovincialis actin cDNA {IVIya2).

to-go DNA labeling kit (Pharmacia Biotech.) and used to screen
400,000 plaques from the cDNA library that has been transferred
to Hybond-N filter membranes (Amersham Corp.). High strin-
gency hybridization was carried out overnight at 65 °C in 5X
Denhardt"s solution, 5X SSPE (Sambrook et al, 1989). 0.1% SDS,
100 (j.g/niL salmon sperm DNA. The filters were washed in a
solution of 0.5X SSC containing 0.1% SDS at 65 °C, followed by
autoradiography. A secondary screening was performed to purify
the positive clones. Phagemids were obtained by in vivo excision
according to the manufacturer's instructions and sequenced on
both strands.

To compare the relative expression of actin messenger in vari-
ous tissues of the mussel (see Northern-blot analysis), a probe
detecting a 18s rRNA, present at the same level in all tissues was
designed. As such, a sense oligonucleotide primer (5'TGAC-
CTCGCGGAAAGAGCGC 3') and an antisense oligonucleotide
primer (5'AGGGGACGTAATCAACGCGAGC 3') were de-
signed from the sequence of the ribosomal RNA small subunit
(Kenchington ci al. 1995) and used in PCR experiments. Five
hundred ng of mussel genomic DNA were submitted to amplifi-
cation in 50 \^.L using 35 cycles consisting of 1 min at 94 C, 1 min
at 60 °C and 1 min at 72 °C with 1.5 mM MgCK and 1 |j.M
primers (Fig. 1 ).

Northern lilol Analysis

The hcmocytes from 4 mussels collected together during winter
(8 X 10'' cells per animal) were centrifuged and resuspcndcd in 1
mL of Tri/ol (Life Technologies). Immedialcly after hemolymph
collection, the mantle, foot, labial palps, gills, hepatopancreas, and
adductor muscle were excised from the same animals and washed
extensively in sterile-riltcred seawater. The tissues (100 mg of
each) were honiogeni/ed in 1 mL of Tri/ol with 30 strokes of a
Potter homogeni/cr to break the cells in 1 ml. of Tri/ol and total
RNA was extracted according to the manufacturer's protocol (Life

Technologies). Five \x.g of total RNA was isolated from each tis-
sue, pooled from each animal (20 jj.g total per tissue) and subse-
quently analyzed.

Total RNA and size markers were electrophoretically separated
on a 1.2% agarose gel containing 17% formaldehyde, transferred
and cross-linked to a Hybond-N filter membrane (Amersham)
which was then stained with methylene blue. The membrane was
hybridized with the |'"Pl-labeled actin cDNA probe in a solution
containing formamide (50%), 5X SSC, 8X Denhardt's solution,
sodium phosphate (0.05 M pH 6.5). SDS (0.1%) and salmon sperm
(100 iJLg/mL) at 55 'C for 12 h. The membrane was washed in 0.2
X SSC. 0.1% SDS at 65 "C and autoradiography was carried out.
After autoradiography, the membrane was stripped by incubating
the blot with a boiling solution of 0.1% SDS for 1 hour and
submitted to a subsequent hybridization with the ['•'P|-labeled
DNA probe revealing I8S rRNA.

Rapid Amplification of 5' cPNA end (RACE-PCR) and PCR

To obtain the complete cDNA sequence corresponding to the
actin mRNA. a 5' RACE-PCR was undertaken. This was per-
formed using the 5' RACE Kit (Boehringer Mannheim) following
the manufacturer's instruction. Briefly. 2 (xg of total RNA from the
pooled hcmocytes were submitted to reverse transcription using
antisense 24 nucleotides primer (5'ATGATGTC TGTTT-
TATAAAGTTAT 3'). deduced from the actin cDNA sequence.
After first-strand cDNA synthesis and addition of a poly(A) tail at
its 5' end. PCR was performed with an oligo d(T)-anchor primer
and a nested antisense primer of 24 nucleotides (5'AGAGGAG-
TATCTCACCCTGACTTC 3) deduced from the actin cDNA se-
quence. Amplification was performed according to the following
program; melting at 94 "C for 1 min. annealing at 50 °C for I min.
elongation at 72 ' C for 1 min (35 cycles). The PCR products were
cloned using the pCR-Script Amp SK (-I-) Cloning Kit (Stratagene)
and several dillerent cDNA clones were sequenced.

Mytilus galloprovincialis 185

1 tcttttacca gtctgttgta gaagtcaggg tgagatactc ctctttagcg

MCDDKVA 7

51 tttagtataa ctttataaaa cagacatcAT GTGTGACGAC AAAGTAGCCG

ALVV DNG SGMC KAG FAG 24

101 CTTTGGTAGT AGACAATGGA TCAGGAATGT GCAAAGCTGG TTTCGCCGGA

NDAP RAV FPS IVGR PRH 41

151 AATGATGCTC CAAGAGCCGT GTTTCCCTCC ATCGTTGGAA GACCAAGACA

QGVMVGMGQKDSYVGD 57

201 TCAGGGAGTC ATGGTTGGTA TGGGTCAGAA AGACTCCTAC GTAGGAGATG

EAQS KRG ILTL KYP lEH 74

251 AAGCCCAGAG CAAGAGAGGT ATCCTCACCC TGAAATACCC AATTGAGCAC

GIVT NWD DME KIWH HTF 91

3 01 GGTATCGTCA CAAACTGGGA CGATATGGAA AAAATCTGGC ATCACACCTT

YNE LRVA PEE HPV LLT 107

351 CTACAACGAA CTCCGTGTTG CCCCAGAAGA GCACCCAGTC CTTCTGACTG

EAPL NPK ANRE KMT QIM 124

4 01 AGGCTCCACT CAATCCCAAA GCCAACAGGG AAAAGATGAC CCAGATCATG

FETF NAP AMY VAIQ AVL 141

451 TTCGAGACCT TCAATGCACC AGCCATGTAC GTCGCTATCC AGGCCGTACT

SLY ASGR TTG IVL DSG 157

501 CTCACTGTAT GCTTCCGGTC GTACCACTGG TATCGTACTC GACTCTGGAG

DGVT HTV PIYE GYA LPH 174

551 ATGGTGTCAC ACACACCGTA CCAATCTACG AAGGTTACGC TCTTCCCCAC

AILC LDL AGR DLSD NWM 191

601 GCCATCCTCT GTCTAGACTT GGCCGGTAGA GATCTTAGTG ATAACTGGAT

KIL TERG YSF TTT AER 207

6 51 GAAAATCCTC ACCGAGAGAG GTTACTCATT CACAACCACC GCGGAGAGAG

EIVR DIK EKLC YVA LDF 224

701 AAATCGTTAG AGACATTAAG GAAAAATTGT GCTATGTTGC TCTTGATTTC

EQEM STA ASS SSLE KSY 241

751 GAGCAGGAAA TGTCAACCGC CGCTTCTTCA TCTTCCCTAG AAAAGAGCTA

ELP DGQV ITI GNE RFR 257

801 CGAATTGCCC GATGGACAGG TTATCACCAT TGGTAACGAA AGATTCAGGT

CPES LFQ PSFL GME SAG 274

8 51 GTCCAGAATC ATTATTCCAA CCATCCTTCT TGGGTATGGA ATCTGCTGGT

IHET TYN SIM KCDV DIR 291

901 ATCCATGAAA CCACATACAA CAGTATCATG AAGTGTGATG TCGATATCCG

KDL YANT VLS GGT TMF 307

951 TAAGGACTTG TACGCCAACA CCGTCTTGTC TGGTGGTACC ACCATGTTCC

PGIA DRM QKEI TAL APS 324 j

1001 CAGGTATTGC CGACAGAATG CAGAAGGAAA TCACAGCACT TGCTCCAAGC

TMKI KII APP ERKY SVW 341

10 51 ACAATGAAGA TCAAAATCAT TGCCCCACCA GAGAGGAAAT ACTCCGTCTG

IGG SILA SLS TFQ QMW 357

1101 GATCGGTGGT TCCATCTTGG CTTCATTGTC CACCTTCCAA CAGATGTGGA

ISKQ EYD ESGP SIV HRK 374

1151 TCAGCAAACA GGAATATGAC GAATCTGGCC CATCCATTGT CCACAGGAAA

(^ p * 3 76

1201 TGCTTCTAAa ctaaattgtt ttctaggact tatattaatt tattttcaaa
1251 tctcgttaaa acaaaaagtt tcgtgcttgg taacatggac tttaatttat
1301 acaaactgtc tttaaccctt tcaaacttca gatctgtact agcattgagc
1351 Caacggtact tgtacaaata taggacagta aattattatt tgttttatgt
1401 gaaaaagtct ggtggttcaa atgcaagaat gtggagagtt gaatgtgaaa
1451 aagacttgta aaaatactaa acaatccgga aacatatttc aggtttccag
1501 gggagataac tttttactaa atttgatgta catgtggaat aaatcatctq
1551 cattattgtg ataaaatgac ctttatacat ccaattatat taaatcttat
1601 aaaaaaaaaa aaaaaaaa
Figure 2. Nucleotide sequence and deduced amino acid sequence of the Mytilus galloprovincialis actin cDNA (lVIya2). Untranslated regions in
lower-case letters. Start codon in boldface letters. Polyadenylation signal is underlined.

RESULTS AND DISCUSSION 1618 bp and codes for a typical 376 amino acids actin. The 5'

RACE-PCR experiment allowed an additional 19 base pairs to be ,

After colony blot of the cDNA library, 5 positive clones among added and helped to suggest the Transcription Start Point ( + 1). j

352 were chosen and submitted to secondary screening for isola- Best homologies in amino acid sequence were found with the i

tion. The corresponding phagemids were obtained by in vivo ex- bivalve Placopeclen magellanicus: 97.8%, the nematode Cae-

cision and the longest was sequenced on both strands (Fig. 2). This norhabditis elegans: 96.27f , the brine shrimp Anemia sp; 96.5%

complete actin cDNA (named Mya2. Genbank accession number and the silk worm Boniby.x inori: 96.2%. For nucleic acid se-

AF157491) shows a potential coding sequence stretching over quence. best homologies are found with the scallop Placopeclen

186

MiTTA ET AL.

12 3 4 5 6 7

Actin
(1750b)

ISsrRNA

Figure 3. Northern blot analysis of RNAs from various tissues of the
mussel. Twenty (ig of total RNAs from various tissues: 1, hemocytes; 2,
mantle; 3, foot: 4, labial palps: 5, gills: 6, hepatopancreas: 7. adductor
muscle. All were separated by 1% agarose-formaldehyde gel electro-
phoresis, blotted and hybridized with '"P-labeled cDNA probe corre-
sponding to actin cDNA. The RNA relative amounts of the various
tissues are evaluated by hybridizing the same membrane with a probe
corresponding to the 18S rRNAs because the actin mRNA probed is
differentially expressed in the different tissues tested.

magellanicus (85%). the zebra mussel Dreissena polymorpha
(84%) and the oyster Crassostrea gigas (83%). According to
Vandekerckhove and Weber, (1978) who described 20 residues
discriminating muscular and cytoplasmic actins, Myal displays

feature of cytoplasmic actin for 10 codons, while 3 of them show
the mark of muscular actins. The cystein in position #2 is a com-
mon feature among invertebrate actins. although some exceptions
are documented. Actin mRNAs were detected in various tissues as
demonstrated by Northern Blot experiments using the M\a2 cDNA
as probe (Fig. 3). The signal is particularly strong in the mantle, the
labial palps, and the adductor muscle. This strong signals, how-
ever, are mainly due to the recognition by the probe of all forms of
actin niRNA. Indeed, conservation is so high (particularly within
the used probe) that both muscular and cytoplasmic forms are
highlighted giving a cumulative signal.

Several isoforms have been reported in higher vertebrates, di-
vided into muscular and non-muscular actins (Rubenstein 1990).
Due to the high level of similarity with the other actin genes, this
sequence may not be suitable for intra- and inter-species phyloge-
netic studies. Nevertheless, the potential availability of intronic
non-expressed sequences within this actin gene would be of inter-
est in developing a selectively neutral marker as has already been
done in other bivalves (Corte-Real et ai. 1994; Ohresser et al.
1997). Furthermore, this complete cDNA sequence can now be
used in regulation studies as an expression level standard as well
as an anchor in the search for the complete gene including the
proximal promoter involved in its expression pattern.

ACKNOWLEDGMENT

We are indebted to Andy Beaumont for critical reading of the
manuscript and correction of English.

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Mitta. G.. F. Hubert. T. Noel. & P. Roch. 1999a. Myticin. a novel cystein-
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Mitta, G., F. Vandenhulcke. F. Huben. F. & P. Roch. 1999b. Mussel defen-
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Joiirmil of Shellfish Research. Vol. 19. No. I. 187-19?. 2000.

GROWTH OF SEED MUSSEL (MYTILUS GALLOPROVINCIALIS LMK): EFFECTS OF
ENVIRONMENTAL PARAMETERS AND SEED ORIGIN

J. M. F. BABARRO, M. J. FERNANDEZ-REIRIZ,* AND
U. LABARTA

CSIC In.stiiitto de Investigaciones Marinas, c/Eduardo Cabello, 6,
E-36208 Vigo. Spain

ABSTRACT Mussel seeds {MyliUis galh)proviiicialis Lmk) of similar weight and length from two different origins (rocky shore and
collector ropes) were cultivated on a raft in the Ria de Arousa (northwest Spain), from seeding to thinning out, for a total period of
208 days (November 1995 through July 1996). Weight increase rates for the seed from collector ropes were higher than those for the
seed from rocky shore, and the growth rate variations during the cultivation period were associated with the environmental parameters
measured (chlorophyll a and temperature). The origin of the seed was also found to be a significant factor. The condition index (CI)
of the seed froin collector ropes was significantly greater than that of the rocky shore seed at the beginning of the cultivation time. Both
mussel seeds showed a similar CI after 70 days and during the rest of the cultivation time. Although allometric coefficient values for
the relation total dry weight-length showed a similar range for both types of seed, no significant differences were observed for this
coefficient in collector rope mussels throughout the cultivation period. Rocky shore mussels showed, on the contrary, a significant
increase for this allometric coefficient value throughout the cultivation period. These preliminary results from the total dry weight-
length relationship obtained here and the change of CI differences serve to strengthen the hypothesis of a physiological basis for the
differences in growth between both types of seed mussel. This finding could be related to the different features of the original habitats
of the two types of seed, in terms of the cycles of availability of food and exposure to the air.

KEY WORDS: Mussel, growth, environmental parameters, condition index, allometric functions

INTRODUCTION

Mussel (Mytilus galloprovincialis Lmk) cultivation in Galicia
and other cultivation zones (Perez Camacho et al. 1995) is depen-
dent on the availability of large quantities of seed, which can be
obtained from two very different origins: coastal stocks from the
rocky shoreline, and collector ropes suspended from cultivation
rafts.

Previous studies about the growth of these two types of seed in
the Ria de Arousa disagree as to their growth potential from seed-
ing to thinning out. On the one hand. Perez Camacho et al. ( 1995)
found differences in growth rates and condition indices of the
mussels that they attributed to the origin of the seed, with collector
rope seed having the highest values. On the other hand, Fuentes et
al. ( 1998) concluded that neither of the two types of mus.sel .seed
(rocky shore and collector rope) has a higher growth potential,
although the authors do recommend that mussel farmers "use seed
from collector ropes due to their significantly larger size at harvest
time."

Dickie et al. ( 1984). Page and Hubbard (1987). and Fuentes et
al. (1992) have all established that the origin of the seed has a
significant effect on mortality rates, although not on growth. How-
ever. Peterson and Beal (1989) and Rawson and Hilbish (1991)
have observed a significant effect of origin on growth, which they
explain as being due to genetic differences.

Bayne and Newell (1983) point to the effect of endogenous
factors (physiological condition, size, and genotype) and the spe-
cific environmental conditions of the area in question as being two
of the factors that most affect growth in bivalve molluscs. In the
case of environmental factors, it has been shown that in areas
where temperature, for example, is not a limiting factor, the avail-
ability of food affects growth to a very large extent (Mallet et al.
1987. Stirling and Okuinus 1994. Sukhotin and Maximovich 1994.
Widdows et al. 1997).

*Corresponding author. E-mail: mjreiriz@iim.csic.es

The aim of this study was to investigate the effects of seed
origin and environmental parameters on different growth indica-
tors (growth rate, condition index [CI|, and the allometric relation
weight-length).

MATERIALS AND METHODS
Experimental Design

Seed of Mytilus galloprovincialis Link, approximately 20 mm
long, was gathered from the rocky coastline and from raft collector
ropes in the mid-to-outer area of the Ria de Arousa (Galicia, north-
west Spain) in November 1995. Both types of seed, from the same
year class, came from the spawning period in the previous spring-
summer, and the sampling locations were 2 km away from each
other. Experimental cultivation, which was carried out in a raft
usually used for the culture in the Ria de Arousa (500 m"). com-
menced in winter in order to minimize any possible advantages
that collector rope seed may have as a result of its being better
adapted to cultivation on the raft. The experiment ran until June
1996 (208 days), thus covering the first stage in mussel cultivation,
from seeding to thinning out (50-60 mm). Sixteen cultivation
ropes (12 m) were used, eight for each type of seed, alternately
placed and having a density of 19 kg of seed per rope ( 1 .6 kg/m of
rope or 2,600 individuals per meter of rope). Sampling was per-
formed by removing mussels from adjacent ropes at an average
depth of 2-4 m for both types of seed.

Initial average lengths (+ standard deviation) were 22.5 ± 1.5
mm for the seed from collector ropes and 19.0 ± 1.9 mm for that
from the rocky shore. Average total dry weights were 0.36 ± 0.06
and 0.27 ± 0.06 g/individual. respectively. No significant differ-
ences were observed for length and dry weight between both types
of seed at the outset of the experiment (analysis of variance
[ANOVA]: P > 0.05),

Environmental Parameters

Natural seston was described as total particulate matter (TPM.
mg/L). particulate organic matter (POM. mg/L). particulate inor-

187

Babarro et al.

ganic matter (PIM, mg/L). total particulate volume (Vol. mm'/L),
and chlorophyll a (ch\-a. p,g/L). The quality of the seston was
expressed as Q, (POM/TPM) and by the chl-a/TPM index.

The values of chl-fl, as well as temperature (°C) and salinity
(%t) of the water column, were supplied by the Marine Environ-
ment Quality Control Centre of the Conselleria de Pesca. Maris-
queo e Acuicultura (Ministry of Fisheries, Shellfisheries and
Aquaculture) of the Xunta de Galicia (Galician Regional Govern-
ment), chl-fl was calculated from the fluorescence data.

Seawater samples were filtered onto pre-ashed (450°C for 4 h)
and weighed GFC filters and rinsed with isotonic ammonium for-
mate (0.5 M). Total dry matter was established and the weight
increment determined after drying the filters to constant weight at
110 °C for 12 h with an accuracy of 0.001 mg. Organic matter
corresponded to the weight loss after ignition at 450 °C for 4 h in
a muffle furnace. Particulate volume per liter of seawater was
determined by counting in the range of 2-56 p.m using a Coulter
Counter Multisizer II fitted with a 100 ixm-aperture tube.

Mussel Sampling

Duplicate samples of 200-350 individuals were taken from
adjacent ropes, which corresponded to both types of seed mus.sel
after 70, 148, and 208 days.

Individual mussel length (L) was measured to the nearest I mm
using calipers, and each sample was divided into 1-mm length
classes. Adjusted length was given by the formula: L = (C, F)/N
(Box et al. 1989), where C, is the individual length class, F is the
frequency, and N is the total number of individuals. Subsamples of
5-15 mussels were each taken from five to six length classes
covering the entire size range and used to determine total dry
weight (DW„„,,|) and organic weight of tissues (OW,|.,.,„^.). After
cutting adductor muscles and allowing intervalvar water to drain
by placing the mussels with their ventral edge on filter paper,
tissues were dissected and both shell valves (DW^heii) ^nd soft
tissues (DW,,.^.,^^.) were weighed after drying at 100°C until con-
stant weight was obtained. We ashed the .soft tissues at 450°C for
48 h to determine OW||.,^„^., with an accuracy of 0.01 g in all cases.

CI was calculated from the ratio of tissue dry weight (DW,,.,,,^^.)
and the dry weight of the valves (DW..,,^.,,) according to the equa-
tion CI = (DW,,.,.,,,^. /DW.,,,^.,,) 100 (Freeman 1974).

Data Analysis

Regression models were calculated for the logarithm ot tiital
dry weight (log DW,^,,,,), tissue dry weight (log DW,,,^.,,,^.). and
tissue organic weight (log OW„.„.„^.) versus logarithm of length
(log L) relationships from data obtained for five or six length
classes covering the entire length class range from 10-15 mm
above and below the mean length: log W = log a -h b log L.
Analysis of covariance (ANCOVA; Snedecor and Cochran 1980)
was used to make a comparison of these functions between both
types of seed mussel and the change of allometric cocfficicnl (b) in
the experiment.

The confidence inlcr\al lor the dillerence in length and weight
between the months of the cultivation period studied that gives the
growth rate for each stage was given by the formula: X, ^ , - X, ±
it (I - .,/2.k, Sp V(l/n,^ , + l/n,)| (Canavos 1988), where X, , , and
X, are the mean values for length and weight al each end of the
intervals, Sp- is the variance at each end ol' the interval, n, _, , and
n, are the nimiher iil samples al each end >i\' (he inler\al. and 1

, I /, ki is the Student /-distribution value with 95'7f confidence and
k degrees of freedom (k = n, ^ , + n, - 2).

Comparison of mean values of growth rate was carried out with
an ANOVA. Homogeneity of variances was tested by the Bartlett
test (Snedecor and Cochran 1980), and correction for heterogene-
ity (when required) was performed by reciprocal or logarithmic
transformation data. In cases in which homogeneity was not ob-
tained after these transformations had been carried out, the
Kruskall-Wallis nonparametric te.st was used.

The effects of environmental parameters and origin of seed
mussel on the growth rate were tested by stepwise multiple regres-
sion. Seed origin was introduced with values 0 and 1 for collector
rope and rocky shore mussels, respectively. Length and dry weight
values of growth rate were transformed by log|||(x -i- I ) to stabilize
variances.

RESULTS

Environmental Parameters

Variation in temperature (°C) took place within a narrow range,
there being a difference of only 2.7 °C between the maximum and
minimum temperatures during the whole of the experimental pe-
riod (Fig. I A). Temperature was high at the beginning of the
cultivation period ( 15.5 °C) and then decreased in zigzag until the
minimum temperature was reached in February (12.9 °C). From
then on, throughout the spring months, there was a steady increase
in temperature.

Salinity (%o) was dependent on rainfall. Average values for the
area (3l.3-.35.2'^() were obtained at the outset, and they gradually
decreased until January, when the minimum value (28.0'^r) was
recorded. Salinity then increased during the spring months and
finally reached its maximum value at the end of the cultivation
period in July (35.6%p; Fig. I A).

High values for TPM were registered in February through April
(0.9-1.4 mg/L; Fig. IB), in contrast with the low values obtained
throughout the winter months. However, the maximum of TPM
occurred at the beginning of January (2.6 mg/L: Fig. IB), which
can be related to maximums in POM (I mg/L) and especially in
PIM ( 1.6 mg/L). With the single exception of this maximum value.

0 30 60 90 120 150 180 210 240
NDEF MAMJJ

IWS 11996

0 30 60 90 120 160 ISO 210 240
ND EF M AMJJ

Figure I. Viiriution of averaut values (nu'an SI)) of tcnipiTalure (°C)
and salinity (',,) (Al: I PM tmii/],). I'OM (mj;/!.), and IMM (mtt/L) (B);
chl-fl Ik/I.) (C): and (|ualit> iif Ihf si'sldn (Q, ti\i I'OMAII'Ml and
chl-rt/Tl'M index (I)), during Ihc experimental period November 1995
through July 1996.

Growth of Mussels from Two Origins in NW Spain

189

POM was higher during the spring O0.5 mg/L) than during the
winter (0.3 nig/L). Fluctuations in chl-i/ produced two peaks in
February and April (1.4 and 2.0 |J.g/L, respectively; Fig. IC) after
the low values recorded during the initial stages of the experiment
(0.3-0.8 M-g/L).

Qi varied between 0.3 and 0.6. showing a greater oscillation in
winter and a narrower range of fluctuation around 0.5 during
spring, which corresponds to the value that is generally obtained
for the Ri'a de Arousa (Fig. ID). The chl-o/TPM index varies to a
much greater extent, with low values being recorded in winter
(0.1-1.1: Fig. ID) and then increasing from February on to reach
a peak in April and June (2.1).

Growth

The growth rate in terms of length (mm/mo) shows minimum
values in winter ( 1.5 and 2.0 mm/mo for collector rope and rocky
shore mussels, respectively; P > 0.05) and maximum levels from
April through June (9.1 and 6.8 mm/mo for the same two mussel
populations, respectively; P > 0.05; Table 1). The average growth
rates for the whole period November through June were thus simi-
lar for both types of mussel seed, at 4.8 and 4.5 mm/nio. respec-
tively {P > 0.05).

Weight growth shows a trend similar to that for length over the
cultivation period, with the minimum in winter (0.07 g DW,„,^|/mo
for both seed types) and the maximum in the April through June
period, when the collector rope mussels showed significantly
higher values (1.60 g DW,^^,,^|/mo) than the rocky shore mussels
(0.86 g DW,„,^,/mo) {P < 0.05; Table I). The overall November
through June values for DW,p,jj| growth rates are 50% higher for
the former (0.61 g DW„„^|/mo) than for the latter (0.41 g DW„„,,,/
mo) (P < 0.05; Table 1 ). The differences between these two groups
of mussels in the final stages of cultivation (April through June)
and in the overall average values (November through June) also
apply to organic and dry weight of tissues (OW,,.,.,,,^. and DW„„^,,^„
respectively), with collector rope mussels once again showing
higher values (see Table 1).

The variation of growth rate in terms of both length and total
dry weight in this study bore a significant relationship to fluctua-
tions in the environmental parameters chl-fl and temperature of the
water column, in this order of importance (Table 2). Both of these
environmental variables show positive and significant coefficients
(P < 0.001 for chl-a and P < 0.05 for temperature vs. growth rate
for length), with chl-a being the major component of the variance

(40.1 and 56.6% for growth rates for dry weight and length, re-
spectively). A significant but residual effect (P = 0.040) was also
noted for seed origin vs. growth rate for total dry weight (Table 2).

Condition Index

CI for collector rope mussels was 33% higher than that for
rocky shore mussels (P < 0.001 ) at the beginning of the cultivation
period (Table 3). After 70 days, similar values of CI were obtained
for both groups of mussels {P > 0.05), and this remained the case
until the end of the cultivation period without differences between
them. The significant increase in CI (P < 0.001 ) for both groups of
mussels between 70 and 148 cultivation days, which corresponds
with the period February through April, is remarkable.

Allometric Functions

Values a and b of the allometric function weight-length (W =
a L"^) for each mussel seed during the cultivation period are shown
in Table 4. No significant differences among the slopes (b) of both
groups of mussels at any time during the cultivation period were
detected when an ANCOVA was performed on the linear trans-
formations of these functions (P > 0.05). However, the intercepts
for collector rope mussels were significantly higher at the end of
the cultivation period (June) in all cases (P < 0.001 for DW,„,^, and
OW,,^,^^, vs. L and P < 0.01 for DW„^,„^, vs. L; Table 4). Signifi-
cant differences were also obtained for the intercepts in February
(P < 0.05) and November (P < 0.01 ) for the relations DW„^,^^, and
OWj.^^jj^ versus L, respectively, in which higher values were once
more recorded for the collector rope mussels (Table 4).

Concerning shell weight, we found no differences at the onset
of the experiment (0.32 ± 0.05 and 0.25 ± 0.06 g for collector rope
and rocky shore mussels, respectively; P > 0.05). The same ten-
dency was maintained during the cultivation period except at the
end (June), when mussels from collector ropes presented heavier
shells (3.63 ± 0.17 g) than rocky shore ones (2.41 ± 0.23 g) (P <
0.001).

A second ANCOVA was performed on the fluctuation of the
values a and b in the relation DW,^„.,,-L over the cultivation period,
for each seed type independently. The results are shown in Table
5. The power b gives similar values throughout the cultivation
period for collector rope mussels (P > 0.05), yet when intercept a
is recalculated for a common power (Rec.a), it gradually increases
over time, with significant differences between November and
April (P < 0.05) and maximums occurring in June (P < 0.001 ). On

TABLE 1.
Grovrth rates of mussels from collector ropes and rocky shore in different periods of culture.

L (mm/mo)

DW„„„

(g/mo)

DW,„,„. (g/mo)

ow„.

sue (g/mo)

Period of

Collector

Roclvv

Collector

Rocky

Collector

Rocky

Collector

Rocky

Cultivation

Ropes

Shore

Ropes

Shore

Ropes

Shore

Ropes

Shore

Nov-Feh

1 .5 ± 1 .4

2.0 ±1.3

0.07 ±0.(17

0.07 ± 0.05

4.10-'±7.10"

6.10"' ±5.10"'

2. 10-' ±5.10"^

4.10-'±4.I0-'

Feb-Apr

4.4 ± 1.5

4.9 ±1.3

0.35 ± 0. 1 1

0.37 ± 0.09

0.12 ±0.02

0. 11 ± 0.02

0.10 ±0.02

0.10±0.02

Apr-Jun

9.1 ±2.0

6.8 ±2.1

1.60 ±0.26*

0.86 ± 0,23

0.37 ± 0.06*

0.19 ±0.06

0.32 ± 0.06*

0.17 ±0.05

Nov-Jun

4.8 ± 0.45

4.5 ±0.5

0.61 ±0.07*

0.41 ±0.06

0.15 ±0.02*

0.10 + 0.02

0.13 + 0.02*

0.09 ±0.01

Percentages

6%

50%

55%

52%

Data are means (n = 5 samples) ± standard deviation. L, length: DW,„,-,|, total dry weight: DW,,^^^, dry weight of soft tissues; OW,,.,,^^, organic weight
of soft tissues.

* Significant differences between both sources of mussels (f < 0.05: ANOVA). Percentage values indicate how much higher is the increment of growth
parameters in collector ropes mussels over total time of culture (November through June, 208 days).

190

Babarro et al.

TABLE 2.

Multiple regression analysis of shell length (L) and total dry weight (DW„„^,) increment on water temperature (in °C) and

chlorophyll-a (in ^g/L).

Parameter

Constant

Chlorophyll-fl

Temperature

Origin

A. L. mm/mo

-2.855 ± 1.131

0.527 ±0.0911 (56.6%)

0.2 10 ±0.078* (70.8%)

-0.01 1 ± 0.008

B. DW,„,,,. g/mo

-2.900 ± 0.392

0.247 ±0.035t (40.1%)

0.197 ±0.030t (84.1%)

-0.053 ±0.026* (87.8%)

A. N = 18; r =

0.708;

F,

1, = 18.155; P <

0.001

B. N = 18; r =

0.878;

F.,

,4 = 33.620; P <

0.001

Mean intercept and coefficients ± SD. Origin is defined with values 0 and 1 for collector ropes and rocky shore mussels respectively. Percentage values
mean proportion of accumulated variance with inclusion of different factors (NS not significant).
* P < 0.05, significant difference from 0.
f P < 0.001, significant difference from 0.

the other hand, rocky shore mussels showed a steady and signifi-
cant increase of slope (b) over time (P < 0.05; Table 5), reaching
maximum values in June (2.507), although significant differences
were already evident between the allometric coefficients for No-
vember (2.276) and April (2.491) (P < 0.01).

DISCUSSION

The variations in factors such as temperature, salinity, and
chl-« in the area studied are consistent with previous descriptions
of the Galician Ri'as (Fraga 1996). Abundant rainfall and low
levels of sunlight until February are the reason for low salinity and
the concentration of chl-t; in the winter months. The maximum
values of TPM and POM that occurred in January constitute an
exception to the winter-spring pattern that characterizes the natural
seston variability and reveal the effect that frequent storms have on
a shallow area such as this at this time of year, leading to a
resuspension of previously sedimented particles. The mainly sedi-
mentary origin of this sudden increase in POM in January is sup-
ported by the low winter values of the chl-(//TPM index. The peak
levels of phytoplankton that occur in the Galician Ri'as can be
related either to an increase in sunlight (the first chl-a peak occurs
in mid-February) or to the upwelling of nitrates/silicates of the
water caused by the appearance of North Atlantic Central Water
(NACW). NACW is the triain reason for the spring upwelling in
the Galician Ri'as, which is represented by a second and higher
chl-(/ peak in mid-April.

Among the environmental parainetcrs studied, the availability
of plankton in the water column in the form of chl-a and water
temperature had a significant effect on the variations in growth

TABLE 3.

Condition index (CI) values for hoth types of seed mussel during
their cultivation on a raft.

Days of
Cultivation

CI

Month

Collector
Ropes

Rocky
Shore

November
February
April
June

0

70

148

208

15.84 ±2.44*
1 3.27 ± 0.87 NS
33.11 +4.10NS
30.08 ± 2.87 NS

11.87 + 0.97
12.08± 1.86
30..19 ± 2.62

28.88 ± 3.26

NS, not significant (N = 12 in all ca.ses).
* Differences highly significant.

rate. Both of these factors have previously been signalled as being
responsible for most of the variation in the growth rate of bivalve
molluscs (Bayne and Newell 1983). and the fact that in the present
study chl-(/ has the greater effect of the two supports earlier results
(Perez Camacho et al. 1995). In temperate waters, such as Ri'a de
Arousa, temperature fluctuations are not as marked as they are in
extreme environments where fluctuations in this factor play a more
important role (Kautsky 1982, Sukhotin and Kulakowski 1992).
Therefore, variations in growth rate in temperate waters have been
associated with the availability of food (Page and Hubbard 1987,
Thompson and Nichols 1988, Femandez-Reiriz et al. 1996).

Growth rate variation, estimated here with low values during
winter and maxitnums in spring, follow a pattern similar to that
found in other studies (Freeman and Dickie 1979. Pieters et al.
1980, Kautsky 1982. Loo and Rosenberg 1983, Skidmore and
Chew 1985, Page and Hubbard 1987, Mallet et al. 1987). The
maximum growth rates for length, which were recorded in spring
(9.1 and 6.8 mm/mo for collector rope and rocky shore mussels,
respectively), agree with the findings described by Perez Camacho
et al. ( 1 995 ) for the same time of year and both types of seed in the
Ri'a de Arousa. The increase in length after the experiinental period
(31-33 mm for 208 days; 4.4-4.7 mm/mo, with both types of seed
included) is comparable to that of a previous paper on the Ria de
Arousa for a similar period of the year, 5.6-5.8 mni/mo (Fuentes
et al. 1998). Perez Camacho et al. ( 1995) found higher growth rates
of 7-9 mm/mo. However, it is necessary take into account that this
experiment began in April and ended 90 days later, which means
favorable conditions froin the beginning with regard to tempera-
ture and seston availability and quality. The lower growth rates
obtained by Fuentes et al. (1992) also with M. gaUoprovincialis in
the Ria de Arou.sa (2.4 mm after 3 mo) can be attributed to the
cultivation technique used (plastic cages).

These differences also appear in the cultivation period needed
before thinning out. which is greater in experiments that com-
mence in winter (5 and 7 mo, respectively, for Fuentes et al. 1998
artd the present study) than in those that start in spring (3 ino; Perez
Camacho et al. 1995).

Although Fuentes et al. ( I99S) recommend that seed from col-
lector mpcs sliould he used for cultivation, since it reaches greater
length and/or weight than rocky shore seed, they differ from Perez
CaiTiacho et al. ( 1995) as to the existence of a difference in growth
rates from seeding to thinning out. Their reasoning is based on the
fact that if more than one cohort were included in the process of
gathering the rocky shore seed, this may well explain the different
growth rates reported by the latter authors. The results of this study

Growth of Mussels from Two Origins in NW Spain

191

TABLE 4.

Results of regression and covariance analysis on data relating weight (W mg) of A/, galloprovincialis from two sources of seed to

length (L mm).

Collector Ropes

Month

Rec. a

a
Common

b
Common

Rockv Shore

Rec. a

DW,„,.,| versus

L:

A

November

0.328 NS

">

Fehruarv

0.152 NS

2.

April
June

0.232 NS
0.644*

0.321

DW,„,„, versus

L

B

November

0.019 NS

2

February

April

June

0.026t
0.047 NS
0.043i

0.036
0.039

2
2.
2.

0W|,^.,„^. versus

L

C

November

0.012+

0.027

-)

Februarv

0.018 NS

1

April
June

0.046 NS
0.037*

0.035

1

2.247 ± 0.092 NS 0.986 10

505 ± 0.068 NS 0.990 1 5

397 ±0.1 96 NS 0.943 11

212±0.118NS 0.978 10

521 ± 0.158 NS 0.970 10

361±0.104NS 0.976 15

464 + 0.161 NS 0.962 11

523 ± 0.260 NS 0.922 10

580 ± 0.208 NS 0.951 10

385 ±0.1 17 NS 0.980 15

429 ±0.1 70 NS 0.958 11

526 ± 0.275 NS 0.914 10

0.325

2.267

0.326

0.171

2.464

0.194

0.207

2.442

0.179

2.383

0.167

0.026

2.398

0.03 1

2.279

0.041

0.040

2.508

0.033

2.546

0.030

2.355

0.028

0.017

2.380

0.016

0.039

2.473

0.032

2.540

0.028

0.273

0.03 1

0.031

0.018

0.027

2.274 ±0.056 0.996 10

2.430 + 0.062 0.992 15

2.491 ±0.054 0.996 11

2.507 ±0.098 0.988 10

2.318
2.200
2.557
2.569

: 0.087 0.988 10

: 0.102 0.972 15

: 0.1 23 0.980 11

: 0.350 0.964 7

2.223 ±0.085 0.980 10

2.374 + 0.251 0.872 15

2.522 ±0.126 0.978 11

2.554 ± 0.232 0.960 7

a and b values are parameters in the equation W = aL*": ANCOVA ANOVA was made after logarithm transformation: log W = log a + b log L. When

there were no differences in slopes (b) of the relationship, a common exponent was therefore calculated and used to recalculate values for the parameter

a (Rec. a). NS, not significant.

*/'< 0.001.

t P < 0.05.

±/'<0.01.

do nevenheless point out a difference in growth rates, especially
for weight, that are also related to vaiiability of environmental
parameters during the cultivation period. This study is also in
concordance with Perez Camacho et al. ( 1995). who showed that
initial size (weight/length) has no effect on the results, either be-
cause the experiment was designed with this condition in mind (as
in this study) or because the statistical analysis (multivariate
ANOVA) performed on the results showed this to be the case
(Perez Camacho et al. 1995). With reference to the study by Fu-
entes et al. (1998), the differences observed in the initial siie of
both types of seed (0.6 cm for rocky shore mussels and 2. 1 cm for
collector rope mussels), as well as the high density of mussels on

TABLE 5.

Results of ANCOVA on data relating DW,„,3| (mg) to shell
length (mm).

Collector Ropes

Rocky Shore

Month

b

a

Rec. a

n

b

a

Rec. a

n

November
February
April
June

2.247
2.505
2.397
2.212

0.328
0.152
0.232
0.644

0.209
0.237
0.249
0.319

10
15
11
10

2.276
2.430
2.491

2.507

0.326
0.194
0.179
0.167

—

10
15
11
10

Collector ropes:

Comparison among slopes. F = 1.091 (DF = 3.38) P > 0.05.

b.„n™o„ = 2.384.
Comparison among intercepts. F = 12.944 (DF = 3.41 ) P < 0.001.
Rocky shore:
Comparison among slopes, F = 3.134 (DF = 3.38) P < 0.05

a and b are parameters in the equation DW,^,,, = aL'' (.see Table 4. top).
Rec. a represents recalculated intercept for common slope.

the ropes (5.000 individuals per meter), which contrasts with the
2.600 individuals per meter of the present study and the 2,000
individuals per meter of Perez Camacho et al. (1995), may have
affected their results, given the effect that both of the above-
mentioned factors (initial size and density) may have on growth
(Sukhotin and Ma.ximovich 1994. Eldridge et al. 1979, Femandez-
Reiriz et al. 1996).

Although our results show a difference in growth rates between
collector rope and rocky shore tiiussels, as was previously ob-
served by Perez Camacho et al. (1995). it should be pointed out
that these differences become more apparent in those months that
most favor growth (April through June), which is precisely the
period in which the experiment by Perez Camacho et al. (1995)
took place, and just as was the case in their experiment, the most
marked differences in our results are those for growth in wet
weight and tissue weight. A higher growth efficiency for collector
mussels when environmental conditions (temperature and overall
quality of food) are more favorable, resulting in a more positive
scope for growth, and the persistence of different metabolic pat-
terns due to immersion-emersion periods that are indicative of
anaerobic pathways for rocky shore mussels, could help us to
understand such different growth responses. Genetic factors could
also explain a significant proportion of the variances in production/
growth of mussels (Widdowset al. 1984. Mallet et al. 1987). since
it has been described that mussels exhibit high levels of genetic
variability measured as enzyme polymorphisms both on a micro-
and a macrogeographic scale (see Hawkins and Bayne 1992). Ad-
ditionally, energy-saving mechanisms related to respiration me-
tabolism have been described for those animals, which live in the
intertidal locations. Metabolic depression and anaerobiosis are
clearly implicated as key factors of energy conservation to with-
stand emersion conditions in order to compensate for reduced

192

Babarro et al.

feeding time with respect to sublittoral animals (de Zwaan and
Mathieu 1992).

Both terms (physiological rates and metabolic patterns) are
being tested for both types of seed mussel cultivated on suspended
conditions in Arousa.

The use of the allometric function weight-length in growth
studies is firmly established (Hickman 1979. Rodhouse et al. 1984.
Sprung 1995, Sara et al. 1998, among others). A correspondence
has occasionally been established between the variation in the
allometric coefficient and local food conditions (Sara et al. 1998).
In this study, we did not measure the original weight-length rela-
tionship of intertidal mussels before putting them on the raft. How-
ever, the fact that the experiment began a few hours after the seed
mussels were gathered from their environments suggests that this
relationship is similar to that which these mussels might show in
their original habitat. An ANCOVA that was performed for this
weight-length relationship in rocky shore mussels with regard to
cultivation time period showed changes in the b parameter value
(more evident between slopes of November and April), whereas
collector rope mussels presented no differences in this value
throughout the cultivation period. This probably means that envi-
ronmental changes for rocky shore mussels, when they are put
under immersed conditions on the raft, might be responsible for
such a response of the allometric functions.

The initial differences in the CI of the two types of mussel seed
can be attributed to their original habitats, which differ greatly with
regard to the availability of food and their respective situations of
emersion-immersion. The disappearance of these differences after
70 days may be related to the changes in physiological responses
resulting from a new environmental situation (Bayne et al. 1984,
1987), although these differences may well persist for some time
(Widdows et al. 1984. Iglesias et al. 1996).

Given the experimental design in this study, any effects on our
results of a genetic nature that have occasionally been used to
explain differences in growth (Peterson and Beal 1989, Rawson
and Hilbish 1991) would be possible when genetic factors play a

part in the choice of substrate (rocky shore or collector rope) by
larvae in the Ria de Arousa or when different cohorts are involved.
As has previously been mentioned by Perez Camacho et al. ( 1995),
an alternative hypothesis would be to consider a physiological
adaptation response of each seed to its habitat of origin, which
would imply that cultivation starts from different physiological
states, which is described by Mallet et al. (1987) as ecological
memory. This ecological memory would condition the physiologi-
cal response of the seed to its new environmental situation, as
shown by an increase in the CI for the rocky shore seed.

We can consider that the aim of slowing down the initial
growth rates in order to minimize any possible advantages for the
collector rope mussels has been achieved. This would explain why
the differences observed in growth rates between the two types of
seed are less marked than those recorded by Perez Camacho et al.
( 1995) during the first stage of the cultivation period. Although the
allometric coefficients for both types of seed need to be tested with
regard to their original habitat for establishing more properly habi-
tat-dependent changes, the CI differences maintained in both seed
types supports the hypothesis that there is an underlying physi-
ological basis for the difference in their respective growth rates.
Moreover, given the experimental conditions under which the
present study was performed and taking into account the CI
changes, the physiological parameters of the two types of seed
could be expected to converge.

ACKNOWLEDGMENTS

We are grateful to Lourdes Nieto. Beatriz Gonzalez, and Sonia
Villar for technical assistance. We also thank Juan Maneiro from
the Marine Environment Quality Control Center of the Consellen'a
de Pesca. Marisqueo e Acuicultura of the Xunta de Galicia for the
determination of environmental parameters. We are also indebted
to the crew of the Jose Maria Navaz from Instituto Espafiol de
Oceanografia. This work was supported by Project CICYT
MAR97-0592. J. M. F. Babarro was funded by a grant from Ex-
cma. Diputacion de Pontevedra.

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I

Joiinwl at Shclljhh Research. Vol. 1^, No. I. 195-201. 2000.

FEEDING BEHAVIOR OF SEED MUSSEL MYTILUS GALLOPROVINCIALIS: ENVIRONMENTAL

PARAMETERS AND SEED ORIGIN

J. M. F. BABARRO, M. J. FERNANDEZ-REIRIZ,* AND

U. LABARTA

CSIC Institiito de Investigaciones Marinas

c/Editardo Cabello. 6. E-36208 Vigo. Spain

ABSTRACT Mussel seed (Mylilus galloprovinciali.'i) from two original habitats (rocky shore and collector ropes) was cultivated on
a raft in the Ria de Arousa (northwest Spain), for a period of 226 days (November 1995 through June 1996), from seeding to thinning
out. during which time the behavior of clearance rates (CR) and ingestion rates (IR) was studied. The study of these two physiological
parameters of energy acquisition (CR and IR) demonstrates that the two types of seed showed significant differences in these
parameters at the start of the experiment and after the first 8 days on the raft. After 15 days, large increases in these physiological rates
were observed for both types of seed, with the increase for the rocky shore mussels doubling that of the collector rope specimens. These
increases led to the disappearance of the significant differences in CR and IR between both seed origins, with this situation being
maintained for the remainder of the experimental period. The variation in CR follows a seasonal pattern, with low values being recorded
in winter and increasing in spring and summer. Minor seasonal variations of total seston concentration are counterbalanced by an
inverse variation in organic content, and so organic IR followed a pattern similar to that of CR. This seasonal variation can be attributed
to fluctuations in the factors food quality (Q, ) and temperature, in this order, as the use of multiple regression analysis has proved. Seed
origin had a significant effect as a factor of interaction with food quality Q,. probably because of differences between the original
habitats of the seed (rocky shore and collector ropes) in the latter factor. Although in this study food quality has been expressed in terms
of organic content (Q, = organic/total particulate matter), the content of phytoplankton as chlorophyll a may have had an important
effect on the variation of both of these physiological rates. A significant exponential relationship has been established between the IR
and the content in total particulate matter, which suggests regulation processes according to the amount of natural food available based
on a decrease of CR.

KEY WORDS: Mytilus gaUoprovinciaUs. mussel seed, clearance rate, and ingestion rate. Ria de Arousa

INTRODUCTION

Clearance rates (CRs) and ingestion rates (IRs) determine the
amount of food that enters the digestive system of bivalve mol-
luscs. The variability observed in these physiological parameters
has been interpreted in terms of the ability of these animals to
adapt to the specific environmental and nutritional conditions of
their habitat (Widdows et al. 1984; Navarro et ai. 1991; Okumus
and Stirling 1994; Iglesias et al. 1996). The relationship between
IR and food concentration depends on CR, which in turn is af-
fected by environmental factors. Hawkins and Bayne (1992) pro-
posed the use of multifactorial analyses to ascertain the relevance
and ecological complexity of the set of environmental variables, as
well as their interaction with physiological parameters.

When attempting to determine the extent of the influence of
habitat, transplant experiments are considered to be the ideal way
of analyzing the effect of the variability attached to the environ-
ment in which the individuals lived previously, in connection with
what Mallet et al. (1987) termed ecological memory. Previous
comparative studies of mussel seed gathered from a rocky shore
and from collector ropes and then cultivated on a raft established
the existence of a significant effect of the seed origin on growth
rate (Perez Camacho et al. 1995; Babarro et al. 2000). with this
effect being associated with physiological parameters.

The extent of time needed for CR and IR to acclimate to new
environmental conditions has been reported in various studies (4.5
mo [Okumus and Stirling 1994] and more than 2 mo [Widdows et
al. 1984], although Hawkins and Bayne [1992] have suggested a
period of less than 2 mo). The aim of the present study was to
determine the extent to which differences in the feeding regime

*Corresponding author. E-mail: mjreiriz@iim.csic.es

and the regime of immersion-emersion in their original habitats
(rocky shore and collector ropes) affects the behavior of CR and IR
during the cultivation period in the raft (20-60-mm shell length).
The study also deals with a set of factors, such as an endogenous
factor (i.e., shell length) and the environmental and nutritional
conditions in the cultivation area, and the effect they have on these
physiological rates for raft-cultivated mussels.

MATERIALS AND METHODS

Harvesting and Maintenance of Mussels

In November 1995, seed of Mytilus galloproviticialis of ap-
proximately 20 mm in length was gathered from the rocky shore
and from collector ropes on a raft, both in the mid to outer area of
the Ria de Arousa (Galicia, northwest Spain). Both types of seed,
from the same year class, came from a spawning period in the
previous spring/summer. Experimental cultivation, which was car-
ried out under production conditions on the raft (500 m"), began in
winter — the season of minimal growth rate — with the aim of mini-
mizing any possible advantages for the collector rope seed as a
result of its better adaptation to raft cultivation conditions. The
experiment continued until July 1996 (226 days) and covered the
first stage of mussel cultivation from seeding to thinning out (50-
60 mm). Sixteen cultivation ropes (12 m) were used, 8 for each
type of seed, disposed alternately and with a density of 19 kg of
seed per rope (1.6 kg/m of rope or 2.600 individuals/m of rope).
Specimens were sampled each time from adjacent ropes from the
stretch of 2-5 m.

The initial length of the seed was 22.55 ± 1 .55 mm for collector
rope seed and 1 9.02 ± 1 .93 mm for the rocky shore seed. Mean
total dry weight was 0.36 ± 0.06 and 0.27 ± 0.06 g/individual for
each type of seed, respectively. These differences in length and dry

195

196

Babarro et al.

weight between the mussels from the two different original habi-
tats were found to be not significant at the beginning of the ex-
periment (analysis of variance [ANOVA]; P > 0.05 in both cases;
n = 96).

Experimental Design

Seawater was pumped from the depth where seed was sampled
(2-5 m) into an open circuit consisting of three rectangular cages
(45 X 40 X 14 cm = length x width x height and 19 L of capacity).
each provided with 16 compartments set in parallel. Seed speci-
mens from the two origins were placed in the side cages while the
middle cage, containing no specimens, acted as the control. The
water flowed independently into each cage from an inlet pipe,
which went all the way around the top of the cage. The water outlet
for each cage consisted of a single pipe leading off from the top of
the cage. The flow in each cage was maintained at a steady rate ot
approximately 3 L/min. so that the concentration of particles at the
outlet would never fall below 50% of that at the inlet. The number
of specimens used in each replica for physiological measurements
varied according to their size (i.e., with the length of cultivation
period). At the outset, six specimens of the 20-mm shell length
class were placed in each compartment, and this number was also
used for 30-mm shell length. From the 40-mm shell length onward,
the number of animals used progressively decreased and at the end
of the experiment there was only one specimen of the 60-mm
length class in each compartment. Physiological measurements
were taken weekly from November to January, fortnightly from
February to May, and monthly in June and July.

Measurements

Natural seston was characterized as total particulate matter
(TPM, mg/L), particulate organic matter (POM. mg/L), particulate
inorganic matter (PIM, mg/L), particulate volume (Vol. mm'/L),
and chlorophyll a (chl-a, |jig/L). The values for ch\-a. as well as for
the temperature ( °C) and salinity (%<) of the water column were
provided by the Centro de Control de Calidade do Medio Marino
da Conselleria de Pesca, Marisqueo e Acuicultura (Xunta de Gali-
cia). chl-rt was calculated from the fluorescence data. Seston qual-
ity was expressed as Q, (POM/TPM), Q, (POM/Vol), and the
chl-(i/TPM index. The same methodology as that used for gravi-
metric analysis of seston was applied to characterize the feces
produced by the mussels in the experimental system: seawater
samples and aliquots of known volumes from each fecal sample
were filtered onto pre-ashed (450 °C for 4 h) and weighed GFC
Alters and rinsed with isotonic ammonium formate (0.5 M). Total
dry matter was established as the weight increment detennined
after drying the filters to constant weight at 110 °C for 12 h.
Organic matter corresponded to the weight loss after ignition at
450 ■ C for 4 h in a muffle furnace. Vol/L of seawater was deter-
mined by counting in the range of 2-56 fji.m using a Coulter
Counter Multisizer II fitted with a lOO-fxm aperture tube. The
variation in these environmental and/or nutritional parameters over
the cultivation period is shown in Table I.

The egestion rates of inorganic matter (mg/h) were determined
for each group of mussels and assumed to represent inorganic IR
(i.e., no absorption of ash in the digestive tract was considered).
CRs were then estimated indirectly, with PIM concentration (mg/L
of seawater) as the reference for available inorganic matter: then
CRIh ' = mgPlM

iy9S). where PIM,

^.„ h'/mg PlM, , L ' (Iglesias et al. 1996:

is the amount o\' inorizanic content \(iided

with the feces in a given unit of time (h) and PIMp„„j is the
inorganic content of the food in a given unit of volume (L). A lag
time of 2 h was allowed between the sampling of seawater and the
gathering of feces, to account for the estimated time for intestinal
transit for mussels from the Ri'a de Arousa (Navan-o et al. 1991).
Before the start of the experiments, mussels were kept in the cages
for 1 h with flowing seawater at the natural particle concentration
to allow for valve opening and acclimation to cage conditions. The
feces obtained on the bottom of cages after this time were refused.

The organic ingestion rate (OIR mg org/h) was calculated as a
product of CR and the organic food concentration (mg POM/L).

For purposes of comparison with this indirect CR estimation.
CR was also calculated by the direct estimation (flow method)
using the Hildreth and Crisp (1976) equation: CR = f([C, - C.,]/
C„), where f is the flow rate, C, and C,, are food concentrations at
the inflow and outflow of the experimental cage, and C, represents
the particle concentration surrounding the mussel. The experimen-
tal design used in this study enabled us to consider C„ concentra-
tion as being close to C,, so C, was subsequently used as the
reference concentration for calculation purposes. C, and C„ were
determined by recording the concentration of particles 2-56 p.m in
water sainples with a Coulter Counter Multisizer.

The degree of correlation obtained between the two calcula-
tions for CR was highly significant for both groups of mussels
together, similar to those observed by Urrutia et al. (1996) and
Iglesias et al. (1998) (Y [biodeposition] = 1.1 18 ± 0.074 x flow
+ 0.043; r- = 0.86; P < 0.001; /; = 300). Once significant rela-
tionships were established for both methodologies, CR here (text,
tables, and figures) refers to indirect measurements by biodeposi-
tion method.

Size Standardization

To preclude variability in physiological rates caused by size
differences, these rates were corrected to a standard-sized indi-
vidual. To this end, once physiological measurements were com-
pleted, shell length of each individual was recorded to the nearest
0.1 mm with Vernier calipers and the soft tissues excised from the
shell, dried at 1 10 °C for 12 h, and weighed. The most commonly
used reference for size is soft body mass; however, the weight
standardization of CR may be somewhat arbitrary, because this
rate is considered to be dependent on filtration (gill) area, which is
closely related to shell length (Hughes 1969; Jones et al. 1992). As
discussed before by Iglesias et al. (1996) and Labartaet al. (1997),
in this study we used shell length (L) to standardize CR following
the equation: Y, = Y^, x (LyL^.)'' where Y, and Y^, are the stan-
dardized and the nonstandardized CRs, respectively; L, is the stan-
dard length of the animal according to shell increment during the
experiment (20-60 mm); L,, is the observed length of the animal;
and b is the power that scales CR with shell length (b = 1.85.
Perez Camacho and Gonzalez 1984). Furthermore, with the aim of
establishing the fluctuation of clearance and IRs over the cultiva-
tion period, 40-mm shell length was chosen as an average size for
the experiment.

Data Analysis

Comparison of means for CR and OIR was carried out by
means of standard ANOVA after data translorniation when nec-
essary. Kruskall-Wallis and Friedman nonparametric tests were
used when homogeneity was lacking (Bartlett's test). Multiple
analysis (stepwise regression) was used to determine the effect ot

Feeding Behavior of Seed Mussel M. galloprovincialis

197

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various factors, both endogenous (shell length) and environmental
(TPM, POM. Vol. Q|. Q., T. and chl-</), and their interactions on
the variation in CR. The factor seed origin was added to this
analysis with values of 0 and 1 for collector rope and rocky shore
mussels, respectively. All of these analyses were performed ac-
cording to the methods described by Snedecor and Cochran (1980)
and Zar( 1984).

RESULTS

The values of CR and OIR during the experiment are shown in
Table 2. CR ranges between 0.20 and 0.26 L/h in the initial stages
of cultivation (20-mm mussel), rising up to 4.37-4.51 L/h for a
60-mm mussel.

CR values for collector rope mussels were significantly higher
than those of rocky shore mussels in days 0 and 8 of the experi-
mental period (P < 0.01 in both cases; ANOVA; Table 2). How-
ever, CR for the rocky shore mussels had increased by 35% by the
end of the 2nd week, compared with only 16% for the collector
rope specimens. From this point on, no further significant differ-
ence in this physiological rate was recorded between the two types
of mussels {P > 0.05; Table 2). In the case of OIR, both groups
of mussels followed the same pattern as that described for CR
(Table 2).

VARIATION OF CR AND OIR IN CULTIVATION TIME

Figure 1 shows the variation of standardized CR and OIR for
the 40-mm length class in cultivation time. CR showed a clear
seasonal pattern with low values during the winter months, in-
creasing in spring and summer. High values recorded in January
constituted the sole exception. These trends were also recorded for
OIR (Fig. 1 ), amplified in this case by the coincidence of high CR
and high POM.

CR versus Shell Length and Environmental Parameters

The multiple regression analysis carried out on the variation
observed in CR during the experiment showed a significant and
positive relation to size (L mm), food quality (Q,), and tempera-
ture (see F-ratio, Table 3). The regression model accounted for
76.7% of the variance for CR, which in turn is mainly accounted
for by size (L mm 67.6%). with a coefficient of 1.762. It is im-
portant to point out the significant negative effect of the interac-
tions of food quality (Q,) with both origin (Q, x origin) and
temperature (Q, x T) (Table 3).

Organic IR versus Natural Seslon (TPM mg/L)

A significant relationship was established between the IR
(OIR) and the variation in seston (TPM. mg/L), that could not be
established in the case of the CR. This response of ingestion to
seston concentration is shown in Figure 2 and fits exponential
functions according to the Ivlev curves IR = a ( 1 - e ):

OIR= 1.29 ± 0.39 [1 -e
n = 14; r- = 0.517; P< 0.0

-a7-'i±0.J6.TPM-i

Rocky shore

OIR = 1.18 :
n= 14; r-

0.36 [1 -e
= 0.481; P< 0.01

0.87±0.4fi-TPM-i

H O

The covariance analysis performed for the linear transforma-
tions of these exponential curves showed no significant differences

198

Babarro et al.

TABLE 2.

Values of physiological parameters (mean ± SD, n = 32) of two sources of seed muscles standardized to shell length (L) according to growth

of Af. galloprovincialis during the experiment.

Cultivation

Source of

OIR

Date

Days

Seed Mussel

L (mm)

CR (L/h)

(mgPOM/h)

11/27/95

0

Collector ropes

20

0.43 ±0.12*

0.16 ±0.04*

Rocky shore

0.34 ± 0.09

0.13 ±0.03

1 2/5/95

8

Collector ropes

20

0.26 ± 0.09*

0.08 ± 0.03*

Rocky shore

0.20 ± 0.09

0.06 ± 0.02

12/13/95

15

Collector ropes

20

0.50 ±0.10

0.16 ±0.03

Rocky shore

0.46 ±0.1 5

0.14 ±0.05

12/20/95

22

Collector ropes

20

0.37 ± 0.06

0.14 ±0.02

Rocky shore

0.35 ± 0.08

0.13 ±0.03

1/3/96

36

Collector ropes

20

0.40 ± 0.09

0.40 ± 0.09

Rocky shore

0.36 ±0.10

0.36 ±0.10

1/17/96

50

Collector ropes

20

0.57 ±0.14

0.26 ± 0.06

Rocky shore

0.60 ±0.1 5

0.28 ± 0.07

1/31/96

64

Collector ropes

30

0.72 ± 0.23

0.20 ± 0.06

Rocky shore

0.71 ±0.25

0.20 ± 0.07

2/15/96

80

Collector ropes

30

0.49 ± 0.09

0.19 ±0.04

Rocky shore

0.52 ±0.15

0.20 ± 0.06

2/28/96

95

Collector ropes

30

0.69 ±0.1 3

0.39 ± 0.07

Rocky shore

0.69 ±0.14

0.39 ± 0.08

3/13/96

110

Collector ropes

40

1.23 ±0.27

0.56 ±0.1 3

Rocky shore

1.25 ±0.38

0.57 ±0.18

3/27/96

125

Collector ropes

40

1.13±0.27t

0.59 ± 0.1 4t

Rocky shore

1.27 ±0.29

0.66 ±0.15

4/10/96

140

Collector ropes

40

1.16 ±0.47

0.74 ± 0.30

Rocky shore

1.17±0.23

0.75 ±0.1 5

4/24/96

155

Collector ropes

50

2.47 ± 0.74

0.97 ±0.29

Rocky shore

2.06 ± 0.64

0.81 ±0.25

6/5/96

197

Collector ropes

60

4.51 ± 1.21

1 .30 ± 0.35

Rocky shore

4.37 ± 1.16

1.26 ±0.33

7/3/96

226

Collector ropes

60

3.92 ±0.89

1.69 ±0.38

Rocky shore

4.09 ± 0.83

1 .77 ± 0.36

CR. clearance rate length-specific (L/h) by biodeposition method: OIR. organic ingestion rale length-specific (mg POM/h).
* P < 0.01. fP < 0.05. ANOVA and Kruskall-Wallis nonparametric test in case of heterogeneity of variances).

between both groups of mussels for OIR (t = 0.037, df = 24. P
> 0.05. and t = 0.358, df = 25. P > 0.05 for analysis of slopes and
intercepts, respectively). Therefore, one exponential curve for both
groups of mussels together is shown in Figure 2:

OIR = 1.23 ± 0.26 [1-e^'^*"-"^™]
n = 28; r- = 0.500; P< 0.01

DISCUSSION

The variation in CR and OIR during the experimental period,
for mussels standardized to 60 mm to compare with the literature
values, covers a wide range ( 1 .46—4.5 1 and 1 . 1 3—4.37 L/h for CR.
0.43-2.23 and 0.33-2.04 mg POM/h for OIR. values for collector
rope and rocky shore mussels, respectively). These data coincide
with those obtained by Navarro et al. (1991) and Iglesias et al.
( 1996) for M. !ialloprovincialis in the Ri'a de Arousa. In the case of
CR. however, these values are higher than those recorded for mus-
sels elsewhere reported by Okumus and Stirling (1994) in their
wide-ranging review. Despite the above-mentioned differences in
CR due to low seston loads, characteristics from Galician Rias in
particular, the values for organic IRs reported in the present study
are similar to those obtained hv Widdows ct al. ( 1979). also under

environmental conditions, and by Bayne et al. ( 1989) in the labo-
ratory, with a higher range of values for seston and organic content
(0.79-7.43 mg TPM/L. 0.43-1.79 mg POM/L. and 0.18-0.71 for

Qi).

The few studies that include CR data recorded over a period of
seasons show that CR follows a clear seasonal pattern, with maxi-
mum values occurring in the spring and summer months and mini-
mum values in winter. This cycle can be observed both under
laboratory conditions, with a constant food supply a\ailable (Wor-
rall et al. 1983). and under natural conditions (Newell and Bayne
1980). Larretxea (1995), taking into account a previous study of
Hawkins et al. (1985). suggests that the seasonal sequence of CR
is persistent to a large extent, although the effect of temperature
could be an important determinant of this seasonal response.

In this study, rates of energy acquisition exhibit a seasonal
pattern, with lower values occurring during the winter months and
slightly higher during spring and summer. The range results
greater in terms of OIR because of the simultaneous decrease in
CR and in organic content of the seston. An exception to this
overall behavior are those values found to deviate largely from the
mean of the season during which they were obtained, namely
unexpectedly high values for CR. and especially OIR. in the
samples taken in January. These may be accounted for by the high

Feeding Behavior of Seed Mussel M. galloprovincialis

199

collector ropes -o- rocky shore

£■21

A

0 30 60 90 120 150 180 210 240

^^■^1

1995 I 1996

Time (days of cultivation/month)

Figure 1. Seasonal changes in CR and OIR standardized to 40 mm of
shell length for both sources of seed mussel M. galloprovincialis.

POM values ( 1 .003 mg/L) and high Q, value (0.386), which can be
considered as a result of a process of resuspension of material from
the bottom in the Ria de Arousa (Babarro et al. 2000). Similar
feeding behavior has been observed by Ki0rboe et al. (1981) and
Larretxea (1995) concerning CR increments associated with an
increase in detritus and sediment resuspended.

With regard to seston composition, the results of this study
show a 33% increase in total organic content in spring/summer
when compared with winter, which can be related to an increase in
mean CR of 30% for collector rope mussels and 40% in the case
of rocky shore specimens.

However, other factors that seem to exert indirect influence on
the energy gain should be taken into account. This is the case for
chl-a values for the period February through July that doubles that
of the period November through February (Babarro et al. 2000).
During the winter months, the proportion of phytoplankton (chl-(()

TABLE 3.

Stepwise multiple regressions of clearance rate of mussels with log

shell length (L), quality of seston (Q, = POM/TPM), temperature

(T, °C), and interactions terms.

Parameter

CoefTicient

SE

F-Ratio

P

r-

Constant

-20.329

LogL

1.762

0.042

1764.516

<0.001

0.676

Q, XT

-1.297

0.085

230.838

<0.00l

0.683

Qi X origin

-0.070

0.023

9.491

<0.01

0.686

T

1.778

0.245

52.512

<0.00l

0.688

Q,

18.948

1.228

238.067

<0.001

0.761

T-

-0.039

0.009

20.776

<0.001

0.767

r- = 0.767: n

= 812; F,.,o5

= 440.729;

/'< 0.001

"1

n

fl

^/^

^^^^^

/On

/a

/ □

i- L

Origin factor has been estimated with values 0 and 1 for collector ropes and
rocky shore mussels, respectively.

TPM (mg/L)

Figure 2. OIR versus TPM relationship for both sources of seed mus-
sel M. galloprovincialis. Both groups of mussels (collector ropes,
squares; rocky shore, circles) were fitted by nonlinear regression ac-
cording to Ivlev curve: Y = atl - e*" ^) (see text for details of fitting
equation).

in the organic content of the diet is 4-23%, rising to 21^1%
during spring and summer, with peaks of 37.4% and 40.9% in
April and February, respectively, which bears a close relationship
to the seasonal variation in CR and OIR (carbon content = chl-«
X 54, Widdows et al. 1979: organic matter = carbon x 1.87,
Fernandez Ri'os 1992). This incidence of phytoplankton (chl-a) can
also be observed in the fact that when the value for POM is not
associated with chl-« (Babarro et al. 2000), no effect on CR is
observed. However, the effect of this factor (chl-«) has not been
tested in the multiple model because of the use of Q, as a factor of
food quality and in order to avoid overlapping of information.

The multiple regression model for CR shows the importance of
shell length, food quality (Q,), and the temperature either as an
independent variable or as a term in interaction (Q, x T). Very
likely the presence of a term Q, x origin could be related to
differences in seston quality between both original locations. Pre-
vious studies carried out with both groups of mussels showed
higher Q, values for subtidal location than that for rocky shore
(unpublished data). Shallow water and stronger tidal waves in the
rocky shore spot seem to affect the relation organic:inorganic frac-
tion, with resuspension processes of the sediment playing an im-
portant role. Mussels seem to adjust their feeding rates in a rela-
tively short time under environmental changes (first 8 days under
culture conditions), and probably when animals are "adapted,"
fluctuations of Q, after this initial period of time do not cause
different CR responses between the two populations.

The effects of the food ration or particle concentration on fil-
tration rates in bivalve molluscs have been widely studied over the
years. A reduction in CR when seston concentration increase has
been reported in several experiments (Foster-Smith 1975: Wid-
dows et al. 1979: Riisgard and Randlov 1981). As was previously
established by Winter (1978). it seems that the ability of bivalves
to adjust CR in response to an increase in particle concentration
allows the regulation of IR.

In fact, the relationship between IR and seston concentration
has been appropriately described by an exponential asymptotic

200

Babarro et al.

function (Ivlev curve) in this study. This behavior suggests to us a
mechanism of regulation of ingestion based, in this case, on ad-
justing CR and taking into account that this saturating increase in
OIR cannot be assigned to the negative organic content versus
seston availabihty relationship, which was not observed in our
study as significant. Although higher CRs are related with lower
seston availability values, significant effects of either TPM or
POM and chl-a on CR were not observed, possibly because of the
reduced range of variation in seston concentration.

As already been mentioned, the results of this study establish
that temperature has a significant effect on CR. The thermodepen-
dence of CR coincides with the observations made by Widdows
(1976), namely that mussels living in a stable thermal environment
(which is the case with a range of temperature variation of 2.73 °C)
have not developed compensation mechanisms, being thermode-
pendent.

Although the effects associated with origin have been consid-
ered by many authors to be indicative of genetic differences be-
tween mussels from different original habitats. Mallet et al. (1987)
offered an alternative explanation. In their study, the authors sug-
gested that these effects would reflect the differential influences
undergone by mussels during their pre-experimental stage, so con-
forming to an "ecological memory" of the individuals with respect
to the conditions experienced in the primary habitat (food avail-
ability and quality, tidal regime, air exposure, etc.). Okumus and
Stirling (1994), Navarro et al. (1996), Iglesias et al. (1996), and
Labarta et al. (1997) all recorded differences in CR for mussels
from different original habitats, which they attribute to this eco-
logical memory.

The present study shows significant differences for CR and
OIR between different original habitats of the mussels (collector
rope and rocky shore) at the outset of the experiment that persisted
after 8 days' cultivation on the raft. These significant differences
concerning the two physiological rates between both types of mus-
sel disappeared 15 days after raft cultivation commenced. The
initial differences in CR and OIR may be the consequence of a
response by the rocky shore mussels to the new conditions found
on raft cultivation (i.e., a lower concentration and higher-quality
Qi of seston in continuous immersion: previous data unpublished)
over a short period of time. This hypothesis is supported by results
of the multiple regression analysis, which indicates that both Q,
and origin, the latter being expressed as a term of interaction with
Qi. account for apart of the variance experienced by CR according
to the model.

ACKNOWLEDGMENTS

We are grateful to Lourdes Nieto. Beatriz Gonzalez, and Sonia
Villar for technical assistance. We also thank Juan Maneiro from
Centre de Control da Calidade do Medio Marifio da Conselleria de
Pesca. Marisqiieo e Acuicultura da Xunta de Galicia. for the de-
termination of environmental parameters. We are also indebted to
the crew of the Jose Maria Navaz from Instituto Espanol de
Oceanografia. This work was supported by Project MAR97-0592
by the Comision Interministerial de Ciencia y Tecnologi'a CICYT.
J. M. F. Babarro was funded by a grant from Excma. Diputacion
de Pontevedra.

LITERATURE CITED

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seed mussel (Myliliis galloprovincialis Lmk): effects of environmental
parameters and seed origin. J. Shellfish Res. 19( 1);187-193.

Bayne, B. L., A. J. S. Hawkins, E. Navarro & J. I. P. Iglesias. 1989. Effects
of seston concentration on feeding, digestion and growth in the mussel
Mytihis ediilis. Mar. Ecol. Prog. Ser. 55:47-54.

Fernandez Rios. A. 1992. El fitoplanclon en la Ri'a de Vigo y sus condi-
ciones ambientales. Tesis Doctoral. Universidad de Santiago de Com-
postela. 416 pp. (in Spanish).

Foster-Smith, R. L. 1975. The effect of concentration of suspension on the
filtration rates and pseudofecal production for Mytihis echilis (L.),
Ceraswderma edule (L.), and Venenipis pullastra (Montagu). / Exp.
Mar. Biol. Ecol. 17:1-22.

Hawkins. A. J. S. & B. L. Bayne. 1992. Physiological interrelations, and
the regulation of production, pp. 171-222. In: Gosling E. (ed.). The
Mussel Mylilus: Ecology. Physiology. Genetics and Culture. Elsevier.
Amsterdam.

Hawkins. A. J. S., P. N. Salkcd. B. L. Bayne. E. Gnaiger & D. M. Lowe.
1985. Feeding and resource allocation in the mussel Mylilus edulis:
evidence for time-averaged optimization. Mar. Ecol. Prog. Ser. 20:
27.^-2X7.

Ilildreth. 1). I. & D. J. Crisp. 1976. A corrected formula for calculation of
fillralion rate of bivalve molluscs in an experi menial llovving sysiem, ./,
Mar Biol. A.mn: U.K. .56:11 1-120.

Hughes, R. N. 1969. A study of feeding in Scrohicularia plana. ,/. Mar.
Biol. A.S.WC. U.K. 46:805-823.

Iglesias, J. I. P., A. P6re/ Camacho. E. Navarro. U. Labarta, R. Beiras.
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Journal of Shellfish Research. Vol. 19, No. 1. 203-212. 20()().

SALINITY TOLERANCE OF BROWN MUSSEL PERNA PERNA (L.) FROM THE GULF OF

MEXICO: AN EXTENSION OF LIFE TABLE ANALYSIS TO ESTIMATE MEDIAN SURVIVAL

TIME IN THE PRESENCE OF REGRESSOR VARIABLES

DAVID W. HICKS,** DOYLE L. HAWKINS," AND
ROBERT F. MCMAHON'

^Department of Biology
Box 19498

The University of Texas at Arlington
Arlington, Texas 76019
^Department of Mathematics
Box 19408

The University of Texas at Arlington
Arlington. Texas 76019

ABSTRACT The nonindigenous brown mussel Periia perna was first recorded in the Gulf of Me.xico at Port Aransas, Texas in 1990.
The association between survival time and chronic exposure to hypo- and hypersaline conditions was examined to estimate the potential
range of habitats that P. pema could invade in coastal North American Gulf of Mexico habitats. A novel application of the discrete
logistic failure time model (DLFTM) was used to estimate covariate-adjusted median survival times from the interval-level survival
data collected. This method allowed factoiial-type comparisons of the covariate-adjusted medians across treatments. This analysis
indicated that salinities ranging from 15-50 ppt are nonlethal to P. perna. under which at least 80% of individuals survived 30 days
(720 h). Chronic exposure to salinities outside 15-50 ppt were lethal to P. perna. Lethality was size-dependent, with both smaller and
larger individuals having reduced survival times. For an average-sized individual (shell length = 40 mm), median survival times were
191, 268. 335, 1 19, and 1 16 h at 0, 5. 10, 55, and 60 ppt, respectively. The 15-50 ppt incipient salinity limits of Texas P. pema suggest
that this species could potentially colonize the majority of marine and estuarine coastal habitats in the Gulf of Mexico.

KEY WORDS: interval-level survival data, mytilacea. nonindigenous species, Perna perna. salinity tolerance

INTRODUCTION

Since its initial discovery in Port Aransas. Texas, in 1990, the
marine brown mussel, Pema perna (L,), has colonized hard shores
at intermittent locations throughout the southwestern Gulf of
Mexico in Texas and Mexico (Hicks and Tunnel! 1993, Hicks and
Tunnell 1995). The majority of the Gulf of Mexico's margins are
sandy beaches, which has historically impeded development of
natural hard-shore communities. Thus, it was not until the con-
struction of jetties, breakwaters, and other coastal structures during
the last century that habitat existed for true intertidal bed-forining
mytilid genera such as Pema. These man-made structures, which
are continuously being constructed to control coastal erosion,
present a relatively open niche for such invasive mytilid species as
P. pema. Generally, considered an open-water species (Berry
1978). Texas P. perna populations have been reported from littoral
and shallow sublittoral habitats of widely varying physiochemical
parameters including the low salinity (20-25 ppt) Lavaca-Tres
Palacios estuary (Davenport 1995) and the hypersaline (35—10
ppt and occasionally higher) Laguna Madre (McGrath et al, 1998).

The endemic range of P. perna (synonymous with Pema pitta
(Bom) and Pema indica Kuriakose and Nair. [Siddall 1980. Va-
kily 1989]) includes India. Sri Lanka. Madagascar, the east coast
of Africa from central Mozambique to False Bay. South Africa,
and the African west coast from Luderiz Bay. Nambia. north into

'Current address: Center for Coastal and Marine Studies, Department of
Biology, Lamar University, P.O. Box 10037, Beaumont, TX 77710.
E-mail: hicksdw@hal.lamar.edu

the Mediterranean from Gibraltar to the Gulf of Tunis as well as on
the Atlantic coasts of Brazil, Uruguay. Venezuela, and in the West
Indies (Berry 1978).

Salinity influences many physiological functions, making it an
important limiting factor in the distributions of estuarine and ma-
rine bivalves (Bayne et al, 1976, Widdows 1985. Dame 1996). As
with most marine mytilaceans. P. perna is incapable of extracel-
lular osmotic control; thus, its extracellular fluids are nearly isos-
motic to ambient seawater over its tolerated salinity range (Salo-
mao and Lunetta 1989). The typical short-term response of osmo-
conforming bivalves to salinity reductions is to close the shell
valves temporarily isolating tissues and body fluids from poten-
tially lethal hyposaline waters, while allowing time for intracellu-
lar volume regulation by adjusting the concentrations of intracel-
lular amino acids and other small organic molecules (Hawkins and
Bayne 1992).

We examined the effects of chronic exposure to hypo- and
hypersaline media on survival times in P. perna. Salinity tolerance
data have proved effective in predicting local distributions in ma-
rine bivalves (Castagna and Chanley 1973). Thus, the incipient
salinity limits determined in this study were used to predict the
potential for P. pema to colonize coastal Gulf of Mexico marine
and estuarine habitats in North America.

We also developed a specialized methodology for analyzing
our survivorship data that allows estimating and comparing co-
variate-adjusted median survival times for grouped lifetime data.
Current methods for analyzing data of this type compare treatment
survival distributions based upon odds-ratios. Our method of using
median survival times, as opposed to odds, provides biologically
more meaningful interpretations of survival data.

203

204

Hicks et al.

MATERIALS AND METHODS

Experimental Protocol

Specimens of Penia perna were collected from intertidal rocks
on the north jetty of Mansfield Pass (26 °34 'N) on the Texas coast
and were transported overnight in cooled insulated containers to
Arlington, Texas. Upon arrival, mussels were maintained in a
284-L aerated holding tank containing artificial seawater (35 ppt)
at a constant temperature of 20 °C on a 1 2; 1 2 hour light-dark cycle
without feeding before experimentation. Experiments were initi-
ated within 30 days of collection.

Individual mussels were excised from mussel clumps by cut-
ting byssal attachment threads with scissors before salinity toler-
ance testing. For each salinity tested, four subsamples of 10 mus-
sels each, visually selected to be of similar size range, were held
for 2 weeks in a constant temperature laboratory at 20 °C (±1 °C)
in 3.5-L plastic aquaria containing 3 L of continuously-aerated. 35
ppt artificial sea water (Fritz Supersalt). Tank medium was re-
placed daily. During the 2 week acclimation period, mussels bys-
sally reattached to tank walls or other individuals. The size range
of the subsamples utilized in each salinity test reflected the size
range in the original sample (shell lengths - 15-70 mm).

After acclimation to experimental conditions, subsamples were
randomly assigned to test salinities of 0. 5. 10. 15. 20, 30, 40, 50.
55, and 60 ppt (created with Fritz Supersalt and City of Arlington,
Texas, dechlorinated tap water), chosen to include the range of
salinities occurring in Texas coastal aquatic habitats. Testing was
initiated by replacing the 35 ppt seawater medium in each tank
with 3 L of test salinity medium. During testing, media were held
at 20 °C (±1 °C), continuously aerated and changed daily. Byssal
attachment, valve opening or closure, and viability of all individu-
als was examined at 24-h intervals. Viability was detennined by
touching the exposed mantle edges of emersed gaping mussels
with the tip of a fine-haired brush. Individuals not closing their
valves in response to this stimulus were considered dead, and were
removed from the aquaria. The time of the observation was re-
corded, and the shell lengths (SL) of dead individuals were mea-
sured as the linear distance from the anterior to posterior margins
of the shell to the nearest 0.1 mm using digital calipers. All non-
gaping individuals were considered alive. Exposure to salinity
treatments and viability testing was continued until either lOOVr
sample mortality was achieved or individuals survived for 30 days
(720 h).

Statistical Methods

The salinity resistance of specimens of P. perna was examined
using a survival analysis strategy designed to determine the effects
of seawater concentration on survival duration: whereas, control-
ling for individual-specific covariates (e.g.. size). Our viability
monitoring at 24-h intervals prevented knowledge of an individu-
afs exact time of death. Thus, survival time was known only to fall
within the interval Ij = |«,_|, ci,]. while «, was the current obser-
vation time, and o^_i was the last observation time. Available
parametric (e.g., Weibull) and nonparametric (e.g.. Kaplan-Meier,
Cox regression) survival estimation nieihods assume that the time
of death is known exactly. However, in the vast majority of such
studies, as in this one. time of death is not recorded exactly, but is
known only to have occurred within a particular interval (Hosmer
and Lemeshow 1989). Applying continuous-data methodology to
such interval-level data can result in serious bias, especially if the

interval length is large relative to the average lifetimes one is
observing.

When survival data are recorded at the interval level, the life
table (i.e., actuarial method) is often used to estimate survival
probabilities. Life tables are essentially frequency tables modified
to deal with censored observations (i.e.. those data for individuals
that survive treatments) (Lawless 1982). The main outcome of life
table analysis is estimation of the survival function Sicij). which is
the probability of surviving to time a,, for all observation times
a^ «j. However, standard life table analysis cannot incorpo-
rate continuous regressors, such as size, which are likely to influ-
ence individual survival times. When such regressor variables are
present, a discrete logistic failure time model (DLFTM), which
generalizes the life table method, can be used to estimate survival
probabilities and allow their adjustment for regressor effects (Cox
1972, Thompson 1977). The ability to include regressor variables
in the DLFTM greatly broadens the scope of life table analysis by
revealing both treatment and individual-specific regressor influ-
ences, allowing more biologically appropriate interpretations of
survival data (Lawless 1982. Hosmer and Lemeshow 1989).

Although survival probabilities, when graphed into the usual
form of survival functions, provide a summary of the survival
experience of a population, these functions are cumbersome when
there are many such populations to be compared (e.g.. levels of
treatments, different values of important regressors). In such cases,
it is useful to have a one-number summary (e.g.. the median sur-
vival time) of each survival function to compare across many
populations. The DLFTM can. as illustrated here, be used to pro-
vide such median estimates for interval level data under some
reasonable assumptions discussed below.

We implemented the DLFTM for our data analysis in a com-
puter program written in SAS's interactive matrix language (IML,
SAS, Gary, NC) available from the authors. The routine was
checked for programming errors using simulations of data from
lifetime distributions with known parameters.

DLFTM and Its Estimation

For our analysis, the 720-h observation period was divided into

24-h time intervals, /, = [o^.,, a,), y = 1 k = 30. where

(( = 24 ■) are times of observation, a„ = 0 and «j+, = ^. The
data for the /th individual, 1 < / s n. consists of the vector G,- =

(G,| G, i^^i ), where G„ = 1 if individual i dies in interval /,

and C„ = 0 otherwise, and a vector x, of covariates describing
treatments and individual-specific characteristics.

Let 7, denote the actual, but unobserved, lifetime of individu-
als. Let S{t I X) = PrtT, > H X) denote the survival function of
individuals with regressor x, which contains indicator variables
describing salinity level and shell length (SL). The goal of
the analysis was to estimate the sur\i\al probabilities Pj(\) =

5((i, I X) at each of the observation limes (/, fl^. By a standard

argument using conditional probabilities (see Lawless 1982. p. 55),

^,(x) = /),(x)- ■ ■ p,i\). 1 ^j ^k.

(1

where /),(x) = Pr {T, > a, I T, >«, ,, x| is the probability of
surviving the time interval /,. given alive at its outset. The /),(x)s
are called "interval-specific" survival probabilities. It is clear from
( I ) thai to estimate P,(x) it is enough to estimate the p^Ws.

The method of maximum likelihood estimation was applied to
estimate the /j,(xIs, because it is known to produce estimates sta-
tislically optimal for large sample si/cs n. In this regard, again by

Salinity Tolerance in Perna perna

205

a standard argument (see Lawless 1982. p.372). the likelihood
function for the above data on n independent indi\iduals. assuming
censoring only at «j. is

^[/'lO /'*c>]=n{n[i-/',<''->]- n /v\>

y=l

(2)

where: /?, is the set of individuals / who are alive just before <;,_,.
and D, is the set of individuals / who die in interval 1^.

The DLFTM is a model for the functional form of ^^(x). Spe-
cifically.

pM)

(1

1

(3)

where p = ((3, P,„)^ is an in x 1 vector of unknown regres-

sion coefficients relating the covariate vector x = (.v.

..V,,,) to

/),. and the a,s are interval-specific parameters (i.e.. the interval
effects). Because (3) implies

I -pj(x + A)

1

■pM)

pM)

. 1 <y<A-

(4)

for any A. it follows that P relates the odds of death in any interval
/, for covariate value x -i- A [i.e., the left side of (4)] to the odds of
death in /, for covariate value x. Thus, if p^ > 0 (<0) for some 1 s
*• < III. the odds of dying in interval /, increases (decreases) as .v,.
increases. The parameter a^ is seen from (3) to equal In {[1 -
Pj{0)]/pjiO)}, the log-odds of death in interval /, when x = 0.

For a particular choice of the covariate vector x (i.e., including
terms to represent treatments, dependence on SL, etc.). the un-
known parameters P and a = (a, a^V in the DLFTM (3) are

estimated by substituting (3) into the likelihood (2). and then maxi-
mizing the resulting "constrained likelihood" (5) with respect to p
and a (Lawless 1982).

L(a,P) = n

n {1

+ e

a,+x,p,-l

The logarithm of this constrained likelihood is

logL(a,P) = 2 2<«y + ".P' - 2l"( I + ''"'"'''*'
7=1

(5)

(6)

The maximum likelihood estimators (mles) d and P of a and P are
obtained by maximizing (6). The maximization is done by the
Newton-Raphson algorithm, which iteratively solves the so-called
likelihood equations (writing x, = (.v,,, . . . ,.v,„,))

fdogL

.v,^

,3 iXv,,-!:,^,

.j+x,P

a;+x,p

: 0. /•= 1 in: and

aiogZ.

r'*a,,

E<i)-E

^oiv+x,p

isD„

isRm

+ e

+x,P

: 0. 1) = 1 k.

(7)

(8)

The interval-specific survival probability estimates, /5,(x) are
obtained by substituting the m/es. a. and P into (3). The survival
probability estimates P,(x) are then obtained by plugging the /5^(x)
into ( 1 ). The issue of which variables to include in the regressor
vector x (e.g.. linear or quadratic functions of SL. treatment by SL
interactions, etc.) was addressed by beginning with a model a

priori deemed sufficiently flexible and then testing a sequence of
nested models until reaching the most parsimonious model that
adequately explained the data. Goodness-of-fit was assessed by
comparing the most parsimonious fitted model to the a priori
model (i.e.. the model containing at most quadratic functions of SL
and treatment by SL interactions) by a Wald statistic.

To conduct inferences using the estimated P,(x)s. their covari-
ance matrix was needed. This matrix was derived by a sequence of
three steps, the first of which was to construct the covariance
matrix of the estimators d and p. An estimate of the covariance
matrix of 6 = (d,P) was obtained as the negative of the inverse of
the matrix of the second-order partial derivatives of log L (6) (i.e..
-H~\ where H is the Hessian matrix). The covariance matrices of
the derived estimates /',(x) and P,(x) were propagated, in turn,
from the covariance matrix of 6 by the Delta method (Sertling
1980). See Appendix A for details.

Estimating Median Siiniial Time

A unique aspect of our application of the DLFTM to analyze
salinity as a lethal factor in P. perna was our incorporation of a
method of estimating, and computing variances for, the covariate-
adjusted median survival times. This method allows factorial-type
comparisons of the covariate-adjusted medians across treatments.
Median survival time. A/(x), which satisfies 5(M(x)lx) = 0.5. was
estimated by assuming that the survival function, S{t\x). was linear
in t (for fixed x) over each time interval [«,_;, a,). Specifically,
given that P,(x) < 0.5 ^Aj-\W for some 1 s j < k. then the
median estimate is (using linear interpolation)

M/x) = aj +

"j-\

1

■ PjW

(9)

P/x)-/^_,(x)_

for the interval index 7 in which P^Cx) < 0.5 < P,_,(x). However,
this interval is itself a random variable, so it is necessary to express
the median estimate as

M(x) = 2'W/x)/[P/x) < 0.5 < P,_i(x)]

(10)

7=1

where M,(x) is given by (10). and the indicator variable [second
factor in the summand in (10)] equals 1 if the parenthetical in-
equality holds and is zero otherwise [note that at most, one of the
terms in the sum in (10) is nonzero]. In plain English, this just says
to "find the interval) where the survival probabilities _Py(x) cross
0.5 and use the estimate (9)." Equation ( 10) defines the estimated
median Mix) only if .Pjix) < 0.5. Otherwise. M(x) is undefined.

To obtain var[M(x)] and cov[A<?(X|). A/(X2)] for x, =^ x,. we
again want to appeal to the delta-method, because, as is apparent

from (10), M(x) = G[P,(x) ft(x)] for an albeit complicated

function C. However, this approach failed in this case, because the
function G is not differentiable with respect to the _Pj s [the indi-
cator functions in (10) are discontinuous and, hence, not differen-
tiable. so that the delta method does not apply]. To avoid this
problem, we used an approximation method detailed in Appen-
dix B.

By manipulating the regressor vector x, estimates (and their
covariance matrix) of covariate-adjusted median survival time
were obtained for each treatment combination. Specifically, let M
denote the vector of the true median survival times for the various
treatments, and for target values of the continuous regressors (e.g..
SL). Let M denote the corresponding vector of estimates, and let

206

Hicks et al.

Var(A/) denote the covariance matrix of M as obtained above.
Comparison of median survival times across treatments was car-
ried out via testing hypotheses of the form H„: CM = 0, where C
is a hypothesis matrix of coefficients having linearly independent
rows, using the Wald statistic

W = (CMflCVar{M)C'']-'(CM).

(11)

Under //„. the statistic W is distributed approximately chi-square
with ni degrees of freedom, where lu is number of rows in C. Large
values of W provide evidence against //„. Type I error was con-
trolled using the Scheffe method for an experimentwise error rate
of a = 0,05.

RESULTS

Survivorship in Penta periui declined in hyposaline and hyper-
saline treatments. Salinities ranging from 15-50 ppt appeared non-
lethal to this species, with at least 80% of individuals surviving the
30-day (720 h) exposure period. Following the .^0-day period,
surviving individuals were cut from byssal attachments and trans-
ferred back to full-strength seawater and allowed 72 h to reattach.
Thereafter, the average number of attached byssal threads per in-
dividual and the associated salinity concentration were as follows:
15 (15 ppt), 1 1 (20 ppt). 13 (30 ppt). 13 (40 ppt). and 8 (50 ppt).
The capacity for byssal reattachment indicated that individuals
exposed to salinities of 15-50 ppt were not physiologically dam-
aged. In contrast, complete sample mortality was observed at the

following times and salinities: 360 h (0 ppt). 408 h (5 ppt). 648 h
(10 ppt), 168 h (55 ppt), and 216 h (60 ppt) (Fig. I).

In the most parsimonious, fitted survival model (Eq. 3). sur-
vival was significantly correlated to both SL and SL". but the SL
effects did not interact with salinity treatment (Xnm = 18.7; P =
0.412). Specifically.

log

PM) J

■■^,.+ ^^r'T + l>SL + y.SL'

where p. 7, and 7, are coefficients for treatment and SL. respec-
tively, and /t- is the indicator variable for salinity treatment T (i.e.,
Ij^ = I for salinity treatment T and zero otherwise).

The estimated survival functions [i.e., the linearly interpolated
_Pj(x)s] for lethal salinity treatments, adjusted to the sample mean
SL (40 mm), indicated decreasing survivorship probabilities as
salinity concentrations departed from full strength seawater (35
ppt) with those in the most saline treatments (i.e., 55 and 60 ppt)
having the lowest median survival times (Fig. 1).

The relationship between survivorship and SL can be shown by
using the model to estimate median survival at the 1 0th, 25th, 50th,
75th. and 90th percentiles of the SL distribution in each salinity
treatment. This analysis indicated that intermediate-sized individu-
als (48 mm SL) had longer survival times (Fig. 2). For an average-
sized individual. SL = 40 mm. the median survival times and
associated salinity concentrations were as follows: 191 h (0 ppt).

1.0 ■{

E
E

e

II

(0

~ 0.6 H

0.8 -

o

>

"E

3

(0

0.4 -

0.2 -

0.0

^^T^\^J^^ — •— 0 ppt
\ t \l \ ~*~ 55 ppt

a

•^ S>4^44^4^xj. .

iJL ^s^ 1

►^

^.^^_^ rt 1 1 hHs

1 1 1—'

100

200

300

400

500

600

700

Hours

Fijjiirc I. Survivorship curves f<ir standard 40 mm shell length specimens of I'cniti piriui clironically exposed to lelhal salinity treatments. Lines
represent linear Interpolalions ot Ihe ciiniiilative Mir\ival pnihahiillies. the /'(S, l)et«een successive 24-h sampling; intervals in each salinity
treatment. Krror hars represent the standard errors of the X',s. .\ median survival time is the hour at which the survival probability curve
crosses Ihe solid hori/ontui line at a survival prohahility of 0.5.

Salinity Tolerance in Perna perna

207

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< '^

/

< V ^

< ^ '^

■s ^

/

^ ^ ^

< N ^

s ^

/

S V ^

< ^ >

< s

/

< V ^

< ^ '^

< ^

1 JL

/
/

h HsA

/

< S ^

< V ^

< ^ '

> V /

} s

/

< s ^

s ^

< s ^

> \ /

) s

/

< V ^

< s ^

5 V ^

5 S /

> v

/

< V -^

< s ^

< ^ ^

? V '^

} s

/

< s ''

s ^

< V '■

? ^ ''

> s

/

< s ^

s ^

< s '^

? ^ ^

> N

/

^ V ''

< s ^

< V ^

> '^ ^

i s

/

\ \ ^

< s ^

2 V ^

> s /

J S

10

55

60

Salinity (ppt)

Figure 2. Median survival times (h) for Texas specimens o( Perna perna in lethal salinity treatments adjusted to the 10th (18 mm SL), 25th (27
mm SL). 50th (38 mm SL), 75th (48 mm SL), and 90th (65 mm SL) percentile of the individual shell length (SL in mm) distribution. Error bars
represent the standard errors of median survival estimates.

268 h (3 ppt). 335 h ( 10 ppt). 1 19 h (55 ppt). and 1 16 h (60 ppt)
(Table 1).

Wald statistics for all-pairwise comparisons of lethal salinity
treatment median survival times adjusted to the sample mean SL
(40 mm) indicated significant treatment differences (Table 1). For
the salinity treatments in which survival curves never fell below
0.5. median estimates could not be computed. However, because
the odds ratio method still works, treatment survival distributions
could be compared directly from their estimated treatment coeffi-
cients in the model (Eq. 3). For example, exponentiation of the
coefficient for the nonlethal 15 ppt salinity treatment (Table 2).

TABLE L

Survivorship of standard 40 mm shell length (SL) Texas specimens

of Perna perna exposed to lethal salinity treatments of 0, 5, 10, 55,

and 60 ppt.

Salinity

Treatment

Median Survival

Sample of 100%

SL Range

(ppt)

Time (h ± SE)

Mortality (h)

n

(mm)

0

5

10

55

60

191.36 (6.27f
268.03 (9.94)"
335.17(10.1)"
119.00(9.68)''
116.22(9.42)''

324
372
636
156
204

46 10.2-94.3

44 10.9-77.5
38 17.6-78.5

45 12.8-87.2
44 10.0-84.6

^-6.002 ^ 0.0025. indicates that the odds of death (in any time
interval) in the 15 ppt salinity treatment were only about 0.25%
(Xfg] = 116.64, P < 0.0001) of the odds of death in the 0 ppt
salinity reference cell treatment (given SL = 40 mm). Pairwise
testing of treatment coefficients indicated that the odds of death
did not differ among the 15. 20, 30. 40. and 50 ppt salinity treat-
ments whose survival probabilities never dropped below 0.5. The

TABLE 2.

Estimated treatment coefficients (P) and standard errors (SE) for

Texas specimens of Perna perna relating the log-odds of death (in

any time interval) to the 0 ppt salinity level reference cell treatment

given shell length (SL) equals 40 mm.

Odds of

SE (Odds

Death

of Death)

Effects

P

SE(P)

efi'

(■'P'SE(P)

Median survival times with the same superscript are not different M P <
0.05xi^i

5 ppt
10 ppt
15 ppt
20 ppt
30 ppt
40 ppt
50 ppt
55 ppt
60 ppt
SL
SL-

salinity
salinity
salinity
salinity
salinity
salinity
salinity
salinity
salinity

-1.302

-2.672

-6.002

-8.194

-7.999

-8.012

-6.782

2.888

2.958

-0.222

0.3 1 1

0.2897
0.3545
0..5557
1.0919

1 .0895

1 .0896
0.7200
0.3757
0.3788
0.0854
0.0614

0.2720
0.0691
0.0025
0.0003
0.0003
0.0003
0.001 1
1 7.9574
19.2594
0.8009
1 .3648

0.07879
0.02450
0.00137
0.00030
0.00037
0.00036
0.00082
6.74658
7.29547
0.06830
0.08370

208

Hicks et al.

odds of death did differ between the 15-50 ppt sahnity level treat-
ments and the extreme hyper- (55-60 ppt) and hyposaline (0-10
ppt) treatments. Among the lethal treatments (i.e.. 0-10 ppt and
55-60 ppt concentrations), the odds of death differed between all
pairs, e.xcept the 55 and 60 ppt salinity concentrations, which is
reasonable, considering their survival curves were very similar
(Fig. 1).

In low-salinity treatments, mussels initially responded by clos-
ing the shell valves (Fig. 3). After 72, 48. 48, and 24 h, individuals
were observed to open the shell valves in the 0. 5, 10, and 15 ppt
salinity treatments. At the lowest salinity treatments (i.e., 0, 5, and
10 ppt), mantle tissues were observed to swell outside of the shell
valves and may actually have forced them apart. Under the ex-
treme hypersaline conditions (e.g., 55 and 60 ppt). individuals
gaped widely from the onset of treatment until death. In nonlethal
salinity treatments, individuals were open normally at the first 24-h
observation period. However, there seemed to be a positive cor-
relation between the percentage of open individuals and salinity in
nonlethal treatments, with fewer individuals displaying open
valves at progressively lower nonlethal salinity exposures (Fig. 3).

The average percentages of open individuals in the 30-day obser-
vation period and their associated salinity concentrations were as
follows: 17.2 ± 7.9 SD (15 ppt), 30.2 ± 7.5 SD (20 ppt), 37.1 ±
10.2 SD (30 ppt), 36.3 ± 8.2 SD (40 ppt), and 38.7 ± 9.6 SD (50
ppt).

DISCUSSION

In this study, we used a DLFTM to estimate covariate-adjusted
median survival times based on interval-level data. We believe that
for interval-level survival data, our strategy of using medians
(where possible) to summarize survival experience is a helpful
complement to the usual survival curves and odds ratios. Unlike
analysis of covariance (ANCOVA) approaches, the DLFTM. like
other regression-type models, can provide meaningful estimates of
median survival time at fixed covariate values even in the presence
of variable interactions.

Texas P. penui survived and maintained normal activity (e.g.,
maintained byssal thread production) over salinities of 15 to 50
ppt, a salinity tolerance range similar to that of 19—1-4 ppt deter-

75

3

■o

>

■TO

c
o

0>

ja
E

3

50
40
30
20
10

0
50
40
30
20
10

0
50
40
30
20
10

0
50
40
30
20
10

0
50
40
30
20
10

0

(0 ppt)

JUIJIllMlj^n.^

(5 ppt)

lillu

(10 ppt)

mill.

T"W"W"W

(15 ppt)

UuPM

yyyLIMI|iyyi|jyyyyyij

(20 ppt)

P|ii|iU|iljlli|yiW

50
40
30
20 •
10

0
50 ■
40 ■
30 ■
20 ■
10 •

0
50
40
30-
20
10

0
50
40
30
20
10

0
50
40
30
20
10

O

(30 ppt)

IllilillillWi

(40 ppt)

lU

IlliMMMyMlMH^hi

(50 ppt)

(55 ppt)

(60 ppt)

0 72 144 216 288 360 432 504 576 648 720

72 144 216 288 360 432 504 576 648 720

Hours I

Figure .^. Shell valve closinj; hehavior In Texas specimens of I'enia pirna exposed In rhronie salinities ranging ()-60 ppt. Histogram bars \
represent numhers of living individuals and the solid portion ol' bars, the numbers of living individuals with shell valves open al each 24-h

sampling interval. ]

Salinit"!' Tolerance in Perna perna

209

mined for similar sized (45-50 mm SL) Brazilian P. perna (Salo-
mao et al. 1980). A similar incipient lov\ salinity value of 16 ppt
was recorded for Perna viridis (L.) from India (Sundaram and
Shafee 1989). Although the salinity tolerance we recorded for
Te.xas P. perna generally agreed with data for South American
specimens, recorded survixal times were markedly different. At
salinities of 4 and 9 ppt, complete sample mortality was observed
after 102 hours in Brazilian specimens of P. perna. In contrast.
Texas specimens oi P. perna persisted for up to 360. 408. and 648
h in salinities of 0. 5. and 10 ppt. respectively. Similar to Brazilian
data for P. perna salinity tolerance, complete sample mortality was
observed to occur in individuals of P. viridis within 96 h upon
exposure to salinities ranging 0-1 1 ppt (Sundaram and Shafee
1989). Median survival times reported for the Brazilian P. perna
were 68. 49. 45. and 96 h on exposure to lethal salinities of 4. 9.
14. and 49 ppt. respectively (Salomao et al. 1980). In our study,
median survival times were considerably greater than those of
Brazilian specimens of P. perna or specimens of P. viridis.

Animal size was found to affect survival duration significantly
in Texas specimens of P. perna. However, although the SL range
of tested individuals was considerable (-15-70 mm SL). the range
of median survival time in different sized individuals varied by
only 22'7f or 59 h maximally. Although the largest individuals in
the sample had reduced survival, the more general trend was in-
creasing survival duration with increasing SL. This result generally
agrees with that of Castagna and Chanley (1973) who demon-
strated, among 29 tested marine bivalve species, that although
smaller specimens generally succumbed more rapidly to lethal
salinity exposures than did larger specimens, variation in indi-
vidual size was not correlated with the minimum tolerated salinity.

Individuals responded to marked salinity reductions (15 ppt or
-43"^ dilution) by closing the shell valves. Shell valve closing
behavior in bivalves allows sur\'i\al of temporarily reduced salini-
ties encountered in intertidal or estuarine environments during pe-
riods of heavy rainfall and/or freshwater run off (Gilles 1972.
Davenport 1981. Akberali and Trueman 1985). Valve closure ex-
tended survival times in Brazilian specimens of P. perna relative
to individuals with artificially propped open valves (Salomao et al.
1980). However, even when the valves are tightly closed, tissues
are not completely isolated from the external environment. Thus,
valve closing behavior provides protection from salinity stress
only over relatively short periods. Indeed, even with the shell
valves closed, P. perna tissues reach osmotic equilibrium within
72 h of exposure to salinities of 19-39 ppt (Salomao and Lunetta
1989). A similar value of 96 h has been reported for Mytilus edidis
L. exposed to salinities ranging from 9-36 ppt (Gilles 1972).

The shell valve closing response is mediated by peripheral
receptors located on the mantle margins and/or siphon surfaces
(Davenport I98I, Berger and Kharazova 1997). These peripheral
receptors are of two types: osmoreceptors, sensitive to osmotic
pressure, and receptors sensitive to changes of specific ion con-
centrations (Berger and Kharazova 1997). Such receptors not only
trigger \ alve closure in response to salinity change, but also enable
detection of relative salinity concentrations. When transferred
from full strength seawater (35 ppt) to lower salinities, the pro-
portion of individuals of P. perna with closed valves was directly
proportional to the seawater concentration: the lower the salinity,
the greater the tendency to close the valves (Fig. 3). This result is
consistent with the valve closure behavior in response to variable
salinity recorded for other marine and estuarine bivalve species
(Akberali 1978, Bailey et al. 1996).

Davenport ( 1981 ) found that increasing salinity is registered by
special detectors located in the tentaculate portion of the inhalant
siphon of Mytilus edulis. These receptors, requiring the presence of
both sodium and magnesium ions, trigger shell valve parting at
salinity concentrations similar to those that induce valve closure
under declining salinity conditions (-17 ppt. Davenport 1981 ). It
is unknown whether similar receptors are responsible for the gap-
ing of individuals of P. perna observed in the highest salinity
treatments (i.e.. 55 and 60 ppt). Exposure to these elevated salini-
ties seemed to have a narcotic effect on this species. Lack of an
appropriate protective valve-closing response suggests that P.
perna rarely encounters hyperosmotic conditions and is an indica-
tion that it is limited to marine intertidal and open-water estuarine
habitats. Given that the tissues of specimens of P. perna will
eventually come into osmotic equilibrium with their external en-
vironment, it seems that P. perna cannot tolerate haemolymph
osmolarities of less than 430 mOsm ( 15 ppt) or greater than 1428
mOsm (50 ppt).

The range of salinity tolerated by marine bivalves in laboratory
experiments is often a good predictor of their distribution in natu-
ral habitats (Castagna and Chanley 1973). Castagna and Chanley
( 1973) observed that the experimentally determined salinity toler-
ance of a number of marine bivalves adequately explained their
distributions in nature. When a species' natural distribution did not
include its entire tolerated salinity range, it was usually attributed
to a species being near its geographic limit where other limiting
factors, particularly temperature, became more important limiting
factors. Within its endemic range. P. perna occurs primarily in
open-water, high-energy hard-shore habitats where salinity re-
mains relatively constant near full-strength seawater (Berry 1978).
However, in Brazil, it is reported from semi-enclosed lagoons
where salinities may be as low as 12.7 ppt (Marques et al. 1991 ).
Although P. perna rarely occurs naturally in enclo.sed-estuarine
habitats, it thrives when relocated to such habitats for culturing in
South America and Sri Lanka (Indrasena and Wanninayake
1994). Indeed, some of the highest growth rates reported for this
species are from culture grow-outs in lagoons where salinities
ranged 20—45 ppt (Indrasena and Wanninayake 1994).

The salinity regimes of coastal environments vary temporally
and spatially over short (tidal or storm induced) and long durations
(seasonal influences). Bivalve salinity tolerance can be influenced
by previous salinity experience (i.e.. acclimation salinity), magni-
tude of salinity change, and/or rate of change (see Remane and
Schlieper 1971 and Kinne 1971 for reviews). In this study, the
incipient salinity limits of P. perna were determined by chronic
exposures of up to 30 days following direct transfer from full-
strength seawater (35 ppt) into one of 10 test salinities. Because of
the long exposure periods involved, individuals had sufficient time
to acclimate to test salinities near this species upper and lower
incipient limits, allowing relatively accurate estimation of P. per-
na'^ incipient salinity range. In addition, direct transfer to test
salinities allowed evaluation of the colonization potential of P.
perna after rapid, anthropomorphically mediated introduction of
adults into new habitats. Pemids foul the hulls of international
vessels (Cariton 1987). Thus, adult mussels byssally attached to
anchor chains and/or the hulls of transoceanic vessels and barges
can be transferred directly into a new habitat whose salinity regime
varies from that of the source population. Our data suggest that if
other environmental conditions are favorable, anthropomorphi-
cally introduced P. perna could colonize waters within a salinity
range of 15-50 ppt.

210

Hicks et al.

The incipient upper and lower salinity limits determined herein
indicate that P. pernu could potentially colonize areas in the Gulf
of Mexico outside of its present Texas/Mexico range (Hicks and
Tunnell 1993, Hicks and Tunnell 1995). The 15-50 ppt tolerated
salinity range of Texas P. pernu suggests that this species could
potentially invade most Gulf of Mexico coastal habitats, including
those from which raw water is drawn by industrial and power
generation facilities, particularly in the Houston ship channel and
Mobile Bay; potentially making them susceptible to the macro-
fouling reported for this species in India (Ragapogal et al. 1995).
Thus, the dispersal of this invasive nonindigenous species should
continue to be closely monitored in the North American Gulf of
Mexico and southern North Atlantic coastal habitats.

ACKNOWLEDGMENTS

We thank Ron Smith and Terry Riggs of Texas A&M Univer-
sity-Corpus Christi for collecting and shipping P. penia samples
to Arlington, Texas. The reported research was funded by Grant
NA56RG0388 from the Texas A&M Sea Grant College Program.

APPENDIX 1: EXAMPLE OF THE DELTA METHOD

The delta method is a general technique of variance propaga-
tion as follows. Let t/ be a p x 1 random vector that is distributed
approximately multivariate normal (MVN) with mean Et/ and co-
variance matrix Var (f/). Let f(z) be an s x 1 vector function of;?
variables; i.e..

fiz) =

'j](zy

Uc),

Let y^ denote the s x p Jacobian matrix of/ whose (ci. b) entry is
dfJSZh- The delta method then asserts that the .s x I random vector
fiU) is also approximately MVN, with mean

£{/({/)) = f(EU) and covariance matrix

WarUiU)] -= J/EU){\/ar(U)}JJ(EU).

As an illustration of the delta method in our problem, we give
the details of obtaining the covariance matrix of the interval-
specific estimates p = \p,{x) ih^^)]' fr"'" the covariance

matrix of 6 (Here or is specified and fixed). The computer program
performs these calculations, as well as those for obtaining the
covariance matrix of the P^a from the PjS. Because /5,(xl is ob-
tained by plugging 0 = (a. P) into (.3). the vectorp is given by
p = f(f)), where

■/,(r)'

Jlz)--

and for A x 1 vector H" = (ir, iij)', m x I vector v = (v

v„y and z - (w, v).

/,(Z) = (I +£>"'«-)-'. I <y< it. (12)

Now, because H is a maximum likelihood estimator, hv trencral

likelihood theory (see Serfling 1980) it holds that 6 is approxi-
mately MVN with mean 6 = (a, (J) [i.e., the true values assumed
to have generated the G,s via (3)] and covariance matrix (as noted
above) -H^'. We applied the delta method to U = 6, £U = 6,
Var(U) = -H"'. and the function fin (12) to get that^ = f(0) is

approximately MVN with E(p) = f(B) = p(\) = |;),(x) P^W]^

(the true value) and Var(p) = J,(e)i-H"' )J,(ei. where the k
X {k + m) Jacobian J^iz) is given by

J,(z) =

fMi'i

1l
fill'.

dv,

V, <lh

(iW,

dVt

Specifically, for any 1 ^ j < k and 1 < r < A:,

— ^ = T— {( 1 + e"^*-"'
ow,. dw,.

f>v,..

.'■=7

and for 1 < .? s m.

-LL - — f( I ^ g"j+nvi+--+-v,„v„,j-i| _ ■■_

dv, dv, ^ ^ ^ ( 1 + (,">^«')2'

APPENDIX 2: CALCULATION OF VARIANCE, VAR[A/(x)] AND

COVARIANCE, COV[M(x,), M(x,)] OF SL ADJUSTED MEDIAN

SURVIVAL TIMES

Writing -, = P,(x) to reduce notation, and writing A/,(x) =
/)(z), where z = (c, Cj), M{\) can be written as /(z), where

Az) = ^f/x)l{Zj<0.5sZj.,).

(13)

y=i

Now the indicator function (the source of the differentiability
problem) is

/(;,-< 0.5 < z,..,) = /(c^_, > 0.5) /(;^ < 0.5) = /i(v, )[1 - Mr,)]

(14)

where

hU) = IU >0.5)

0, 1 < 0.5
1 . r > 0.5 '

But h(i) can be clo.sely approximated (as e approaches zero) by the
function h,.U) = cl)(f - 0.5/e), which is the cdf of the N(0.5, e")
distribution (Fig. 4). In fact, /;,.(/) -^ hit) as e ^ 0 for every r e
[0,1 1 - (0.5). Furthermore. h^M) is, unlike /;(/), differentiable.
Thus, we may closely approxiinate l{Zj < 0.5 S;^_|) = /z,.(;^_|)(l -
/;,.(,-,)], and, hence, approximate /Iz) by the differentiable function
fjz) for small k;

f[z) =/„(z) H ^f,WhJz,_,)[ 1 - h,iz,)].

(1-5)

Thus, we approximated the estimate M(x) =f{P, P,^) by fJP,.

■ ■ ■ . P/,). to which the delta method applies. Details of this analy-
sis appear in the computer code available from the authors upon
request.

Salinity Tolerance in Perna perna

211

o

^^^^_^^^

^

hit)

1.00-

(kw

0.75-

0.50 "
0.25 -

J

"A

0.00 -

0.00

— I —
0.25

0.50

—I —
0.75

1.00

Figure 4. Illustration depicting the approximation of tlie indicator function li(t) by tlie cdf of the N(0.5. f ^) distribution, h,(l). as e ^ 0.

Akberali. H. B. 1978. Behavior oi Scrobicularia phmu (da Costa) in water

of varying salinities. / Exp. Mar. Biol Ecol. 33:227-249.
Akberali. H. B. & E. R. Trueman. 1985. Effects of stress on marine bivalve

molluscs, pp. 108-198. In: J. H. S. Blaxter. F. S. Russell and M.

Younge (eds.). Advances in Marine Biology, vol. 2. Academic Press,

New York.
Bailey, J., J. Parsons & C. Couturier. 1996. Salinity tolerance in the blue

mussel, Mytilus edulis. Bull. Aquacult. Assoc. Can. 96:74-76.
Bayne, B. L.. R.J. Thompson & J. Widdows. 1976. Physiology 1. pp.

121-206. In: B. L. Bayne (ed.). Marine Mussels: Their Ecology and

Physiology. Cambridge University Press, London.
Berger. V. J. & A. D. Kharazova. 1997. Mechanisms of salinity adaptations

in marine molluscs. Hydrobiologia 355:115-126.
Berry. P. F. 1978. Reproduction, growth and production in the mussel.

Perna perna (Linnaeus), on the east coast of South Africa. Investl. Rep.

Oceanogr. Res. Inst., Durban 48:1-28.
Cariton. J. T. 1987. Patterns of transoceanic marine biological invasions in

the Pacific Ocean. Bull. Mar. Sci. 41:452-465.
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valves from inshore and estuarine environments in Virginia waters on

the western mid- Atlantic coast. Malacologia 12:47-96.
Cox. D. R. 1972. Regression models and life-tables (with discussion). J. R.

S!aii.s!. Soc. B. 34:187-220.
Cox, D. R. & D. Oaks. 1984. Analysis of survival data. Chapman & Hall

Publishers, London, England. 201 pp.
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CRC Press. New York. 254 pp.
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exposed to rising seawater concentrations. J. Mar. Biol. Ass. U.K.

M:bbl-bli.
Davenport. R. 1995. Perna perna enters the bays. Tex. Conch. 31:92.
Gilles. R. 1972. Osmoregulation in three molluscs: Acanthochitona dis-

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JiHiriwI «j Shellfish Research. Vol. 19, No. 1, 21.^218, 2000.

A POLYMERASE CHAIN REACTION ASSAY FOR THE DETECTION OF GENOMIC DNA OF A
RICKETTSIALES-LIKE PROKARYOTE ASSOCIATED WITH WITHERING SYNDROME IN

CALIFORNIA ABALONE

KARL B. ANDREE,* CAROLYN S. FRIEDMAN,' "
JAMES D. MOORE,- AND RONALD P. HEDRICK'

Department of Medicine and Epidemiology
School of Veterinary Medicine
University of California
Davis, California 95616
'California Department of Fish and Game
Bodega Marine Laboratory
Bodega Bay, California 94923

ABSTRACT The 16S rDNA from a Rickettsiales-like procaryote (RLP) infecting postesophageal tissues of blaclc abalone Haliotis
cracherodii Leach exhibiting signs of withering syndrome (WS) was amplified, cloned, and sequenced. The I6S rDNA sequence for
the RLP was similar to that of species found in the genera Ehrlichia. Anaplasma. and Wolbachia. A polymerase chain reaction (PCR)
test was developed that specifically amplifies a 160 bp segment of the 16S rDNA from the RLP associated with WS. Positive reactions
were obtained for all black abalone samples of digestive gland or postesophagus known to be infected with the RLP by microscopic
examinations of stained tissue sections. The PCR worked equally well for infected tissues of black and red abalone H. nifescens Leach.
There was no amplification of genomic DNA from four other microbial species isolated from cultures of intestinal flora of abalone or
from abalone deemed free of the RLP by microscopic examinations. This PCR test greatly increases the ability to detect the bacterium,
because to date no means to grow the organism from marine invertebrates on synthetic media or in cell lines have been developed. This
PCR test should allow detection of the RLP before the onset of clinical signs of withering syndrome in cultured or wild abalone stocks.
Furthermore, the test may be useful in identifying reservoirs or other related RLPs in other marine invertebrates.

KEY WORDS: Polymerase chain reaction, ribosomal DNA. Rickettsiales. abalone. Ehrlichia. Haliotis

INTRODUCTION

MATERIALS AND METHODS

The black abalone Haliotis cracherottii Leach, a species once
common along much of the intertidal zone of southern California's
rocky beaches, has suffered severe negative impacts because of the
disease termed withering syndrome (WS) (Alstatt et al. 1996.,
Friedman et al., 1997, Haaker et al. 1992, Tissot et al. 1991,
VanBlaricom et al., 1993, 1996). Affected populations of black
abalone have been reduced to 1-10% of population densities ob-
served before the onset of WS (Haaker et al. 1992). The disease
has now been observed among fanned red abalone (H. rufescens)
(Moore et al. in press), and this has prompted the California De-
partment of Fish and Game to place a partial ban on movement of
cultured red abalone from locations where WS is endemic to lo-
cations free of this disease. The epizootic manner in which the
disease has spread throughout black abalone in the Channel Islands
and the mainland indicate a role for an infectious etiologic agent
(Lafferty and Kuris 1993). Although not completely proved, evi-
dence suggests that WS is caused by a previously undescribed
Rickettsiales-like prokaryote (RLP), which infects gastrointestinal
epithelia (Friedman et al. 1997, Gardner et al. 1995). Withering
syndrome and the associated RLP have also been documented in
wild and cultured red abalone, Haliotis rufescens Leach, (Haaker
et al. 1992, Moore et al. in press). Because of the inability to
culture most marine RLPs, differentiation of these organisms is
difficult and is based upon morphological characteristics using
light and electron microscopy. Sensitive and accurate detection of
the WS-as,sociated RLP is critical to our understanding and control
of the spread of WS. The goal of the current study was to develop
a polymerase chain reaction (PCR) test to improve our detection
and understanding of the biology of the RLP causing WS in aba-
lone and other as yet unidentified hosts.

DNA E.xtractions, Sequencing, and Alignments of rDNA

DNA isolation from whole tissue and from bacterial cells was
performed as described by Sambrook et al. ( 1989) and Friedman et
al. (in press). Briefly, rinsed tissues were homogenized in lysis/
proteinase K buffer. After 1 h at 55 °C, the DNA was extracted in
a phenol-chloroform solution. Isoamyl alcohol was added, mixed
for 10 min, and centrifuged. The top aqueous phase was removed
and l/IO volume of 3 M sodium acetate was added. Cold absolute
ethanol was added to precipitate the DNA. The pellet was washed
in 70 % ethanol. air-dried, and resuspended in Tris-EDTA (TE)
buffer. Alternatively. DNA was prepared using the QIAmp Tissue
Kit {Qiagen Inc.. Valencia. CA) following the manufacturers
■"mouse tail protocol". Bacterial I6S rDNA from infected abalone
tissue was amplified using EUB A and EUB B universal eubac-
terial I6S rDNA primers (Giovannoni 1991). The RLP 16S rDNA
gene was cloned into pCR2.1 using a TOPO Cloning Kit (Invit-
rogen. San Diego. CA) following manufacturer's protocols. Clones
were screened using PCR with primers that flanked the multiclon-
ing site of the vector. Positive clones were selected from among
those that had an insert of the appropriate size (-1550 bp). The 16S
rDNA sequencing and alignments of sequences were performed as
described previously (Andree et al. 1997). The completed se-
quence was used in a BLAST search of GenBank to confirm the
similarity of the sequence to other Rickettsiales. The species ap-
pearing in the results of the BLAST search were compared with
other closely related bacterial species in a pairwise analysis of
sequence similarity (Table I ) in addition to a phylogenetic distance
analysis (Friedman et al. in press).

213

214

Andree et al.

TABLE 1.
A pairwise comparison of the 16S ribosomal DNA sequence similarity seen among closely related species.

1

8

10

11

12

13

14

15

1 WFS-RLP

2 W. pipientis

3 E. sennetsu

4 E. risticii

5 A. marginale

6 C. ruminarnium

7 E. phagocylophila

8 E. bovis

9 C. cuiyophila

10 C hurnettii

1 1 R. prowesekii

12 R. rickettsii

13 NHP

14 P. salmonis

15 E. coti

74.0

73.3

73.2

77.3

75.4

75.5

75.8

71.3

69.7

73.4

73.3

70.9

69.8

69.0

77.8

11.6

80.7

80.5

81.5

81.0

741

73.6

77.3

77.5

73.7

73.3

73.0

94.5

78.4

11.9

79.7

78.9

74.7

73.5

76.3

76.6

74.2

72.6

71.3

78.2

77.5

79.5

78.7

74.9

73.5

76.0

76.4

74.3

73.3

71.0

85.8

89.4

88.2

76.9

75.1

77.1

77.6

75.9

73.6

73.7

86.2

85.4

76.7

73.8

77.5

77.8

74.1

72.7

71.5

91.2

76.8

74.3

78.3

78.5

75.7

74.0

73.1

76.1

74.1

74.5

77.6

77.3
74.4

77.6
77.8
75.0
93.0

75.6
78.8

72.5
76.7
77.1

73.2
74.6
80.5
74.3
74.3
72.3

72.9
72.3
78.1
71.8
71.8
70.3
81.5

Primer Selection and Oligonucleotide Synthesis

A BLAST search of GenBank indicated three species. Ana-
plasma marginales. Ehrlichia bovis, and Wolhachia pipientis.
were most similar to the 16S rDNA sequence amplified from in-
fected abalone (clone designation pl6RK3; GenBank accession
number: AFl 33090). The four sequences above and that of Pis-
cirickellsia salmonis and a Rickettsiales-like prokaryote from
shrimp (Freiier et al. 1993) were aligned to identify those se-
quences most unique to the RLP from infected abalone. Several
primers were selected for synthesis and testing. In an initial trial,
two primers designated RA5-I [""'GTTGAACGTGCCTTCAGTT-
TAC"] and RA3-I ["CTGAGGCCATCTGTTAAAATGG"],
were .synthesized (Gibco BRL, Inc.. Gaithersburg. MD) and used
in an initial screening of samples containing enriched RLP from
abalone tissues. The best results were obtained using these primers
in conjunction with an annealing temperature of 55 °C. An ampli-
fied product of 946 bp was obtained from all RLP-enriched
samples tested (data not shown). Subsequent tests with DNA
samples extracted from digestive gland tissues of diseased animals
showed a poor correlation with the histology results (e.g.. samples
from known positive abalone were negative by PCR). We specu-
lated that the I6S rDNA of the normal gut flora could be hybrid-
izing with the primers for the PCR, reducing efficiency of ampli-
fication. Accordingly, we designed new primers for the WS-PCR
based on a second alignment using the I6S rDNA sequences from
pl6RK3, E. hovis, A. mariiinah's. \V. pipicnlis, P. salmonis. and E.
coli as a representative of intestinal flora. This alignment showed
the sequence of primer RA3-I to be completely conserved in the
168 rDNA of £. coli, and this could have been contributing to the
poor results observed in the PCR by nonspecific hybridization of
this primer to bacterial DNA in the sample. We, therefore, de-
signed three additional primers designated RA5-6 |'"GAAG-
CAATATTGTGAGATAAAGCA"!, RA3-6 l^'ACTTGGACT-
CATTCAAAAGCGGA"!. RA3-8 (^'CCACTGTGAGTGGT-
TATCTCCTG"] for testing as potential primers for the WS-PCR.
The primers RA5-1 and RA3-6 were designed to amplify a se-
quence of -160 bp from the 5' end of the I6S rDNA (Fig. I ). The
primer pair RA5-6 and RA3-8 was designed to amplify a sequence
of -230 bp from the 3' end of the I6S rDNA (Fig. I ), The previ-

ously extracted DNA samples were then retested with these new
primer sets.

We also examined the possibility of using crude cell lysates
rather than purified DNA in the assays for the WS-PCR. Tissue
samples from 26 abalone were prepared by homogenization in TE
buffer and boiling for 5 minutes at 100 °C. From this solution, 3 |jil
was added to a PCR cocktail. After amplification, DNA was sepa-
rated on 1.5 % agarose gels.

PCR Amplification of rDNA

All amplifications were performed in standard 50 pil reactions
containing 10 mM Tris-HCI pH 8.3 (at 25 °C), 50 niM KCI, 1.5
niM MgCI, , 0.001 % w/v gelatin, 400 |j.M dNTPs, 5 jjlM tetram-
ethyl ammonium chloride. 40 pmoles of each primer, and 2 U Taq
polymerase. The PCR thermal cycler used was a model PTC-200
(MJ Research, Watertown, MA).

A 160 bp fragment (using primers RA5-I and RA3-6) of the
I6S rDNA from the RLP was amplified using 40 cycles of I min
at 95 °C, followed by 30 sec at 50 °C, followed by 30 sec at 72 °C.
The amplification cycles were preceded by a denaturation step,
where samples were held at 95 °C for 5 min and followed by an
extended elongation step where samples were held at 72 °C for 10
min.

Specificity of the PCR

Adult black abalone were collected from the Vandenberg Air
Force Base, CA (in July 1996) where WS is epidemic and from
Monterey, CA (in December 1998) where the RLP has been re-
cently detected but where no signs of clinical WS have yet become
apparent (Finley and Friedman, unpubl. obs). Additional samples
of adult abalone were collected from the following two locations
where neither WS nor the RLP had been observed: black abalone
from Carmel Point in August 1997 and red abalone from Shelter
Cove in December 1998. Farmed red abalone obtained in January
1999 from a facility in Central California were also examined.
Visual as.sessment of WS was determined according to Friedman
et al. (1997). Digestive gland, postesophagus and/or epipodium
were collected and stored at -80 °C until DNA extraction. The
hemolymph of abalone contains no blood-clotting factor; there-

ETS

Withering Syndrome in California Abalone

16S

ITS-1

215

EUBA

EUBB

1525 bp

16S rDNA

RA5-1

RA3-6

RA3-1

RA5-6

RA3-8

160 bp

Figure. 1. Diagrammatic representation of the approximate location and orientation of primers used for development of the PCR assay to detect
the Rickettsial-like prokaryote among abalone with withering syndrome (WS). Primers EUB A and EUB B were used in the initial amplification
of the 16S rDNA from infected tissues.

fore, collection of samples from internal organs is fatal for the
animal. DNA samples obtained from different abalone tissues were
tested to identify those best for use in PCR assays and to determine
if the RLP could be detected in nonlethal samples of the epipo-
dium.

Genomic DNA of endogenous gut flora was tested for possible
nonspecific amplification of DNA using the WS diagnostic prim-
ers. To obtain cultures for DNA extraction, postesophagus tissue
(0.8-2.0gm) was dissected from three black abalone (Vandenberg
Air Force Base. CA) and held separately in sterile vials containing
10 mL of 0.2 (jLm-filtered seawater, on ice. Vials were shaken
vigorously to dislodge and suspend bacterial flora associated with
epithelial surfaces, and the tissue was removed from each vial. The
bacterial suspensions were spread onto marine and TCBS agar
plates that were held at 15 °C. Colonies appearing after 7 days
were streaked on marine agar plates. Four colonies from the TCBS
plates and two from the marine agar plates were selected and
passed several times on marine agar. Based on biochemical (API
NFT Biomerieu.x Vitek. Hazelwood, MO) tests, supplemental
tests, and colony morphology on TCBS and marine agar, four
distinct isolates were obtained (Table 2). Cultures grown in TSB
broth (supplemented with 2 % additional NaCl) were centrifuged
(3.200 g. 10 min, 4 °C), resuspended in TE buffer (10 niM Tris-

HCl. pH 7.5. 0.1 mM EDTA). and frozen at -80 °C. The presence
of any RLPs among the colonies tested was precluded by the
multiple passages of the isolates on artificial media. DNA was
extracted from the bacterial cultures, as described above. Approxi-
mately 150 ng of genomic DNA from each of these isolates was
tested with the primers RA5-1 and RA3-6.

Histology

Several 3-mm cross sections that included mantle, epipodium,
postesophagus. digestive gland, and foot muscle were excised
from each abalone, placed in Invertebrate Davidson's solution
(Shaw and Battle 1957) for 24 h and processed for routine paraffin
histology. Deparaffinized 5 [o-m tissue sections were stained with
Harris's hematoxylin and eosin (Luna 1968) and assessed for the
presence of RLPs, and condition of the foot muscle and digestive
gland were evaluated (Friedman et al. 1997). The digestive gland
was scored as 0 if normal, with terminal acini occupying most of
the tissue present. A score of 1 represented a moderate degenera-
tion of or replacement of terminal acini with transport ducts or
connective tissues: whereas, a score of 2 represented a severe loss
of acini. Similarly, condition of the foot muscle was scored as 0
when muscle bundles were tightly packed, I when a moderate loss

TABLE 2.
Characteristics of four postesophagous bacterial isolates grown in culture and tested for reactivity using the WS-RLP PCR protocol.

Isolate

Gram

Shape

Sucrose Reaction on TCBS

0/129 Sensitivity

H2S Production

PCR Reaction

I50-A

Negative

Rods

150-B

Negative

Rods

213

Negative

Rods

239

Negative

Rods

Positive
Negative
Negative
Negative

Sensitive

Negative

Negative

Sensitive

Negative

Negative

Resistant

Positive

Negative

Resistant

Negative

Negative

216

Andree et al.

of muscle bundles and concomitant increase in connective tissue
was observed, and 2 when such loss was severe.

RESULTS

PCR Detection of the Putative Eliologic Agent of Withering Syndrome

The DNA from tissues of abalone with naturally acquired in-
fections was screened for the presence of RLP DNA. Tissues from
abalone collected from geographic regions where signs of WS and
the associated RLP were absent served as negative controls. In
addition, epipodial tissue was tested to determine if nonlethal
samples could serve as diagnostic material for PCR analysis.

In total, we examined 23 animals by PCR (Table 3). We com-
pared epipodium, digestive gland, and/or postesophagus for some
individual animals. The 160 bp amplicon was present only in
tissues from those animals that came from WS enzootic areas
(most of which had proved to be positive by microscopic exami-
nation). The identity of the amplified DNA was confirmed by
automated sequencing (data not shown). In addition, the identity of
the species being detected was confirmed by in situ hybridization
experiments in which the PCR primers were used as probes (An-
tonio et. al. in press). The yield of amplified DNA was greatest
from postesophageal tissue (Fig. 2). This agrees with microscopic
observations that demonstrated the postesophagus was more
highly infected than the digestive gland. A weak amplification of
DNA was obtained from some samples of epipodium from a subset
of animals that tested strongly positive using the digestive gland.

Specificity of the PCR

Of the primer pairs tested, RA5-I and RA3-6 gave the best
results at an annealing temperature of 50 "C. Retesting of samples
with this new primer produced a 160 bp amplicon from all tissues
known to be positive for the RLP by microscopic examinations. In
addition, all samples considered free of the RLP by microscopic
examination were negative by PCR. There was no amplification of
genomic DNA from the selected bacterial isolates from the intes-
tine of black abalone or from a recently isolated Piscirickettsia-
like organism isolated from white sea bass Atractoscion nobilis
Ayres in California (unpubl. obs.). Only DNA extracted from aba-
lone tissues known to contain RLP yielded a 160 bp amplicon. All
samples of postesophagus and digestive gland from RLP-infected
red and black abalone tested positive. There was a 250 bp ampli-
con present from the epipodial tissue of six black abalone collected
from Vandenberg. Monterey, and Shelter Cove. However, two of
these six samples yielded both amplicons (160 bp and 250 bp).
Those with only the 250 bp amplicon were scored as negative
based on the difference in the molecular weight and sequencing of
the larger amplicon that indicated it was not related to bacterial
I6S rDNA.

DISCUSSION

Diagnosis of infectious disease during the past century has
generally relied on such methods as culture, direct observation of
parasites or. more recently, antigen-based assays (Sethi et al.
1996). These methods may involve expense and time for sample

TABLE 3.

Detection of a Rickettsiales-like prokaryote by PCR and histology in digestive gland, postesophagous and epipodium tissues in black and red

abalone in various stages of withering syndrome.

Animal # Species

Source/Date

WS
sign"

Digestive
gland PCR"

Digestive gland
Histology'

Postesophagus
PCR"

Postesophagus
Histology'

Epipodium
PCR"

Epipodium
Histology

1

Black

Carmel Pt. 8-97

0

-

0

nd

0

-

2

Black

Carniel Pt. 8-97

0

-

0

nd

0

-

3

Black

Carmel Pt. 8-97

0

-

0

nd

0

-

4

Black

Carmel Pt. 8-97

0

-

0

nd

0

-

5

Red

Monterey 12-98

0

-

0

nd

0

-

6

Red

Monterey 12-98

0

-

0

nd

0

-

7

Red

Monterey 12-98

0

-

0

nd

0

-

8

Red

Shelter Cove 12-98

0

-

0

nd

0

-

9

Red

Shelter Cove 12-98

0

-

0

nd

0

-

10

Red

Shelter Cove 12-98

0

-

0

nd

0

-

11

Black

Momerey 12-98

0

-f-+

1

nd

1

-I-

12

Black

Monterey 12-98

0

-I-+

1

nd

1

-

13

Black

Momerey 12-98

0

++

2

nd

2

-

14

Black

Vandenberg 1997

0

++

1

nd

2

-

15

Black

Vandenherg 1997

1

+

0

nd

2

+

16

Black

Vandenberg 1997

1

■n-

2

nd

3

+

17

Black

Vandenberg 1997

1

+++

3

nd

3

+

18

Red

Farm A 1 -99

1

-H- +

2

nd

2

-

19

Black

Vandenherg 1997

y

-

0

+-H-

3

nd

20

Black

Vandenberg 1997

3,

++

3

+++

3

nd

21

Black

Vandenberg 1997

y

-^++

3

nd

3

++

22

Red

Farm A 1-99

y

+++

2

++++

3

-

2.1

Red

Farm A 1-99

y

+++

1

nd

1

-

" WK sign: Degree of body mass shrinkage 10 = vvilhin normal range, 1, 2, 3 = slightly, moderately, severely shrunken).

'' Relative intensity of 160 bp amplicon hand in elhidium bromide stained gels ( - = absent, ++++ = brightest, nd = no data).

' RI.P intedion intensity by microscopic examinalion (0 = absent. 1 - low density. 2 = moderate, .^ = high).

Withering Syndrome in California Abalone

217

12 3 4 5

7 8 9 10

Figure. 2. Detection of a Rickettsial-like prokaryote in tissues from abalone with withering syndrome (WS) using a newly developed PCR assay.
Approximately 300 ng of genomic DNA was used for each sample assay. Lanes land 10: 100 bp ladder molecular weight standard; lane 2: positive
control sample ( 10 ng of plasmid pl6RK3 containing cloned 16S rDNA); lane 3: negative control sample ( uninfected black abalone postesophagus
tissue); lanes 4 and 5, respectively: digestive gland and postesophagus of infected black abalone; lanes 6 and 7, respectively: digestive gland and
postesophagus of infected red abalone: lane 8: negative control sample (150 ng of genomic DNA from prokaryote cultured from the gut of
abalone): lane 9: epipodial tissue from infected black abalone displaying the 250 bp amplicon.

preparation or may lack sensitivity in detecting low numbers of
parasites. Furthermore, many parasites, including the RLP from
abalone. have not been cultured outside the host animal (Arnoldi et
al. 1992. Mari et al. 1995. Sethi et al. 1996). Molecular approaches
to parasite detection such as the PCR are rapid, reproducible, and
relatively easy to conduct (Andree et al. 1998. Muuel et al. 1996.
Sethi et al. 1996). Early and accurate detection of parasites in
marine invertebrates is critical, because therapeutic approaches are
limited, and avoidance becomes a principal means of disease man-
agement and resource protection.

Withering syndrome is a fatal, infectious, bacterial disease of
both wild and cultured abalone in California (Friedman et al.
1997). Diagnosis of WS, like many diseases, currently relies on
observation of gross signs of affected abalone and detection of the
RLP in target tissues by microscopic examination of stained tissue
sections. Unfortunately, inclusions of other intracellular bacteria
are frequently found in shellfish and are often morphologically
indistinguishable from the RLP associated with WS (Friedman and
Hedrick. unpubl. obs.). We have confirmed the PCR test differen-
tiates among RLPs in abalone tissues by in situ hybridization ex-
periments that utilize the PCR primers as probes (Antonio et al. in
press). The probes only hybridize to the RLPs that correlate with
the observation of disease symptoms (data not shown). The RLP
is. therefore, more easily and rapidly detected by the application ot
such DNA diagnostic tests as PCR than by microscopy.

This PCR test should prove to be a useful tool for the study of
WS, especially for epidemiological investigations of the modes of
transmission and reservoirs for the parasite in the marine habitat.
In addition, experimental exposure studies combined with this
PCR assay should help to identify naturally resistant populations
of black abalone. The PCR assay provides direct visualization of
specific bands on ethidium bromide stained gels, more rapid di-
agnosis than microscopic examination of tissue sections, and ob-
viates the need for cell culture isolation of the parasite. Further-
more, we presume the PCR test will detect organisms at lower
levels of infection than microscopic examination of stained tissue
sections.

The primers designed for PCR detection of the RLP were cho-
sen after alignments showed sequences obtained from Anuplasma
marginales. Ehrlichia bovis. Wolbachia pipientis, Piscirickettsiu
salmnnis. Esclierichio coli. and an unnamed Rickettsia-like bacte-

rium from shrimp found no homologous sites for hybridization.
The lack of hydridization of the RLP primers with genomic DNA
isolated from bacterial flora cultured from the postesophagus of
abalone and from P. salmonis genomic DNA from cells grown in
tissue culture was indicated by the absence of any amplified prod-
ucts following the PCR. The assay performed equally well on
RLP-infected black and red abalone. There was some nonspecific
amplification from epipodial tissues of a small number of the black
abalone tested but the product (250 bp) was clearly different in
molecular weight and sequence from the expected 160 bp ampli-
con. This 250 bp product may be the result of surface contamina-
tion of the epipodium. because it was not observed in red abalone.
or black abalone from all locations.

Some heavily infected animals gave positive test results with
epipodial tissues; however, in general, most samples of the epipo-
dium were negative by PCR. The relatively weak positives among
the epipodium samples may represent cross contamination (with
more heavily infected tissues) during sampling or the adherence of
RLPs on the epipodium as shed from infected animals in crowded
tanks during transport or holding before sample collection. This
concern combined with the appearance of the 250 bp amplicon in
nonlethal epipodial biopsies discourages us from recommending
this sampling approach for detection of the RLP. A more reliable
approach is to collect postesophagus or digestive gland tissues
from each animal, which requires sacrificing the animal (as do
current microscopic procedures).

As mentioned above, an additional application of the PCR
primers is their use for in situ hybridization (ISH) to visualize the
parasite in various tissues or alternate hosts and to differentiate this
bacterium from other RLPs commonly observed in marine species
(Elston 1986. LeGall et al. 1988). Future work utilizing ISH may
also identify portals of entry and eariy developmental forms not
easily seen by standard microscopic examinations, as shown with
other parasites of aquatic hosts (Antonio et al. 1999; Antonio et al.
in press).

ACKNOWLEDGMENTS

We thank Thea T. Robbins for the culture and partial charac-
terization of the black abalone postesophagus bacterial isolates.
This work was supported by the Saltonstall-Kennedy Program of

218

Andree et al.

the National Oceanic and Atmosplieric Administration. U.S. De-
pailment of Commerce under Grant NA76FD0046. Additional
support was provided by the California State Resources Agency,
California Department of Fish and Game. The views expressed

herein are those of the authors and do not necessarily reflect the
views of NOAA or any of its subagencies. The U.S. Government
is authorized to reproduce and distribute this work for governmen-
tal purposes.

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Antonio D. B., K. B. Andree, T. S. McDowell & R. P. Hedrick. 1999.
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Joimuil of Shellfish Research. Vol. 19. No. 1. 219-228. 2(){)().

MICROSTRUCTURE, CHRONOLOGY AND GROWTH OF THE PINTO ABALONE, HALIOTIS

KAMTSCHATKANA, IN ALASKA

SCORESBY A. SHEPHERD,' DOUGLAS WOODBY,"
JANET M, RUMBLE,- AND MIGUEL AVALOS-BORJA^

^ South Australian Research and Development Institute,

Henley Beach 5022. South Australia
'Alaska Department of Fish and Game.

Douglas. Alaska 99824-0020
^Centro de Ciencias de la Materia Coudensada. Universidad Nacional

Autonoma de Mexico.

Ensenada 22800. Baja California. Mexico

ABSTRACT The microstructure. deposition of rings, and growth checks in the pinto abalone. Haliotis kaimschalkana Jonas, were
examined at seven sites in southeast Alaska. Rings were of calcium carbonate with prismatic or block-like structure or were of organic
material (called brown rings); sometimes both types were juxtaposed in a compound ring. Rings alternated with nacreous layers having
a brick-like or laminar structure. Laminar thickness was correlated with ambient sea temperatures and provided internal evidence of
periodicity of ring deposition. One ring a year appears to be deposited in the spire of this abalone in about mid-summer, and a growth
check is laid down at the growing edge of the shell in about mid-winter. Rates of deposition of the rings and growth checks were
validated by comparison with known growth rates from mark-recapture experiments at one site, and modal analysis of length frequency
data at others. Examination of a sample of shells of known age confirmed a deposition rate of one ring a year. Brown rings appear to
be laid down adventitiously and were excluded from ring counts for aging purposes. The three independent techniques, rings, growth
checks, and modal analysis, gave consistent juvenile growth rates at 7 sites of 14-18 mni/y dunng the first 4 y. Thereafter, growth rates
followed a declining exponential curve. Growth rates differed little between sites, and mean parameters of fitted von Bertalanffy growth
parameters for seven sites were: K = 0.20; L^ = 125.9 mm.

KEY WORDS: Chronology, growth rings, growth checks, shell-aging, growth rates, abalone. Halioris kamtschatkana

INTRODUCTION

The molluscan shell is known to cryptically encode, within its
microstructure. information on seasonal growth, age, and even
habitat relations (Bandel 1990). Knowledge of the microstructure
of the abalone shell is accumulating. It is known that the abalone
shell deposits prismatic rings in the spire that are useful for aging,
and that the shell's aragonitic laminae reflect seasonal temperature
changes that confirm the periodicity of ring deposition (Shepherd
et al, 1995, Shepherd and Avalos-Borja 1997).

The pinto (or northern) abalone, Halioris kamtschatkana. so
named because of its striking color pattern on the epipodiuin.
occurs from southeast Alaska to northern California and was the
basis of small commercial fisheries in British Columbia and
Alaska until declining stocks caused their respective closures in
1990 (Farlinger and Campbell 1992) and 1995 (Woodby et al. in
press). However, recreational and subsistence harvest of the pinto
abalone continues in Alaska, and an understanding of the species"
population dynamics, growth rate specifically, is necessary for the
management of the existing stocks and the rehabilitation of those
that have declined.

In this paper, we first describe structural features of the pinto
abalone shell. This shell lays down nonpigmented. and occasion-
ally pigmented, rings in the spire, which are clearly visible in
horizontal shell sections, and periodic growth checks at the shell's
growing edge. We interpret microstructural and ultrastructural
properties of the shell to show the periodicity of ring deposition in
the shell. We obtained samples of shells from wild populations and
estimated the rates of deposition of the rings with size, and the
sizes at which checks were laid down. Then we used shells of
known age and independently obtained field data on the growth
rate to confirm the rate of deposition of the rings and checks. We

show that rings are laid down in summer and checks in winter and
apply the techniques to estimate the growth rate of this abalone at
a number of sites in southeast Alaska.

MATERIALS AND METHODS

Data Collection

Shell samples of the pinto abalone were collected by diving
from seven sites in southeast Alaska (Fig. 1). At each site, divers
searched intensively from the shallow sublittoral to the lower
depth limit of the abalone at a depth of 10-15 m and specifically
under boulders for all abalone in the size range 0-100 mm shell
length (SL). and at some sites above 100 mm as well. We also
collected the dead shells of abalone encountered.

Using the method of Shepherd et al. ( 1995). we took horizontal
sections of each shell by grinding the spire with a disk grinder until
a minute hole appeared in the shell, then polishing the section with
sequentially finer abrasive (500-1,200 grit), and finally etching the
surface with dilute HCl. The horizontal section reveals a series of
concentric prismatic layers (called rings), laid down alternately
with aragonitic nacre. The number of rings in each section was
counted under a low-power binocular microscope. The prismatic
layers are opaque and sometimes faintly honey-colored. Examina-
tion of the section showed clearly whether the most recently laid
material was nacreous or prismatic. A few shells (2% of the total),
bored around the spire by boring bivalves or annelids, were un-
readable and were discarded.

Vertical sections of a subsample also were cut across the mid-
point on the spire with a slow-speed electric saw with a diamond
disc, were polished and were etched as described above, and then
were cleaned in an ultrasonic bath for further analysis.

219

220

Shepherd et al.

/Magic I.
\-^ Battery I.
Galankin I.

ALASKA

Southeast Q

%^

Thimble Cove
Ridge

/
Jumbo I.

Figure. 1. Map of southeast Alaska showing sampling sites. Locations:
Magic Island, 57=05'457135 2407": Battery Island, 57 03'267
135 22'55": Galankin Island, 57 (12(147135 2(»'05": Thimble Cove,
55 18'467133°34'42": Ridge Island, 55 16267133 1218": .lumho Is-
land, 55 I4'377132°39'55"; and Gravina Island, 55°21'13713r 51'23".

To exumine the variation in crystalline ultrastructure across the
inner nacreous layer of vertical sections, we made a transect from
the outer to inner shell surface at right angles to the prismatic
layers and a series of micrographs (x5,0()()) were taken under a
scanning electron microscope (SEM) (JSMS^OO, JEOL). The
thickness of aragonitic laminae was measured at two sites on each
micrograph with three replicate measurements per site. Each mea-
surement was of 10 adjacent laminae from which a mean laminar
width was calculated for each site. We chose a transect location on
the vertical section where the rings were evenly spaced and took
3-5 micrographs at about equal intervals between each ring. The
main elemental composition of rings was examined by X-ray mi-
croanalysis with energy-dispersive spectroscopy, in all. nine ver-
tical sections were examined in detail with SEM.

We compared seasonal variation in sea surface temperatures
with laminar widths to elucidate the periodicity of ring deposition.
Monthly mean temperatures were derived from optimally interpo-
lated weekly surface observations and satellite measurements at 1°
resolution (Keynolds and Smith I994|. The data set was provided
by the National Center for Atmospheric Research and was devel-
oped at the National Center for Environmental Prediction of the
National Oceanic and Atmospheric Administration.

Pinto abalone. presunipti\ely 0— t y old. lay down growth
checks (described in Results), which are best seen with transmitted
light (Fig. 7) in shells to about 7()-S() nmi SL. Larger shells ap|iear

also to lay down growth checks in the shell, which may be seen by
incident light; however, they are not clear, and their interpretation
was difficult and outside the scope of this study.

We measured SL to the 1st. 2nd. 3rd. and 4th growth checks
(SL|, SL,, SL,. and SL4. respectively) where these checks were
visible for shells from four sites with sufficient data. Occasionally,
a double check or another secondary, less conspicuous check was
observed between these checks. In the former case, length to the
checks was averaged, and in the latter case where we were uncer-
tain as to which was the primary check, we measured the SL to the
former of the two. In order to estimate when the growth checks
were laid down, we first estimated for each site (except for Battery
Island and Jumbo Island, for which there were insufficient data)
the mean length at a presumed age of 1 y. These lengths were
extracted from Table I for three sites and from the mean length of
shells with one ring at Galankin Island. We then estimated the
mean proportion of the annual growth achieved before deposition
of the first growth check. By assuming that the growth checks were
laid down exactly 1 y apart, and that the site-specific growth rates
were those calculated in Table 2. we calculated the proportion of
the 2nd, 3rd, and 4th year's growth achieved before deposition of
the respective growth check for that year.

We examined seven shells of known age. These abalone were
taken from the wild at Sitka at a size of about 50 mm SL in the
summer of 1978 and were maintained in aquaria at Seward. The
shells were estimated from growth checks to have been about 3 y
old at capture and were assigned a birth date of July 1. 1975. The
abalone died between 1 985 and 1 994, and the year of death was
recorded on the shell. We assumed that each abalone died on July

1 of the year marked on the shell. In addition, there were two shells
marked at 82 and 92 mm SL, respectively, and at liberty off Demp-
ster Island in British Columbia for 5 y. We estimated the number
of rings present at the date of tagging, from length, in the light of
the known growth rate for that site, and by deduction from the
number present at the date of capture, and the estimated number of
rings deposited during the period between dates of lagging and
recapture. Growth checks in the shells were counted, and the pres-
ence of checks at lengths near those recorded for SLj-SL^ in Table

2 was inferred where not visible.

Sladslics

We estimated the growth rate by two methods, both indepen-
dent of interpretation of ring counts and growth checks. First,
where there were adequate length frequency data, we used the
EMMIX program to separate modes. The procedure fits Gaussian
curves and uses maximum likelihood methods to separate them
(MacLachlan and Peel 1998, MacLachlan et al. 1999). The modes
were assumed to be annual year classes because the pinto abalone
has a nanow summer spawning season lre\iewed by Sloan and
Breen 1988). Modes are conspicuous for at least the first 4 y. but
less so after that. We followed Fournier and Breen (1983) and
Breen and Fournier (1984) and considered that the first mode at
15-25 mm SL appearing in summer samples was the 1-y-old co-
hort and that each succeeding mode was 1 y older. We estimated
growth rates troin modal intervals at .several sites. Second, we
examined a subset of mark-recapture data from Gravina Island
where the period between dates of lagging and recapture was about
a sear (Woodby et al. in press) and derived mean juvenile growth
rales lor that site.

In the regressions o\' length \ersus number of rings, there is an
unknou n measurement error in the count of rings, suggesting that

The Pinto Abalone

221

TABLE 1.

Regression equations of SL versus presumptive age in years (A) for

data on modal means of length-frequency distributions and

presumptive age."

Figure. 2. Types of rings in H. kamtschatkana. The deposition of shell,
and the descriptions below, go from top to bottom in each micrograph.
a) Successive layers of nacreous laminae, a minor ring (arrows), more
laminae, and the first major ring of block-like prismatic structure, b)
Successively, a major ring, nacreous laminae, and a brown ring of
organic material (arrow) followed by irregular material and lastly
nacreous laminae, cl Nacreous laminae, followed by a double ring, and
then more laminae, d) A compound ring comprising block-like prisms
followed sequentially by an organic brown ring, irregular material
and, last, nacreous laminae.

the regression should be formulated as an "error-in-variables"
model (Model II. see Ricker 1973). However, the regressor vari-
able is not normally distributed, hence the methods of solution are
complex (Fuller 1987). Acknowledging this problem, and assum-
ing that the measurement error is small, we note that the slopes
(growth rates) at each ring interval may be slightly biased toward
zero when formulated as a standard linear regression.

On the assumption that rings were laid down annually, von
Bertalanffy growth parameters and standard errors were estimated
with a Gauss-Newton nonlinear regression (SAS 1996).

RESULTS

Microstructure

Length at Age

Site

Regression Equation (SE)

R'

1 y (mm)

Gravina Island

SL = 17.0 ■!■ 11.0(0.8) A

0.99

h

Magic Island

SL = -},A+ 18.5(1.3) A

0.99

16.3

Thimble Cove

SL = 7.9 + 13.8 (0.2) A

1.00

21.5

Ridge Island

SL = 12.2 + 13.1 (0.8) A

1. 00

22.3

' The age at one year is the modal mean of the initial mode in the length-
frequency distributions.
•^ No I-y-old animals were found.

rings were of two kinds. Most were of calcium carbonate, were
unpigmented. were of simple, prismatic, or block-like structure
with very little organic material (Fig. 2a, c), and were presumed to
be composed of aragonite and/or calcite. as is found in other aba-
lone species (Dauphin et al. 1989. Hawkes et al. 1996). The second
type of ring contained relatively little calcium but was high in
carbon, sulphur, oxygen, silicon, and sodium, indicating a likely
organic composition (Fig. 2b, d); these rings were pigmented when
\ iewed under the optical microscope and are termed brown rings.
The brown rings were sometimes isolated from other rings and
sometimes juxtaposed on one side or the other of a prismatic ring
to form a compound ring (Fig. 2d).

The width of minor rings was 4-9 |xm, and they were readily
distinguishable by size from major rings, which were 10-46 |j.m
across. Brown rings were lO-l.'i p.m across and were visible even
under low-power optical microscopy by their honey color. They
could be confused easily with compound rings.

Mean laminar thickness measured along transects running
transverse to the rings varied in a cyclic manner between rings. A
plot of the change in the thickness of laminae along a transect (Fig.
3a) shows a decline in thickness soon after deposition of the ring
and a later increase prior to deposition of the next ring. This pattern
was repeated between rings and was the typical pattern in the
sections examined. The thickness of laminae was significantly
correlated with sea temperature, assuming that the rings were laid
down in mid-summer (see below). The highest correlations oc-
curred when a lag period of 1 mo (/■ = 0.59; P < 0.001 ) or 2 mo
(r = 0.69; P < 0.001 ) were applied to sea temperature data.

We also examined a parasitized shell in w hich a brown ring and
two compound rings were present. Assuming the same relationship
with temperature, we estimated the timing of ring deposition from
the laminar thickness. The results (Fig. 3b) show that the brown
ring interrupted the pattern of seasonally changing thickness of
laminae, which is consistent with our conclusion that they are
adventitious. In this shell, the highest correlation between tem-
perature and laminar thickness occurred with a lag period of 2 mo
for sea temperature (/• = 0.67: P < 0.001 ) compared with ;■ = 0.64
(P < 0.001 ) for a I -mo lag period and ;■ = 0.43 {P < 0.05) with no
lag.

The rings of H. kamtschatkana show variation in microstruc- ^,„g Deposition
ture. The initial minor ring seen in a proportion of shells under

optical microscopy was usually separate from but occasionally At low magnification, horizontal and vertical sections of the

juxtaposed to the first major ring overall or to part of its length in shell show distinct growth rings. Shells of 13-28 mm SL were

the section (Fig. 2a). so that it may not be distinguishable from the considered to be 1-y-olds (see below), and a proportion of them

major ring at low magnification. SEM microscopy revealed that showed a fine outer ring (termed minor ring) as well as the sub-

222

Shepherd et al.

TABLE 2.
Lengths to growth checks SL,, SL,, SL„ and SL4 for shells of H. kamtschatkana at six sites.

Mean Growth

Site

A-

SL, (SE)

N

SLj (SE)

N

SL, (SE)

N

SL4 (SE)

Rate SE (mm/y)

Gravina Island

26

12.0(0.5)

39

27.5 (0.4)

40

42.7 (0.6)

28

57.5 (0.9)

15.1 (0.2)

Magic Island

27

11.4(0.6)

29

26.1 (0.8)

23

44.6 (0.6)

18

61.4(0.6)

16.9(0.5)

Thimble Cove

42

11.5(0.3)

42

26.2(0.51

28

41.6(0.6)

12

56.3(1.1)

15.0(0.1)

Galankin Island

13

11.8(0.5)

11

27.6(0.9)

13

44.1 (0.9)

13

57.8 (0.9)

15.5(0.4)

Ridge Island

22

10.9(0.4)

46

26.6(0.5)

52

42.5 (0.5)

31

55.3 (0.8)

14,9(0.5)

Battery Island

3

14.0(1.7)

6

25.5(1.1)

8

47.1 (I.I)

8

61.4(1.0)

16.4(1.3)

Jumbo Island

7

11.8(0.6)

16

25.3 (0.6)

21

40.6 (0.8)

16

53.6(0.8)

14.1 (0.3)

Mean values

11.9(0.4)

26.4(0.3)

43.3(0.8)

57.6(1.1)

15.4(0.4)

Individual lengths are not independent within a site because individual shells have multiple growth checks.

sequent thicker rings (termed major or prismatic rings). In H.
kamtschatkwM. the spire is unusually elevated some 2-5 mm
above the surrounding shell, compared with Australian or Mexican
species (unpublished observations) and. hence, more susceptible to
erosion than those species. The incidence of minor rings did not
decline in shells to a presumed age of about 4 y but thereafter
declined rapidly, and none were seen in shells > 5 y old. We
presume that the decline in their incidence was due to erosion,
because with increasing age the minor ring disappeared from the
face of the horizontal section but could still be seen on the eroded
lateral margin of the spire.

The incidence of separate minor rings also varied between sites.
At Galankin Island, 62% of shells had minor rings, and at Magic
Island, 48%, but at Thimble Cove, 17%, at Ridge Island, 13%, and
at Gravina Island only 2% had minor rings. It is possible, of
course, that minor rings may have occurred juxtaposed to their
neighboring major rings (Fig. 2a). in which case we would not
have distinguished them. As some presumptive 1-y-old shells had
both a distinct minor and a major ring, we concluded that both
were laid down in the first year, the minor ring possibly during the
first winter when the first growth check was deposited (see below).
We examined a sample of presumptive 1-y-old shells from the two
sites with the largest proportions of shells with minor rings to see
if deposition of a distinct minor ring was related to length. The
mean length of shells that deposited a distinct minor ring was 21.0
mm (SE 1.6 mm), and of those that did not. was 15.2 mm (SE 1.1
mm). The differences were significant (/ = 2.4: P < 0.05). This
suggests that individuals, which either grew faster or settled earlier
in the summer (or both), were more likely to lay down a distinct
tninor ring than those that grew more slowly or settled later. As the
presence of minor rings was variable, and they were in any event
.superfluous for aging, we excluded them from further consider-
ation.

Examination of the shell nacre on the ventral surface at the
spire indicated whether nacre or a ring (visible as an opaque layer
with a nonretlective surface) was the last layer deposited there. In
all, at four sites 91.4% of shells (N = 358) had most recently
deposited a ring over a broad area near the spire. In the remaining
ca.ses. some nacre had been more recently laid down at the spire.
We concluded that a ring must have been laid down during the last
episode of shell deposition before the collection of the samples in
mid to late July at all our sites.

A practical problem that we experienced in estimating the age
of juvenile shells from ring counts, mainly at the Magic Island and
Thimble Cove sites, was the difficulty in distinguishing minor
from major rings. Thus, it was possible to interpret a shell with two

rings as a 1-y-old with a minor ring or a 2-y-old without one. The
presence of two growth checks (see below) in 2-y-old shells helped
to resolve this dilemma. Another problem was ambiguity in ring
counts. This happened where two rings merged or where there
were false rings, i.e., where one ring divided into two and then
merged again into one (see Fig. Id in Shepherd et al. 1995). In
these cases of uncertainty as to whether there was one or two rings
(6% of all shells), the ring count was revised in the light of the
number of growth checks observed. In another 2% of all shells
examined, there was a clear discrepancy of I year in age estimated
from ring counts and from growth checks. In these cases, we
adopted the ring count for the purpose of the regressions, although
they were not consistent with the counts of growth checks. In very
few cases (1.4% of all shells), the exposed section was milky, and
no rings were visible.

The growth rate of many species of abalone is linear or nearly
so for the first 3-5 y of life, and thereafter is curvilinear. The linear
pha.se can be fitted with a linear regression, and the curvilinear
phase with a von Bertalanffy curve (Shepherd and Heam 1983).
Hence, a regression of SL versus the number of rings during the
linear phase of growth will give an estimate of the rate of depo-
sition of rings with age if the growth rate is known (Shepherd and
Triantafillos 1997).

Plots of length versus number of rings showed a linear or nearly
linear relation with length to about 80 mm SL at each site after
which the curve approaches an asymptote as expected for von
Bertalanffy growth. Linear regressions were fitted to data for
each site for the linear phase of growth. The regression equations
are given in Table 3 and are plotted in Figure 4 for these sites
where we found the most juveniles. The deposition rate of rings
with length was sitnilar at all sites. Assuming that the deposition
rate of rings is age-related, then the growth rate appears to be
fastest at Magic Island and Jumbo Island and slowest at Gravina
Island.

Mark-recapture data from Gravina Island o\cr the initial length
range of 45-75 mm SL (Fig. 5) show that the growth rate declined
linearly from about 15 mm/y at 50 mm SL lo about 5 mm/y at 75
mm SL ( Woodby et al. in press). The mean growth rate of marked
individuals over this length range was 10.3 mm/y (SE 0.9 mm/y).
This is close to the mean growth rale of I 1 . 1 mm/y (SE 0.7 mm/y)
given in Table 3 for Gravina Island. Although the two estimates
cannot be compared statistically, their SEs overlap, suggesting that
they are not significantly different. Tagged abalone at Gravina
Island showed von Bertalanffy lather than linear growth (Fig. 5).
which is inconsistent with our hypothesis of linear juvenile growth
rale and with our analysis of length-frequency data. This incon-

The Pinto Abalone

223

TABLE 3.
The seven sampling sites, with regression equations of SL versus number of major rings (R) of H. kamtschatkana for all shells <80 mm SL."

Length at

Age

Brown

Site

N

Regression Equation (SE)

R^

1 y (mm)

Rings ( % )

Gravina Island

56

SL = 19.8 (2.7) + ll.l (0.7) R

0.80

29.9

27

Magic Island

45

SL = 2.5 (2.5) + 16.7 (0.8) R

0.90

19.1

13

Thimble Cove

86

SL = 8.2 (1.6)+ 13.7 (0.5) R

0.90

21.8

14

Galankin Island

32

SL = 8.3 (2.9)+ 13.8 (0.8) R

0.90

22.1

14

Ridge Island

70

SL = 10.0 (2.4) + 13.3 (0.7) R

0.84

23.3

33

Battery Island

9

SL = 3.2 (7.2) + 18.6 (2.4) R

0.90

21.8

17

Jumbo Island

28

SL = 24.9(5.5)+ lO.I (1.4) R

0.67

35.0

19

•' Estimated mean growth rates and length at a presumed age of 1 y were derived fom the regression equations. The mean incidence of brown rings in
shells >70 mm SL are given for each site. The slope of the regression equation is the mean annual growth rate (mm per year).

sistency could be an artifact of insufficient recaptures of tagged
abalone that were tagged at < 50 mm SL.

Length-frequency distributions for four sites are shown in Fig-
ure 6 with Gaussian curves fitted to the prominent modes. Linear
regressions of modal means versus presumptive age gave estimates
of the mean growth rates (Table 1). These were 13-19 mm/y, and
length at 1 y of age was 16-23 mm. according to the site. High R'
values of > 0.99 for every site indicate that the growth rate was
uniform and very nearly linear. The two sets of e.stimates of growth
rates for the four sites, summarized in Tables 1 and 3, were very
close with overlapping SEs. so we accepted that one ring per year
was laid down at these sites. Mean lengths at one ring (Table 3)
and at 1 y (Table 1 ) were also very close (we found no 1-y-olds at
Gravina Island), which is consistent with the hypothesis that one
ring per year is deposited.

Growth curves for all length-age couplets for each site and for
all sites combined are given in Table 4. Length-age couplets and a
growth curve for all sites combined are plotted in Figure 9.

Brown Rings and Shell Erosion

As shown by SEM micrographs and energy-dispersive spec-
troscopy, brown rings are qualitatively different in structure and

a) shell length=93 mm

Brown ring

6/93 12/93 6/94 12/94 6/95 12/95 6/96 12/96 6/97 12/97 6/98

Sea surface temp
■ Laminae width

8/92 2/93 8/93 2/94 8/94 2/95 8/95 2/96 8/96 2/97 8/97 2/98 8/98

Date (m/yr)

Figure 3. Change in laminar width in vertical sections in the spire of
H. kamtschatkana for: a) a 5-y-old shell (93 mm -SLl that had a brown
ring between the first and second major rings, and h) a 6-y-oId shell
(107 mm SL). Arrows indicate the location of rings in the sequences,
and the first arrow in each sequence is a minor ring.

composition from prismatic rings. An examination of a series of
vertical sections showed that brown rings were laid down only in
the presence of infestation by endobionts and that the number of
brown rings increased with increasing infestation. Further, they
appeared to have been laid down independently of the usual regu-
lar pattern of deposition of prismatic rings, and so they were con-
sidered adventitious when alone rather than substitutional for a
prismatic ring. Accordingly, we excluded brown rings from the
count of rings for the purpose of aging shells when they occurred
alone but not when they occurred as compound rings, i.e., when
they were juxtaposed to a prismatic ring. Shepherd and Huchette
(1997) similarly found that brown rings in Haliotis scalaris were
adventitious. In horizontal sections, brown rings were usually rec-

Figure. 4. Plots of length (mm) versus the number of rings of H.
kamtschatkana at Gravina Island, Magic Island, and Thimble Cove.

224

Shepherd et al.

25

20

E

E. 15 H

c

E

£
u

c

o

o

10 -

-5 -

-10

40 50 60 70 80 90

Initial shell length (mm)

100

110

Figure. 5. Plot of annual increment (mm per year) versus the initial
length of marked H. kamtschatkana at Gravina Island.

ognizable because the process of grinding and polisining caused
cavities on the surface where the softer organic material was pref-
erentially excavated.

Brown rings were rarely present in shells < 70 mm SL. Above

a) Gravina Island

-.lifyiMdiJiyr.

- 6
4

2

b) Magic Island

^<lii?feJl Lntrr-. ^n^nnOOOnnolwtililni

c
3

(D

20 30 40 50 60 70 80

Shell length (mm)

Figure. 6. Plots of longth-lrit|uenc\ data (vertical l)ars are running
means of 3-nini size intervals), rings at length (solid circles! and (iaus-
sian modes fitted bv KMMIX Idashed lines). Modal means are: a)
(iravina Island: 40..1. 48.7. .>y.S. and li.y mm, variance l.^.ll mm: (h)
Magic Island: 16.,^, .^0.5,.=;6.6. and 72.7 mm, variance 16.1: c) Ridge
Island: 2.1.1.41.(1, 52.0, 64.8. and 76.7 mm, variance 15.8; and d)
Thimble Cove: 21.5, .V5..1. 4<).9, 62.7, and 76.5 mm, variance 14.9.

Figure. 7. Growth checks of three shells of H. kamtschatkana from
Thimble Cove seen by transmitted light. A) SL = 66 mm. Three checks
indicated by arrows, a) SL, not visible; SL, = 28 mm; SL, = 42 and 46
mm (a typical double check); and SLj = 60 mm. B) SL = 25 mm (left);
and SL = 26 mm (right). For both shells SL, = 10 mm (indicated by
arrows) and SL^ = 25 mm (faintly visible at the edge of the shell).

that size, the incidence of brown rings in the spire sections of shells
in samples varied from 13-33% according to site (Table 3). Over-
all, however, only 3% of shells > 70 mm SL were unreadable
because of the effect of parasites alone or were compounded by
erosion of the shell.

Shell erosion was slight (2-5%) at our study sites, which were
all in places sheltered from ocean swell. Generally, erosion of the
shell was associated with attacks by parasitic endobionts, which
were provisionally identified as polydorid annelids, and occasion-
ally the bivalve, Penitella sp., which caused excavations around
the elevated spire. In most cases, the eroded rings could still be
seen and counted, not in the horizontal section, but at the eroded
margin of the spire near the prominent suture line that joins the
older shell with the later deposited shell.

Growth Checks

Growth checks, which are lines marking an interruption in shell
growth, were characterized by at least one, but usually more, of the

Figure. 8. Lateral view of two shells of H. kamtschatkana showing a
suture line in the shell (hat marks a growth check continuing below the
line of pore-holes to the margin of the shell, a) SL = 50 mm (SL,
indicated by arrov\). h) SL = 41 mm (SL, indicated bv arrow).

The Pinto Abalone

225

following features as seen by transmitted light (Fig. 7): a indge up
to 0.5 mm high dorsally on the shell; and a fine suture line in the
shell or a discontinuity in the pattern of pigmentation or sculpture
of the shell. The growth checks were apparently formed at the
growing margin of the shell at the time of interruption to shell
growth. The growth check continued past the pore-holes to the
margin of the shell where the discontinuity or ridge was often more
conspicuous (unless eroded) than elsewhere (Fig. 8). The first
check, SL|, is lost almost always by age 3 y. and the next check,
SL-,. is lost a year later as new nacre is deposited on the ventral
surface of the shell, preventing transmission of light through the
shell. Checks SL, and SL4 are often visible dorsally only as dis-
continuities in the shell.

The estimation of age from growth checks alone has some
limitations or ambiguities. In 8% of the shells that are > 5 y old,
one or more checks were missing, apart from the incremental loss
of the earliest ones. In 6% of the shells over the same age range,
dual growth checks, i.e., two checks very close together, were
seen, most often at SU and SL,. In these cases, we measured length
to the check that persisted to the shell margin or to the former of
the two checks, if they were indistinguishable. In about 1 % of the
shells, no checks could be seen at all.

The mean lengths at which the growth checks SL1-SL4 were
laid down are given in Table 2. These were compared to estimates
of the lengths at age 1 y from the regressions in Table 1 for the four
sites listed and from the mean length of shells with one ring for
Galankin Island. For these five sites, a mean value of 60% of the
first year's growth was achieved before the growth check was laid
down, declining to a mean of 41% in the fourth year. The decline
was not significant (t = 0.20). Overall, for the five sites 53% of
the annual growth during the first 4 y was achieved before depo-
sition of the growth check for the respective year. Assuming a
uniform birth date in July, we conclude that the growth check was
laid down about half way through the year, i.e., in about January,
which is about mid-winter with minimal sea temperatures.

The mean lengths at which growth checks were laid down
(Table 2) are very nearly linear with age (r > 0.99 for all five
sites), indicating a linear growth over the length range of -10-60
mm SL. The growth rates derived from growth check analysis
(Table 2) are highly correlated with those calculated from ring
analysis (Table 3) {r = 0.88; P < 0.01 1, and the mean growth rates
for all sites combined derived from the two independent methods
did not differ significantly (t = 1.24). The consistency in growth
rates between the ring and growth check analyses supports the
hypothesis that growth checks are deposited annually.

TABLE 4.

Parameters of von BertalanfTy growth curves fitted to length-age
data for each site.

Site

iV

K(SE)

L. (SE)

Gravina Island

86

0.21 (0.02)

119.7(5.6)

Magic Island

103

0.20(0.01)

129.6(4.4)

Thimble Cove

110

0.16(0.01)

136.9(7.7)

Galankin Island

41

0.22 (0.03)

113.0(7.7)

Ridge Island

79

0.16(0.02)

134.9(13.7)

Batterv Island

32

0.25 (0.02)

118.9(4.2)

Jumbo Island

58

0.19(0.02)

128.6(5.5)

Mean value

0.20(0.01)

125.9(3.4)

All sites combined

509

0.18(0.01)

131.9(2.4)

120

E

£ 100 ■

1 ! .^^ ' ' :

hell length

000

/

^

^'' ■ ■ ■

CO

20

n

/'

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Rings and estimated age

Figure. 9. A plot of a von BertalanfTy growth curve fitted to length-
at-age data for all sites combined, and length-increment data from
Gravina Island.

The number of rings and growth checks present in shells of
known or estimated age are given in Table 5. Of the seven shells
of known age, three were eroded and/or parasitized at the spire and
the rings could not be counted. The remaining four deposited a
mean number of 1 .03 rings per year (SE 0.02 rings per year), after
deducting the number of brown rings, over a mean period of 14.5
y/shell. The seven shells also laid down an estimated 0.92 growth
checks per year. The two shells from British Columbia each laid
down an estimated five rings during the 5 y between tagging and
recapture, but growth checks were not visible in these shells.

DISCUSSION

Microstructure

The microstructure and pattern of changes in thickness of ara-
gonitic laminae show striking similarity to those of Haliotis cor-
rugata and Haliotis fulgens Phillipi. The block-like prisms and the
brown rings (described by Shepherd and Avalos-Borja 1997 as
being of granular structure) are similar in the three species, al-
though in H. corrugata the rings were compound, with prismatic
rings and brown rings regularly juxtaposed. The similarity between
the three species is perhaps not surprising, given their recent com-
mon ancestry (Brown 1993, Lee and Vacquier 1995).

The cyclic change in the thickness of the laminae between rings
and the correlations with temperature provide internal corrobora-
tive evidence that laminar thickness is temperature-dependent and,
hence, that ring deposition is annual. The significance (if any) of
the 1-2-mo lag correlation is unclear. We arbitrarily assumed that
laminar deposition occurred continuously throughout the year. If,
however, there was a pause in laminar deposition for 1-2 mo after
sea temperature reached a maximum, as could happen around the
time of spawning, then an apparent lag would be observed. This
question cannot be resolved until laminar deposition can be accu-
rately pinpointed in time as with a time stamp (see Hawkes et al.
1996). The dependence of laminar thickness on temperature has
been previously noted for the abalone H. corrugata (Shepherd and
Avalos-Borja 1997) and H.fulgens (Shepherd et al. 1995), as well
as some bivalves (reviewed by Lutz and Rhoads 1980). The effect
is analogous to the control of width of tree rings by air tempera-
tures (Briffa et al. 1995) and to the differential density banding in

226

Shepherd et al.

Shell Length
(mm)

TABLE 5.
Number of rings and growth checks in shells of H. kamtschatkana of known or estimated age.'

Age (y)

No. of No. of Checks

Rings (Inferred + Counted)

Comments

103

10

10

4 + 8

103

12

12

1 + 12

106

15

8?

2+12

98

17

18

3+10

96

18

13?

2+11

98

19

2+17

102

19

20

2+15

99"

10.2 +

0.3

10

114"

10.8 ±

0.3

11

Two extra brown rings excluded
Two extra brown rings excluded
Spire partly destroyed by erosion and parasites
Two extra brown rings excluded. Eroded in part
One extra brown ring excluded. Spire partly destroyed
Spire completely destroyed
Two extra brown rings excluded
Shell from Dempster Island. B.C.

Shell from Dempster Island, B.C.. remnants of two rings seen at eroded
margin of spire near suture line

" The first seven shells were held in aquaria at Seward, Alaska until they died. The presence of growth checks (where not visible in the expected position
to age 3.5 years) was inferred; i.e. 4 + 8 means 4 rings were inferred and 8 later ones counted.

" Both tagged abalone at liberty for 5 y. Age at tagging was estimated from a mean growth rate of 15.9 nim/y derived from the regression of the number
of rings versus SL for a sample of shells (N = 21) from the tagging site, assuming that one ring per year was laid down.

corals due to seasonal sea-temperature changes (Dodge and Lang
1983).

Shell Aging

A number of studies have shown that rings can be reliably used
to age the shell of various species of abalone (Prince et al. 1988.
Erasmus et al. 1994, Shepherd et al. 1995a, Shepherd et al. 1995b.
Shepherd and Avalos-Borja 1997, Shepherd and Huchette 1997.
Shepherd and Triantafillos 1997, Shepherd and Turrubiates 1997).
On the other hand, only four species are known to lay down growth
checks that are useful for aging (reviewed by Day and Fleming
1992, Shepherd et al. 1995b). Previously Shepherd and co-authors
(cited above) have considered that ring counts may not give a
precise age for each individual shell due to apparent random vari-
ability in the deposition rates between individuals, although they
do give a valid estimate for a population. The low incidence of
clear inconsistency (-2%) in estimates of age between the two
methods increases confidence in each and suggests that the rate of
deposition of rings is relatively precise for individuals of this spe-
cies, although slight ambiguity in interpreting structures exists
with both methods. Thus, ring deposition satisfies the criteria pro-
posed by Day and Fleming (1992) that deposition rates must be
consistent and that deposition is at about the same time each year.
Similarly, in species thai lay down growth checks, a small per-
centage of shells fail to do so in any year. The use of both inethods
in the pinto abalone has the advantage that each method provides
an independent test of the accuracy of the other, since both rings
and growth checks are laid down according to different external
cues.

On the question of whether horizontal or vertical sections at the
shell spire give belter readings, we note that, although the two
kinds of section show spatial effects in different planes, the read-
ings from each are comparable (see Shepherd et al. 1995). We
prefer horizontal sections for the practical advantages of ease of
preparation and reading (especially fine rings). An advantage of
vertical sections is that brown and compound rings are more easily
distinguishable under higher magnification.

Rings appear to be laid down during maximum summer tem-
peratures or spawning (or both) as Shepherd and co-workers have
found for Mexican and Australian species. Conversely, growth
checks are apparently deposited during mininiuin temperatures, as

is known for other cold-temperate species (Forster 1967, Sakai
1960, Poore 1972) and one curious subtropical species (Shepherd
et al. 1995b).

The exclusion of brown rings from counts for aging purposes is
rarely problematic. Brown rings can most readily be distinguished
in horizontal sections by the cavities that commonly surround them
(caused by the rasping process, which preferentially excavates
softer organic matter) and by their rupture of the pattern of depo-
sition of rings. Color alone is ambiguous because of the frequent
occurrence of brown rings in juxtaposition to prismatic rings at
least over some part of the length of the latter. Usually, the pris-
matic and organic elements of a compound ring can be seen at
about x20 magnification or more.

We do not exclude the possibility of using growth checks to
estimate the age of shells > -4 y, but we simply point out that
ambiguity in identifying checks seems to increase with age and
may nullify the benefit of an independent aging method. We note
that a few shells of known age had fewer than the predicted num-
ber of growth checks (Table 5), suggesting the possibility of some
bias. We also observed that growth checks were less conspicuous
in shells from British Columbia than in those from Alaska. Pos-
sibly higher sea-temperature minima in British Columbia cause a
briefer recession or no cessation of winter growth. Another source
of bias in aging abalone is through the (usually) combined effect of
parasites, which cause deposition of brown rings, and shell ero-
sion. This was not problematic in this study becau.se few pinto
shells had brown rings in the length range of interest.

Growth Rates

Our study provides the most detailed information on the growth
of the pinto abalone in Alaskan waters. The growth rate of this
abalone is poorly known in the first 4 y of life but is better known
for older individuals in British Columbia waters (reviewed by
Sloan and Breen 1988). Paul et al. (1977, Fig. 4) estimated a
growth rate of -17 mm/y in the laboratory for individuals main-
tained at 12-I5°C, a rate later confirmed in the laboratory by Sloan
and Breen ( 1988). The latter authors presented an equation relating
the growth rate of .30 mm SL abalone with water temperature. The
mean annual sea surface temperature range at Sitka is ~4.5-l4°C
(Standley 1987), and the monthly mean is 8.2°C. This would be
somewhat higher than the mean temperature at 5-10 m depth

The Pinto Abalone

227

where this abalone lives. Application of their formula gives a mean
growth rate of 18.0 mni/y. which is slightly greater than those
found in this study. A study by Larson and Blankenbeckler ( 1980).
which is cited in Sloan and Breen (1988). found that the growth
rate decreased with increasing size. Annual length increments av-
eraged 19.1 mm for individuals having < 50 mm SL. 12.6 mm for
those of 50-74 mm SL, 6.2 mm for those of 75-99 mm SL. and 4.3
for those of > 100 mm SL. Quayle (1971) estimated that the pinto
abalone had a 35-mm SL at age 2 y and thereafter grew at a rate
of 10 mm/y. Fournier and Breen (1983) estimated that growth rates
from 1-5 y ranged from 11-16 mm/y at two sites; these estimates
w ere based on the decomposition of "snapshot" length-frequency
data into annual modes. Our data are more accurate than these
estimates but are within the same range.

Like Fournier and Breen (1983), we do not have direct infor-
mation on the growth rate of this species in the first year. An
alternative hypothesis is that the initial mode we found is of 2-y-
old abalone. We discount this possibility because of known labo-
ratory growth rates in the first year cited above, and also because
we could detect animals down to at least 10 mm SL on crustose
corallines. If another mode were present, we should have seen it.

Von Bertalanffy growth parameters for Alaskan abalone are
remarkably close to those in British Columbia. Sloan and Breen
(1988) reviewed growth at nine sites in British Columbia. Exclud-
ing the values for Lyell Island because there were few tag recov-
eries there, they recorded for eight sites a mean K value of 0.24
(range 0.16-0.24) and a mean L-_. value of 120.1 mm (range 95.2-
137.3 mm) compared with our mean K value of 0.20 and L^_ value
of 126.7 mm for seven sites (Table 4). While the two sets of values
are not strictly comparable because the values of Sloan and Breen
are derived from length-increment data, whereas ours are from
length-at-age data (see review of Day and Fleming 1992), they
clearly show close similarity.

What is the cause of the differences in growth between sites?
Breen (1980) considered that the growth rate of the pinto abalone
was related to the food supply. Abalone in sheltered to semie,\-
posed habitat in Macrocystis or Nereocystis forests grew faster and
to a larger size than those in habitats dominated by the unpalatable
kelp Pterygophora and exposed to ocean swell. All of our sites
were in moderately to highly sheltered habitats, usually in places

of moderate tidal current. The habitats were variously dominated
by Macrocystis, Nereocystis, Agarum and Laminaria cf saccha-
rina. Grant Cove at the northern end of Gravina Island was ex-
posed to local seas to the north, and Jumbo Island, far up Hetta
Inlet, was the most sheltered. There was little variation in growth
between our sites, and we attribute this to the overall similarity of
habitat.

Ring counting is an appealing alternative to tag-and-recapture
methods for obtaining growth rates, which are important for regu-
lating fisheries (Troynikov and Gorfine 1998). Ring counting is
cost-effective, requiring only one sampling event, and does not
negatively affect growth as tags may (reviewed by Day and Flem-
ing 1992). Ring counting also provides length-at-age data, and not
simply increments at size, and. with catch-curve analysis can pro-
vide estimates of the total mortality rate of a population. The
present application has provided the first broad geographic depic-
tion of abalone growth rates in Alaska. These data are an important
component of the stock assessment program that must precede any
future reopening of the Alaskan fishery after its collapse in the past
2 decades (Woodby et al. in press).

ACKNOWLEDGMENTS

The first author is grateful to the Alaska Department of Fish
and Game for the provision of accommodations, facilities, and
logistic support during his stay in Alaska and to Centro de Ciencias
de la Materia Condensada for the provision of accommodations
and laboratory facilities at Ensenada, Mexico. Dr. H. Echavarria
kindly provided computing facilities at Centro de Investigacion
Cientifica y Escuela Superior de Educacion. We thank Robert
Larson, Craig Sempert, Scott Walker, Marc Pritchett, Kyle Hebert,
and Dave Barto for diving assistance, Tom Brookover for field
assistance, Peter Hagen and Kristen Munk for technical advice,
and Cori Cashen for preparation of Figure I . Israel Gradilla gave
generously of his time for SEM studies and took the micrographs
in Figure 4, and G. Vilchis took the photographs in Figures 3 and
8. Dr. A. J. Paul of Seward Marine Centre made available shells
for aging, and Dr. A. Campbell provided shells from British Co-
lumbia. Dr. Rob Day and referees helpfully criticized the manu-
script.

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Journal of Shellfish Reseiuvh. Vol. 19. No. 1, 229-231. 2000.

PREDATION OF THE INVASIVE FRESHWATER MUSSEL LIMNOPERNA FORTUNEI

(DUNKER, 1857) (MYTILIDAE) BY THE FISH LEPORINUS OBTUSIDENS VALENCIENNES,

1846 (ANOSTOMIDAE) IN THE RIO DE LA PLATA, ARGENTINA

P. E. PENCHASZADEH,' G. DARRIGRAN," C. ANGULO,'

A. AVERBUJ,' M. BROGGER,' A. DOGLIOTTI,' AND N. PIREZ'

'De/7. C. Biologicas, FCEyN, UBA:

Ciudad Universitaria, Pab. II, Nunez,

Piso 4o., Buenos Aires. MACN-CONICET. Argentina
^FCN y Museo de La Plata.

Paseo del Bosque s/niimero,

La Plata. Argentina

ABSTRACT A study of the presence of the freshwater mussel Limnoperna foitunei in the diet of the native fish Leporiims obtusidens
was performed in Costanera Norte. Buenos Aires. Fish were collected monthly for a 1-year period to analyze their digestive tracts.
From a total of 157 tlsh collected (20-55 cm total length), 21 had empty digestive tracts. Of the remaining 136 individuals. 98 (72.1%)
contained fragments o{ Limnoperna shells. The mussel represented 14.5% of the stomach content dry weight and 44.4% of the intestinal
content dry weight. Limnoperna was present in almost all fish over 30 cm (total length). The largest number of mussels ingested by
a Leporinus individual was 77, estimated by mussel beak count. Mussels 15-20 mm in shell length to be the most abundant in
Leporinus digestive tract.

KEY WORDS: Invasive mussel. Limnoperna control, fish predation

INTRODUCTION

Limnoperna forlnnei(DunkeT 1857) arrived in Ri'o de la Plata,
Argentina, in the early 1990s (Pastorino et al. 1993). The mussel
attaches to any available hard substrate using byssal threads and
can form dense aggregations. The rapid expansion and the high
densities achieved in the Rio de la Plata and the Parana basin
(80.000 individuals per square meter recorded in 1993, Darrigran
and Pastorino 1995; and more than 100.000 in 1995, Darrigran et
al. 1998) give the impression that L. fortunei has not encountered
local competitors, predators, or parasites.

A counter example is the Zebra mussel, Dreissena polymorpha
(Pallas 1754), which invaded North America in the mid- 1980s. In
laboratory conditions small Zebra mussels (< 8 mm in shell length)
are preyed upon by Crayfish (Love and Savino 1993, Maclsaac
1994). and by the blue crab in estuarine conditions (Molloy et al.
1994). Several fish have been reported as significant predators of
the Zebra mussel (French and Bur 1993, Marsden 1997, Tucker et
al. 1996).

In Argentina, Darrigran and Colauti ( 1994) reported the impor-
tance of the native fish Pterodoras granulosus (Valenciennes
1833) as a predator on Corbicula fluminea (Miiller 1774), another
invasive freshwater species.

To identify possible predators of Limnoperna fortunei in the
Rio de la Plata, an analysis of fish gut contents caught in Costanera
Norte, Buenos Aires was performed. Preliminary results showed
that native Leporinus obtusidens, as well as being the dominant
catch was a relevant predator of Limnoperna fortunei (Penchasza-
deh et al. 1998). This paper is a specific study of the presence of
the L. fortunei in the diet of L. obtusidens.

Leporinus obtusidens ("boga" is the common local name) has a
small conic head, rounded snout, well-developed lips, and small
frontal teeth in the mouth. Each maxillary has a single row of
chisel-shaped teeth pointing forward, the two anterior being longer
than the others: the name of the genus refers to this characteristic,
this kind of dentition is reminiscent of leporinid rodents.

MATERIAL AND METHODS

Fish were collected form sports fishermen from March 1998 to
February 1999 in Costanera Norte, Rio de la Plata, 34°34'S,
58°23'W; a location heavily colonized by Limnoperna. The tidal
regime is mixed, although predominantly semidiurnal with a tidal
range of 0.63 to 1.07 m. Water levels and currents are strongly
influenced by meteorological conditions, mainly wind direction
and intensity, the most important of which is the "Sudestada." This
is characterized by a gradual and persistent increase in wind speed
blowing from the SE to the SSE, during which time, winds can
pick up to around 25 m/sec (Guerrero et al. 1997). Heavy rain in
the Parana basin can also strongly influence water levels. Mean
surface water temperature values for Costanera Norte are: summer
(January to March) 24.07 + 2.01 °C; fall (April to June) 13.83 ±
4.68; winter (July to September) 14.63 ± 2.00 and spring (October
to December) 2 1 .04 ± 0. 1 1 .

Fish length was measured to the nearest 1 cm and then dis-
sected in situ. The entire digestive tract was removed and pre-
served in a 10% formalin solution. In the laboratory, stomach and
intestine contents of each Leporinus were analyzed under a dis-
secting microscope and separated into Limnoperna shells and other
material. Then, the dry weight (80 °C until constant weight was
achieved) of the two groups was taken.

Limnoperna beaks (anterior portion of the valve containing the
umbonal region) were counted, the total number then divided by
two was the estimated number of Limnoperna contained in each
fish. To estimate the length of the mussels consumed, Limnoperna
individuals were collected from the same fishing site in January,
1999. These mussels were then measured and separated into four
size ranges. Ten mussels from each category were weighed, and a
proportion of shell length to shell weight was obtained for each
category. The total shell weight in the digestive tract of each fish
was then divided by the number oi Limnoperna eaten, estimated by
the beak count.

229

230

Penchaszadeh et al.

RESULTS

DISCUSSION

Of the !57 Leporiniis analyzed, the digestive tracts of 21 were
empty. Of the remaining 136 individuals, 98 (72.1%) had frag-
ments of Limnopenia shells in the stomach, the intestine, or both
(Table 1). Limnopenui shells represented 14.5% of the stomach
content dry weight and 44.4% of the intestinal content dry weight.
Considering the entire digestive tract, 33.4% of the content dry
weight was Limnoperna shells. The digestive tracts did not show
damage or bleeding, although they were sometimes fully packed
with shell fragments.

Limnoperna was present in the diet oi Leporiniis during almost
the entire year of sampling. Lowest values occurred during winter
(June-August) when mussel shells were found in only 40. 14. and
0% of analyzed fish, respectively (Table 1 ). Winter was also the
season of lowest Leporiniis catch in the study area.

Except for six individuals, Limnoperna was present in all fish
(47) over 30 cm (total length) with material in their digestive tract.
The presence of Limnoperna in the Leporiniis digestive tract was
maximum during the period from October to February (83 to
100%).

The largest number of mussels ingested by a Leporiniis indi-
vidual was 77 (beak count method). According to the shell length-
weight proportion found (Table 2), the 10 fish with the largest
number of ingested mussels contained various sizes of prey. The
mean shell weight ranged between 0.01 2-0. 020g (<I0 mm of shell
length. 20%), 0.0.V0.068g (10-15 mm of shell length: 70%) and
0.175 g (15-20 mm of shell length; 10%). These results indicate
that Limnoperna of between 10-15 mm in shell length were the
most abundant in Leporiniis digestive tract.

In 36 cases, entire Limnoperna individuals with unbroken
shells and intact soft tissue were found in the digestive tract of
Leporiniis. Mussels measured between 1.5 and 5.7 mm in shell
length and fish ranged 26.0 to 33.0 cm (total length).

Aquarium observations (M. Brogger, pers. comm.) show that
Leporiniis do not always remove the entire mussel from its sub-
strate; on several occasions fragmented mussels with the beak area
still attached to the substrate were observed. Other mussels >30
mm had bite marks on the periostracum.

Before the Limnoperna invasion there were few published re-
ports on Leporiniis obtiisidens diet, and almost all of these are
confined to the middle Parana river region (Mastrarrigo 1950, De
Occhi and Oliveros 1974).

Mastrarrigo ( 1950) defined the alimentary regime oi Leporiniis
as mainly omnivorous, with a high proportion of aquatic vegeta-
tion (in the case of at least 72% of the fish analyzed in Rosario,
middle Parana), but also mentioned the presence of a small pro-
portion of fragmented river snails in the gut. Snails were referred
to as the main food for Leporiniis in the Uruguay river (Guale-
guaychii, Entre Ri'os), where their digestive tracts are often full of
shell fragments. Mastrarrigo (1950). commented that local fisher-
men called them "bogas caracoleras" ("snail-eating bogas").

According to De Occhi and Oliveros (1974), mollusks were
present in 32% of the examined Leporiniis (5% bivalves and 27%
gastropods, basically Helobia sp.; however, seeds and fruits were
always dominant (37%). They believe that in Leporiniis ohliisidens
because of the disposition of the pharyngeal teeth, these are used
only to crumble soft material, because they lack crushing surfaces;
whereas, fragmentation is achieved by action of the oral teeth.

Aquarium observations show that Leporiniis did not always
ingest the entire prey; instead, they bit off only a portion of the
mussel (the bivalve was not completely removed from the sub-
strate on many occasions). These observations suggest that there
could be a underestimation in the amount of ingested mussels
calculated by the beak counting method. This also could lead to an
overestimation in the calculated size of ingested mussels.

The weight oi Limnoperna in the fish digestive tracts could also
be underestimated, because the soft material was weighed as a
whole (with certain contribution of mussel tissue).

Differences observed in the amount of Limnoperna shell found
in the stomach content (14.5%) and intestine content (44.0%)
could indicate that Leporiniis is preferentially an early-morning
feeder, because all of the studied material was captured between
12:00 m. and 5:00 p.m.

The most abundant Limnoperna sizes in Leporiniis digestive
tracts were 10-15 mm in length. According to Boltovskoy and
Cataldo (in press), who estimated Limnoperna growth in experi-

TABLE 1.

Presence of the freshwater mussel Limnoperna fortune! in the digestive tract of the fish Leporinus obtiisidens in Costanera Norte, Buenos

Aires (1998-1999).

Fish Length (cm)

Digestive Tract
with Contents ( % )

Presence of L. fortunei
in the Contents (%)

Month

n

Mean

SD

Range

March

14

26.8

3.6

2U-33

92.86

69.23

April

13

28.9

8.6

20-55

76.9

60

May

41

26..5

5.1

10.5-43

75.6

70.96

June

6

27.1

3.2

21-30

83.4

40

July

8

26.5

1.7

24-29

87.5

14.28

August

2

27.5

4.9

24-31

50

0.0

.September

16

28.6

4.0

22-37

86.7

53.85

October

8

31.6

2.7

28-35

87.5

100

November

7

27.1

i.A

17-33.5

85.7

83.3

December

18

30.8

4.2

22..'>-37

100

94.4

January

17

31.8

4.4

24-39

100

88.2

February

7

27.9

4.1

20-33

100

85.7

Total

157

28.4

5.1

10..'i-55

86.6

72.1

LlMNOPERNA FORWNEI PREDATION BY NATIVE ARGENTINEAN FiSH

231

TABLE 2.

Proportion of shell length to shell weight for four size-range groups
of Limnoperna fortunei.

Shell length (mm)
(anterior to posterior)

5-10

10-15

15-20

20-25

Shell weiehi {&)

0.022

0.067

0.172

0.317

mental conditions, these sizes correspond to mussels 3 to 6 months
old.

The presence oi Limnoperna. with its extraordinary abundance,
occupying a seemingly empty niche in the Plata basin, has intro-
duced a new element in the diet of some fish and constitutes the

main food item for Leporimis obtusidens. Although other predators
have yet to be identitled, the "boga" has proved to be an important
natural enemy of the invading mussel. Limnoperna fortunei.

ACKNOWLEDGMENTS

Our thanks are given to the following persons for their invalu-
able assistance: Dr. Hugo Lopez. Museo de La Plata, who kindly
identified the fish; the sports fishermen who donated their catch;
Nora Brignoccoli who helped in the laboratory; Dr. Paula
Mikkelsen who had kindly read an earlier version of the manu-
script; and Claudia Penaloza for her English translation. We also
thank the two anonymous reviewers who improved upon an earlier
version of this paper. Project PICT 07-03453 and Fundacion An-
torchas, Argentina, partially supported this research.

LITERATURE CITED

Boltovskoy, D. & D. H. Cataldo. 1999. Population dynamics of Limno-
perna forumei. an invasive fouling moUusk, in the lower Parana River
(Argentina). Biofouling I4{3):255-263.

De Occhi & O. B. Oliveros. 1974. Estudio anatomico-histologico de la
cavidad bucofaringea de Leporinu.s obuisidens Valenciennes y su rela-
cion con el regimen alimemario (Pisces. Tetragonopteridae). Physis
Secc. B, Buenos Aires 33:79-90.

Darrigran, G., S. Martin, B. Gullo & L. Armendariz. 1998. Macroinverte-
brates associated with Limnoperna fortunei (Dunker 1857) (Bi-
valvia. Mytilidae) in Rio de la Plata. Argentina. Hydrobiologia 367:
223-230.

Darrigran. G. & G. Pastorino. 1995. The recent introduction of the Asiatic
bivalve. Limnoperna fortunei (Mytilidae) into South America. Veliger
38:183-187.

Darrigran. G. & D. Colauti. 1994. Potencial control del molusco invasor
Corhicula fluminea (Miiller. 1774) en el Ri'o de la Plata. Comunica-
ciones Sociedad Malacologica del Urngiiay 7:368-373.

French. J. R. P. Ill & M. T. Bur. 1993. Predation of the zebra mussel
(Dreissena polymorpha) by freshwater drum in western Lake Erie.
pp.453^64. In: T. E. Nalepa and D. W. Schoesser (eds.). Zebra Mus-
sels: Biology. Impacts, and Control. Lewis Publishers. Boca Raton. FL.
810 pp.

Guerrero. R.. C. Lasta. E. Acha. H. Mianzan & M. B. Framiiian. 1997.
Atlas Hidrografico del Rio de la Plata. Comision Administradora del
Rio de la Plata-INIDEP. Buenos Aires-Montevideo. 109 pp.

Love. J. & J. F. Savino. 1993. Crayfish (Orconectes virilis) predation on

zebra mussels {Dreissena polymorplia). J. Freshw. Ecol. 8:253-259.
Maclsaac. H. J. 1994. Size selective predation on zebra mussels I.Drei.^sena

polymorpha) by crayfish (Orconectes propinquus). J. Nortli Am.

Bentholog. Soc. 13:206-216.
Marsden, J. E. 1997. Common carp diet includes zebra mussels and lake

trout eggs. / Fre.sim: Ecol. 12:491-492.
Mastrarrigo. V. 1950. La boga. contribucion a su conocimiento biologico.

Almanaque del Ministerio de Agrictdtura y Ganaderia. Buenos Aires

25:417^26.
Moloy. D. P.. J. Powll & P. Ambrose. 1994. Short-term reduction of adult

zebra mussels {Dreissena polymorpha) in the Hudson River near

Catskill, New York: an effect of juvenile blue crab iCallinecles sapi-

dus) predation. J. Shellfish Res. 13: 267-371.
Pastorino. G., G. Darrigran. S. Martin & L. Lunaschi. 1993. Limnoperna

fortunei (Dunker 1857) nuevo bivalvo invasor en aguas del Ri'o de la

Plata. Neotropica 39:34.
Penchaszadeh. P. E.. A. Dogliotti. N. Pirez & G. Darrigran. 1998. Depre-

dacion del bivalvo invasor Limnoperna fortunei (Dunker) (Mytilidae)

por el teleosteo autoctono Leporimis obtusidens Valenciennes (Anos-

tomidae). en el Ri'o de la Plata. Argentina. Libro Res. XII Congreso

Nacional de Malacologia, Malaga, Espaiia:44-45.
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(Cyprinus carpio). J. Freshwater Ecol. 1 1:363-372.

iJ

Journal of Shellfish Research. Vol. 19. No. 1. 233-240. 2000.

EMERSION AND THERMAL TOLERANCES OF THREE SPECIES OF UNIONID MUSSELS:

SURVIVAL AND BEHAVIORAL EFFECTS

MICHELLE R. BARTSCH,' DIANE L. WALLER,'
W. GREGORY COPE/ AND STEVE GUTREUTER'

'(7.5. Geological Sun^ey
Biological Resources Division
Upper Midwest Environmental Sciences Center
2630 Fanta Reed Road. La Crosse. Wisconsin 54603

'Department of Toxicology
North Carolina State University
Box 7633. Raleigh. North Carolina 27695

ABSTRACT We evaluated the behavior and survival of unionid mussels after emersion in air temperatures across a range that is
likely to be encountered during status surveys or relocations. Five laboratory tests were performed with pocketbook Lampsilis cardium
Rafmesque {2 tests), pimpleback Quadrula pusrulosa Lea (1 test), and spike Elliptio dilataia Rafmesque (2 tests) mussels, each
conducted in a completely randomized, nested experimental design. For each mussel species (except Q. pustulosa). treatments tested
included two water temperatures (25 and 10 °C). five air temperatures (ranging within ±20 °C of the water temperature), three aerial
exposure durations (15, 30, and 60 min), and a no emersion control. All treatments were duplicated, with 10 organisms per emersion
time and aerial exposure temperature (n = 320 mussels per test). Behavioral response (ability to upright) and mortality were measured
daily for 14 d postemersion. Both water and aerial exposure temperature (air shock) were important predictors of times to first
uprighting. The intensity function of first uprighting differed among species (/> < 0.01 ), and there was a significant interaction between
E. dilatara versus the other species and water temperature (P < 0.01). Over-all mussel survival after emersion was high (939c ); however,
E. dilatata experienced significant treatment related monality at the 25 °C test water. 45 °C aerial exposure temperature. Because of
the high incidence of uprighting and survival of mussels in our study, emersion at moderate temperatures (15 to 35 °C) and durations
(15 to 60 min) does not seem harmful to mussels, and, therefore, conducting relocations and status surveys under these conditions
should not impair mussel survival and over-all success.

KEY WORDS: Unionid mussel, conservation, emersion, temperature, behavior, mortality

INTRODUCTION

The imperiled status of unionid mussels (Williams et al. 1993)
has prompted conservation efforts by public and private natural
resource agencies that include status surveys, restocking, and re-
location. The effects of collection and handling on mussels in field
studies are generally considered benign and inconsequential to
mussels relative to most threats (construction, zebra mussel infes-
tation, habitat loss). However. Cope and Waller (1995) reviewed
the success of relocation projects and found that mortality of mus-
sels after relocation can be significant (>70% in 30% of projects
reviewed). Mortality was highest within 1 year of the event, sug-
gesting that effects of collection, handling, and displacement of
mussels may be greater than were previously considered. The en-
vironmental conditions that mussels experience during collections
and surveys may contribute to low survival, but can also be con-
trolled to some extent. Determination of the emersion and thermal
tolerances of unionid mussels would provide guidelines on the
conditions in which surveys and relocations should occur to en-
hance mussel survival and over-all success.

Past studies suggest that mussels can tolerate emersion for
hours or even days (Byrne and McMahon 1994. Dietz 1974, Hol-
land 1991, Schanzle and Kruze 1994, Waller et al. 1995). How-
ever, survival of mussels is related to such environmental condi-
tions during emersion as relative humidity and air temperature. For
example. Waller et al. (1995) emersed Amblema plicata plicata
Say and ObUquaria reflexa Rafmesque for a maximum of 8 h and

found that mussels had greater survival when handled during the
fall (water temperature -15 °C: air temperatures ranged from 12 to
25 °C) compared to those handled during the spring (water tem-
perature -23 °C; air temperature ranged from 18 to 29 °C). In the
present laboratory study, we augment these data by evaluating a
range of extreme air temperatures and water-air thermal differen-
tials. We selected the minimum and maximum water and air tem-
perature and emersion times based on conditions likely to be found
in field collecting situations. In addition to survival, the uprighting
behavior of mussels after emersion was selected as a potential
indicator of emersion stress; presumably, the ability to upright and
burrow into the substratum indicates normal functioning. Waller et
al. (1999) found significant species and water temperature related
differences in the uprighting and movement intensity of four mus-
sel species after displacement. Thus, displacement, coupled with a
thermal and emersion challenge, may also produce significant be-
havioral changes.

In this study, we evaluated the effects of emersion and tem-
perature on the survival and behavior of three mussel species
Lampsilis cardium Rafinesque (pocketbook). Quadrula pustulosa
Lea (pimpleback), and Elliptio dilatata Rafinesque (spike), and
examined the variation in survival and behavioral response within
and among the three species. These mussel species represent two
subfamilies (Lampsilinae and Ambleminae) and two contrasting
life history strategies (long-term and short-term brooders) within
the Unionidae. Additionally, L. cardium and Q. pustulosa served
as surrogates for two U.S. Federally Endangered species, the L.

233

234

Bartsch et al.

higginsi Lea (Higgins" eye) and Q. fragosa Conrad (winged
mapleleaf). both found in the Upper Mississippi River basin. El-
liptio dilatata was chosen as a second surrogate for Q. fragosa.
because too few Q. pustulosa were available for testing at low
(10 °C) water temperature.

MATERIALS AND METHODS

Test Organisms

Three species of unionid mussels were collected from the Wolf
River at Shawano, Shawano County. Wisconsin. Mussels were
transported in holding tanks, containing Wolf River water (25 °C),
to the Upper Midwest Environmental Sciences Center, in La
Crosse, Wisconsin. Holding tank water temperatures were main-
tained at 25 ± 3 °C (with addition of nonchlorinated ice as needed),
and the dissolved oxygen concentration was maintained at >60%
saturation with aeration. Water temperature and dissolved oxygen
(Yellow Springs Instrument Model 58 oxygen meter) were mea-
sured at 1-h intervals. At the laboratory, mussels were placed into
submerged cages held in the Black River (water temperature,
27 °C), near La Crosse, Wisconsin until study initiation. The mus-
sel cages (122-em length x 122-cm wide x 46-cm height) were
constructed of angle and strap iron frame with netting (1.9-cm
diam. polyethylene) attached to the iron frame by tie wraps and
nylon rope. One species of mussel (111 total; density of 75/m-)
was placed into each cage. During collection, transport, and allo-
cation to cages, mussels were continually immersed in river water.

Experimental Design and Exposure System

Five laboratory tests were performed with L cardiiim (2 tests),
Q. pustulosa (1 test), and E. dilatata (2 tests), each conducted in a
completely randomized design as a nested experiment. For each
mussel species tested (except Q. pustulosa), there were two water
temperature treatments (25 and 10 °C), five air temperatures (rang-
ing within ± 20 °C of the water temperature), three aerial exposure
duration treatments (15, 30, and 60 min), and a no emersion con-
trol treatment (Fig. 1 ). Because of limited availability, Q. pustu-
losa was tested only at 25 °C, the treatment we assumed to be more
lethal. All treatments were duplicated, with 10 organisms per em-
ersion time and temperature (n = 320 mussels/test), for a total of
32 experimental units. Ten mussels were placed into a flow-

AcclimaOon water temperature
25°C(10)

Emersion air temperature {"C)

15(0) 20(5) 25(10) 35(20) 45(30)

rr

Emersion duration (min)

15

30

60

Control

no emersion

Figure \. Experimental design for the thermal and emersion exposure
tests with three species of unionids. The numhers in parentheses rep-
resent the 10 °C test water-aerial exposure regime.

through, stainless steel tank (61 -cm length x 30-cm wide x 36-cm
height) containing sand (13 ± 0.5-cm depth) and 42 ± 0.5 L of
overlying well water. Each tank was placed into one of six water
baths (305-cm length x 84-cm wide x 46-cm height) maintained at
the test temperature (10 or 25 ± 1 °C) with a thermostatically
controlled, liquid circulation pump (Remcor Model CFF-501,
Remcor Products Co., Franklin Park, IL) connected to the water
bath. Tanks were aerated to maintain dissolved oxygen concentra-
tions at >60% saturation. The flow rate of water into each tank was
200 mL/min with a turnover rate of seven times per day. The
photoperiod was 16-h light and 8-h dark.

Laboratory tests began when the water temperature of the
Black River reached the desired test water temperature (25 °C:
July/August and 10 °C; November/December). Each mussel spe-
cies was tested individually, and a given species was transported in
coolers containing Black River water to the laboratory for testing.
Ten mussels were randomly selected for each experimental unit.
Plastic mesh netting was placed on top of the sand substrate to
prevent mussels from burrowing into the substrate before aerial
exposure. Mussels were acclimated in their respective tanks at a
water temperature of 25 ± 1 °C for 2 d; mussels were not fed
during the acclimation period. Mussels within a replicate were
numerically marked (1 to 10) on their right valve with a permanent
marker. To enable identification of mussels after burrowing into
the substrate, each mussel was uniquely tagged with a numbered
fishing bobber (3.81-mm dia.) that was attached to a 22.9-cm piece
of cotton thread and secured to the umbonal region of the right
valve with cyanoacrylate (Krazy Glue® Gel. Borden, Inc., Colum-
bus, OH). Both siphons (incurrent and excurrent) remained im-
mersed while bobbers were being attached to the shells; total han-
dling time was less than 3 min per mussel.

For each treatment, 20 mussels (10 from each replicate) were
removed from the test water (25 or 10 °C), transported in water
(held at test temperature), and placed into an environmental cham-
ber (Hotpack® Biological Chamber. Hotpack Corp.. Philadelphia,
PA) at a given air temperature (25 °C water temperature; 15, 20,
25, 35, 45 °C air temperature; 10 °C water temperature; 0, 5, 10,
20, 30 "C air temperature) for a duration of 15. 30. or 60 min.
Treatments were conducted in order of increasing air temperature
and emersion duration. The target relative humidity in the envi-
ronmental chambers was 60 ± 57c. This relative humidity was
selected based on average ambient air conditions experienced in
our geographic region (Steve Thompson. National Oceanic Atmo-
spheric Administration. La Crosse. WI, pers. comm.). Following
emersion, mussels were removed from the environmental cham-
ber, transported in well water (held at the test temperature, 25 or
10 °C), and returned to their respective tanks. Each mussel was
placed directly on top of the sand substrate, with the right valve
(tagged side) facing upward. Test organisms were fed a mixture of
C4 algae diet (Coast Seafoods Co., South Bend, WA; 0.2 niL per
mussel) and dry Chorclla (0.013 g dr> weight per mussel) daily.
Mussels were monitored for mortality and uprighting response for
14 d postemersion. At test termination (14 d postemersion), mus-
sels were recovered from each tank and measured for total length
and whole mussel wet weight. Sex of L. cardiwn was determined
by shell dimorphism. Elliplio dilatutu and Q. pustulosa are not
sexually dimorphic; thus, we examined histological sections of
half of the mussels from each replicate in the 25 °C test to deter-
mine the sex ratio, and assumed animals tested at the 10 °C water
temperature had a similar sex ratio, because all mussels came from
the same population and were randomly sampled.

Emersion and Thermal Effects on Mussel Survival

235

Statistical Analyses

For each mussel species, we examined patterns between two
response variables, times to first uprighting and death, as a func-
tion of water temperature (°C). duration of aerial exposure, and air
shock temperature CO. which we define as the difference between
w ater temperature and air emersion temperature. We refer to both
first uprighting and death as events, and our primary data consist
of elapsed times to occurrences of those events for each mussel.
Some event durations may have exceeded the study duration ( 14 d)
and, therefore, went unobserved; these events are said to be "right-
censored" (Hosmer and Lemeshow 1999). Proper accommodation
of censoring is critical to valid interpretation of time-to-event data.
For both events (first uprighting and death), we used the Cox
proportional hazards regression model (Cox 1972. Newman 1995.
Hosmer and Lemeshow 1999) to identify factors that explained the
pattern in uprighting and survival. We arbitrarily selected L car-
diiim as the baseline species for our analysis; this choice does not
affect the over-all results. The ba.seline temperature was 0 °C. and
other temperatures were coded as deviations from this baseline.
Denote E = U and E = D for the events uprighting and survival,
respectively. Our full regression models for both first uprighting
and survival are

\^,(,) = \£(,(r)exp(5, -t- P,r-H P^A + P,M + PjA- + (3,M- + P,,r
+ ^2.A + Ps.,-^ + Pe^A + ^-,TM + PsAM -I- P^rA" + ^^,A'
+ Pft',TA -I- pV.TM + ^sAM + ^uTAM + P, ..JAM). ( 1 )

where; X^/r) is the hazard function for event type E for the /th
species at time f; XfoW 's the corresponding baseline hazard; 5,/ =
I. 2. are two fixed-effects parameters for identification of the three
species (5, denotes E. dilatata. and S, denotes Q. piistiilosa): T
represents water temperature (°C) with coefficient P,; A represents
air shock temperature (°C). which we define as the difference
between water and air emersion temperatures with coefficient p,;
M represents air exposure duration (min) with coefficient p,; A'
and M- are quadratic (u or n-shaped) effects of air shock and
exposure duration, respectively; P|,r represents the species x wa-
ter temperature interaction; P-,y4 represents the species x air shock
interaction; P^.M represents the species x air-exposure duration
interaction; TA. TM, and AM are two-way interactions among 7". A
and M: TA' is the interaction between T. and the quadratic effect
of A; P4.,A- is the interaction between species and A;
Pf, ,TA. ^j,TM, and Pg/IM represent three-way interactions
among species, A. T, and M: TAM is the three-way interaction
between T.A. and M: and p,, ,7AM is the four-way interaction
among species. 7". A, and M. In this model, "interactions" are on
the log scale. Although in such terminal events as death, it is
customary to refer to X(f) as the hazard function for deleterious
events such as death, the term intensity is more appropriate than
hazard for events such as first uprighting. Therefore, we refer to
\(t) as either the hazard or intensity function, depending on wheth-
er we are addressing survival or first uprighting. respectively. We
fitted Eq. (1 ) to the uprighting and survival data by maximizing the
partial likelihood (Cox 1972). and constructed likelihood-ratio and
Wald chi-square tests for each parameter (Hosmer and Lemeshow
1999) with the SAS PHREG software (SAS Institute 1997). For
each event type, we began with our full regression model [Eq. ( 1 )]
and. one-by-one. deleted terms for which the corresponding like-
lihood-ratio chi-square test was not significant at the a = 0.05
level, except we did not delete terms for which a higher-order
interaction was statistically significant. This model reduction pro-

TABLE 1.

Physical characteristics of three mussel species after aerial exposure
at various water-air temperature differentials.

Water

Temperature

Mean Length

Wet Weight

(°C)

Species

(mm)

(g)

25

Elliptio dilatata

80.57(9.7)

53.79(184)

Quadrula pustiilosa

61.82(13.2)

78.27 (42.0)

Lampsilis card'niin

100.32(10.7)

185.59(54.0)

10

E. dilatata

78.64(10.4)

52.21 (19.6)

L. cardium

99.71 (11.6)

179.74(33.1)

Numbers in parentheses are the standard deviation of the mean.

cess identifies the simplest model for each event type that pre-
serves the hierarchical structure of Eq. ( 1 ). Our recorded event
times were based on observations at fixed times rather than exact
measurements of event times, and, therefore, contained ties. We
used Efron's method to adjust for tied event times, which has been
shown to perform better than alternatives (Hertz-Picciotto and
Rockhill 1997). We assumed that events occurred at the observa-
tion time rather than, for example, the temporal midpoint between
successive observations to produce conservative estimates of the
intensity of first uprighting or the hazard of mortality.

A particularly desirable feature of proportional hazard regres-
sion models is that the parameters have natural interpretations that
provide informative descriptions of the event times. Because these
models are still unfamiliar in ecology, interpretation requires some
explanation. The hazard function \{t) quantifies the number of
events per interval of time at time /. From Eq. ( I ), the dimension-
less hazard ratio (risk ratio) at time t is given by HR(r) = \,(r)/
\o(?) = exp[5, + ■ ■ ■ -^ P^jfAA/]. For such categorical variables
as species S, in our analysis, the hazard ratio for species ; relative
to the baseline species is exp(S,), and the hazard ratio for species
1 relative to species 2 is exp[S, - 5,] in the absence of higher-order
interactions. If. for example. exp(S,) = 0.5, we say that the relative
hazard (or intensity) for species / is only 50% of that for the
baseline species. For continuous covariates such as water tempera-
ture, the statistic 100[exp(P|) - 1] is the estimated percentage
change in the hazard (or intensity) ratio for each unit change in
temperature. If, for mortality, P, = 0.5, then the hazard ratio
increases by approximately 65% for each 1 °C increase in tem-
perature (Allison 1995). These interpretations extend to more com-

TABLE 2.

Proportional hazards (intensity) model fitted to the time to first
uprighting for Elliptio dilatata, Quadrula puslulosa. and Lampsilis
cardium. Lampsilis cardium at 0 C constitute baseline conditions.

Parameter (Effect)

Estimate (SE)

Wald x"

P-Value

5| (species: E. dilatata)

0.3142(0.1965)

2.6

0.11

5, (species: Q. pustiilosa)

-0.3031 (0.0821)

13.6

<0.01

p, (water temperature T)

0.1491 (0.0083)

319.0

<0.01

p, (air shock A)

-0.0677(0.0146)

31.4

<0.01

P4 (A')

0.0055(0.0011)

26.9

<0.01

P,i(S, xD

-0.0562(0.0096)

34.1

<0.01

P^fT'xA)

0.0027(0.0007)

16.2

<0.0I

P,(7-x/l^)

-0.0003(0.0001)

30.9

<0.01

See text for explanation of the model and parameters.

236

Bartsch et al.

B

''''°'^n°g

Lampsilis cardium

'^^°c.,o,;

Elliptio dilatata Air s/,^

c<r^og

Air cA '"

Quadrula pustulosa

Figure 2. Estimated probabilities of times to first uprighting, T, were less than times on the axis marked Time for L. cardium, E. dilatata, and
Q. puslulnsa at five air shock temperatures obtained from proportional hazards model (Table 2). Air shock was defined as the difference between
water and air emersion temperatures. Letters A and B refer to the 25 and 10 C" water exposure treatments, respectively.

plicated models having significant interactions. For example, from
Eq. ( 1 ) the hazard ratio for species I to species 2 at water tem-
perature T. air shock temperature A, and exposure duration M is
given by expl.V, - S, + (P, , - (3, ,)r + (3,., - (3, ,)/\ + O, , -
P,.,)M + (P, , - p^,)A- + (p^, - 3^,)rA + (P, , - <^,2)TM +
(ps., -P«.,)/IW + (P,, , -P,,,)7-/1M|.

Although hazard ratios have natural interpretations that provide
the means to assess the relative importance of the explanatory
variables, we display model features using graphs of the "survivor"
functions (Hosnicr and Lemcshow 1999). which are probabilities
that times until events exceed some time /. We computed product-
limit survival estimates (Kalbtleisch and Prentice 1980) of the

Emersion and Thermal Effects on Mussel Survival

237

survivor function and used those to display model features for both
first uprightings and deaths.

RESULTS

Physiochemical Characteristics of Water

Temperature, dissolved oxygen (Yellow Springs Instrument
Model 58 oxygen meter), and pH (Beckman Model Oil meter)
were measured daily in each tank during each test. Averages and
standard deviations (SD, in parentheses) for physiochemical char-
acteristics of water in all tanks at each water temperature (25 and
10 "C. respectively) were as follows: temperature 24.5 °C (0.8),
10.1 (0.4); dissolved oxygen 8.2 mg/L (0.4), 1 1.5 mg/L (0.6); pH
8.10 (0.06), 8.13 (0.09). Un-ionized ammonia concentrations (mg/
L) were measured in six randomly selected tanks for each test
(range. 0.0013 to 0.0054 mg/L). and were well below the concen-
trations reported to adversely affect mussel growth (0.036 mg/L at
6 weeks, Sparks and Sandusky 1981) or survival (96 h LC50 =
1.1 mg/L, Arthur et al. 1987). The mean relative humidity over all
five tests during aerial exposures was 63.6 (2.19).

Mussel Characteristics

The average length and wet weight of mussels were similar
within a species between water temperatures (Table 1 ). The sex
ratios (male:female) of E. dilatata and Q. pustulosa in the 25 °C
treatment were similar (66 male:67 female and 74 male:65 female,
respectively); however, the sex ratio for L. cardium was approxi-
mately 2:1 males to females (209 male: 1 1 1 female) in the 25 °C
water treatment and approximately 3:1 males to females (239
male:80 male) in the 10 °C water treatment.

Uprighting Behavior

The intensity of first uprighting differed among species, water
temperatures, and air shock temperatures in a complex way in-
volving multiple interactions (Wald x" = 633.2, <//= 8, P < 0.01 ).
There was a significant interaction between E. dilatata versus the
other species and water temperature (P < 0.01; Table 2). Both
water temperature and air shock temperature were important pre-
dictors of times to first uprighting. As expected, the intensity of
uprighting was greater at the higher water temperature. Moreover.
the effects of air shock differed with water temperature and
showed a significant quadratic response (u or n-shaped. Fig. 2).
At the 10 °C water temperature, the uprighting response was
U-shaped; whereas, at the 25 °C water temperature, the response
was n-shaped. Although air exposure duration had no statistically
significant effect, it is important to note that, by definition, any
response to air shock temperature requires exposure. In this ex-
periment, the briefest air shock duration was apparently sufficient
to affect uprighting intensity, and longer durations showed no
additional effect.

In addition to uprighting, we also observed other behavioral
responses to emersion. Shell gaping behavior was observed in L.
cardium during emersion in >25 °C air for 30 min and in E.
dilatata during emersion in 45 °C air for 15 min. Also, the occur-
rence of foot extension increased with emersion time in £. dilatata
at 45 °C (-70% at 15 min duration to -100% in the 60-min ex-
posure duration). All three species extruded mucus from the si-
phonal region after emersion in 45 °C air for 60 min.

Sun'ival

Survival of mussels differed among species and with water
temperature, air shock, and air exposure time in a complicated way
involving both two- and three-way interactions (Wald x" =
253.82, df= 14, P < 0.01). Survival of Q. pustulosa did not differ
significantly from L. cardium, the baseline species, at any water or
air shock temperature, or with air exposure time (Table 3. Fig. 3).
Eliiplio dilatata differed significantly from L. cardium up through
interactions with the linear and quadratic effect of air shock tem-
perature, and the three-way interaction among species, water tem-
perature, and air shock temperature (Table 3). The parameters
(effects) for E. dilatata (S,). air shock (A), squared air shock (A").
and the E. dilatata x air shock (S, x A), water temperature x air
shock (T X A), air shock x exposure duration (A x M), E. dilatata
X squared air shock (S, x A"), E. dilatata x water temperature x air
shock (5, x r X A), and water temperature x air shock x exposure
duration interactions (7" x A x M) were significantly different from
zero (Table 3). Through the last day of the experiment, survival
varied only slightly except for the £. dilatata in the 25 °C water
temperature treatment that were exposed to large positive air
shocks (Fig. 3). For £. dilatata in the 25 °C water treatment,
survival probabilities decreased significantly in the 60-min air ex-
posure duration treatments (Fig. 4).

DISCUSSION

Over-all mussel survival after emersion was high (93%) and
indicated that these mussel species are remarkably resistant to
emersion and thermal shock. For example, in the 10 °C water tests,
both L. cardium and £. dilatata survived the air shock treatments
despite a 20 °C air-water differential and emersion in subzero air.
However, variations in tolerances to water-air treatments were
evident among species at the higher water temperature. Elliptio
dilatata died within 24-h postemersion to the 45 °C air temperature
treatment, with 100% mortality at the 60-min aerial exposure du-
ration and 50%' mortality at the 30-min duration. Surprisingly, the
other two species survived the highest air shock treatment. Several
studies have documented that mussel survival, during and after
emersion, is directly related to relative humidity (Byrne and Mc-

TABLE 3.

Final fitted proportional hazards model (equation 1) for Elliptio

dilatata, Quadrula pustulosa, and Lampsilis cardium survival data.

Lampsilis cardium at 0 °C constitute baseline conditions.

Parameter (effect)

Estimate (SE)

Wald X-

P-Value

S| (species: E. dilatata)

-1.8979(0.8667)

4.8

0.02

5i (species: Q. pustulosa)

-0.0261 (0.4375)

<0.1

0.95

Pi (water temperature T)

-0.0352(0.0433)

0.7

0.42

p, (air shock A)

0.2702(0.0731)

137

<0.01

(5, (exposure duration M)

0.0266(0.0163)

2.6

0.10

P4(A')

-0.0034(0.0018)

3.7

0.05

(3,.i(S, xr)

0.0253(0.0471)

0.3

0.59

P2i(S, xA)

-0.3527 (0.0633)

31.0

<0.01

P^'T-xA)

-0.0148 (0.0034)

18.4

<0.0l

f,T(TxM)

-0.0004(0.0009)

0.2

0.63

Ps (A X M)

-0.0042(0.0014)

9.4

<0.01

Pm (5| xA-)

0.0126(0.0029)

19.2

<0.0I

P6,(S, xTx/l)

0.0164(0.0032)

25.8

<0.01

Pi, {TxAxM)

0.0003(0.0001)

17.1

<0.01

See text for explanation of the model and parameters.

238

Bartsch et al.

B

""KCC)

"Oct- /o

Lampsilis cardium ( Q

Elliptio dilatata

'°"^(°C)

Quadrula pustulosa

Figure 3. Kstimatcd survival probiihilitii's. T, were greater than times on the axis marked Time for /,. cardium. K. dilatata. and Q. pustulosa at
five air shocl\ temperatures and three exposure durations obtained from proportional hazards model (Table 3|. Air shock was defined as the
difference between water and air emersion temperatures. Letters A and B refer to the 25 and III C water exposure treatments, respectively.

Emersion and Thermal Effects on Mussel Survival

239

Elliptio dilatata

Figure 4. Estimated survival probabilities, T, were greater than time
on the axis marked Time for E. dilatata at three exposure durations for
the 25 °C water, 45 °C aerial emersion test obtained from proportional
hazards model.

Mahon 1994. Dietz 1974. Holland 1991). Dietz (1974) reported
that Ligiimia subrostrata Say could survive >40 d in air, if water
loss was retarded; however, survival was reduced to 6-10 d when
mussels were exposed in air with a low relative humidity (45-
55'7f) at 25 °C. Relative humidity was held constant (60 ± 5%)
across all treatments in our study. Survival probabilities are ex-
pected to vary directly with relative humidity and should not be
considered absolute.

Uprighting behavior was more sensitive to species and treat-
ment effects than survival. Lampsilis cardiiiin uprighted before Q.
pustulosa (25 °C water test only) or E. dilatata (25 and 10 °C
water tests) regardless of air shock exposure or duration. In addi-
tion, both L. cardium and E. dilatata required more time to upright
when held at the cooler water temperature than those held at the
wanner water temperature. Waller et al. (1999). similarly found
that mussels displaced in cooler water temperatures (7 °C) required
up to tenfold more time to upright than those displaced in higher
water temperatures (21 °C). In addition, the uprighting intensity
for L. cardium and E. dilatata in this study showed a significant
quadratic response to air shock temperature (Fig. 2). This is par-
ticularly noteworthy, because at the lower water temperature (10
°C), air shocks that differed greatly from water temperatures in
either direction seemed to stimulate uprighting; whereas, at the
higher water temperatures (25 °C), large positive or negative air
shocks tended to inhibit uprighting slightly.

In addition to the uprighting behavior, we made qualitative
observations on other sublethal behavioral responses to emersion
and thermal stress, which included shell gaping, foot extension,
and mucus production. These behaviors were elicited during em-
ersion in air >25 °C; whereas, mussels closed their valves in air
temperatures between -10 to 20 °C. Elliptio dilatata exhibited all
three responses (shell gaping, foot extension, and mucus produc-

tion) at the higher air shock temperatures in contrast to partial
responses (shell gaping and mucus production only) in both L.
cardiiitn and Q. pustulosa. There was an apparent correlation be-
tween the occurrence of these behaviors with survival. Byrne and
McMahon (1994) reviewed emersion capacities of freshwater bi-
valves and found that dessication resistance was inversely related
to the degree of mantle exposure behavior of a species. We suggest
that these behavioral responses, along with uprighting, are valu-
able sublethal indicators of stress and could be further quantified.

Emersion tolerance in freshwater bivalves may also be affected
by shell characteristics. For example, both L. cardium and Q.
pustulosa are spherical in shape; whereas, E, dilatata is elliptical.
A sphere has the largest volume per outside surface area of any
geometrical configuration (Kreith 1973). Conversely, an ellipse
has a larger surface area per volume, which may have allowed the
£. dilatata to reach a higher internal temperature more quickly and
for a longer duration than the two spherically shaped species.
Other physical characteristics of the shell that may affect emersion
tolerance are shell thickness and over-all shell size. Shell thickness
was similar among the three species that we tested; Q. pustulosa
had the thickest shell, followed by E. dilatata and L. cardium.
which have comparable shell thickness. In general, we expect
thick-shelled species, such as amblemines, to be most tolerant to
emersion and thinner-shelled species, such as anodontines, to be
least tolerant. In addition, smaller individuals of a given species
will be more sensitive to emersion than larger individuals because
of the larger surface area to volume ratio. The rank of the average
size of mussels in this study was E. dilatata (smallest), Q. pustu-
losa. and L cardium (largest). Thus, these additional physical
factors, shell shape, thickness, and over-all size, may also explain
the greater sensitivity of E. dilatata to emersion.

One of the primary objectives of this study was to establish
temperature guidelines for collection and handling of two federally
endangered species. Q. fragosa and L. higginsi. Quadrula pustu-
losa and L. cardium were chosen as potential surrogates, because
they are congeners. Elliptio dilatata was chosen as a second con-
gener of Q. fragosa, because it is also an amblemine, but was more
abundant at the study site than Q. pustulosa. Surprisingly, Q. pus-
tulosa, and L. cardium, although in different subfamilies, re-
sponded more similarly than Q. pustulosa and £. dilatata, the two
amblemines. These results demonstrate the difficulties associated
with the use of surrogates and extrapolation to other mussel spe-
cies, particularly when there is no sound biological basis for their
selection. Therefore, guidelines should err on the side of the most
sensitive species for which data are available, because results are
seldom validated with threatened and endangered species.

In conclusion, these data provide baseline information for de-
veloping management guidelines for handling the species tested.
Generally, we found that these species were tolerant of emersion,
but may experience mortality in extreme conditions. Our survival
estimates are conservative, because they were derived from labo-
ratory studies conducted under controlled conditions rather than
under field conditions in which air temperature and relative hu-
midity can vary significantly within several hours. In addition, the
microclimate to which the mussels are exposed can be very dif-
ferent from measured air temperature and relative humidity. Mus-
sel survival can be enhanced by conducting field work in moderate
conditions (10 to 25 °C air temperature), protecting the mussels
from extreme changes in air temperature, and covering them with
damp cloths during emersion to maintain high relative humidity.

240

Bartsch et al.

ACKNOWLEDGMENTS

This research was partially supported by the U.S. Fish and
Wildlife Service, Twin Cities Field Office in Bloomington, Min-
nesota. We thank Jim Luoma, Ron Hayden, Dennis Wasley, Mark

Hanson, and the staff of Ecological Specialists, Inc. for assistance
in the field and laboratory during this study. We also thank Chuck
Kjos and Bob Hay for assistance with the planning and execution
of this project.

LITERATURE CITED

Allison. 1995. Survival analysis using the SAS system: a practical guide.
SAS Institute, Gary, NC.

Arthur, J. W., W. W. Coriis, K. N. Allen & S. F. Hedtke. 1987, Seasonal
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Joiimol of Shellfish Rcscanh. Vol. 14. No. I. 241-245. 2000.

ELEMENTAL SULFUR IN THE GILLS OF THE MANGROVE MUD CLAM ANODONTIA

EDENTULA (FAMILY LUCINIDAE)

JUNEMIE HAZEL L. LEBATA

Aqitaciiltiire Department

Southeast Asian Fisheries Development Center

P.O. Bo.x 256

lloilo City 5000

Philippines

ABSTRACT Different sizes of the mangrove mud clam .Anodontia edenuila were collected from the mangroves in Bgy. San Roqiie
in Estancia. lloilo, central Philippines, and the mantle, gill, and foot tissues were analyzed for elemental sulfur content. Mangrove mud
(substrate) was aKso analyzed for total sulfur content to establish the possibility of clam-bacteria symbiosis in this lucinid clam. Sulfur
analysis showed highly significant (P < 0.0001 ) amounts of elemental sulfur in the gills (247.64 + 63.28 |j.moles/g FW) compared with
the quantities observed in the mantle (0.84 ± 0.22 (j.moles/g FW). Elemental sulfur was absent from the foot tissues. Results also
showed a .significantly ^P < 0.05) decreasing elemental sulfur from the newly collected clams (mean = 461.18 ^.moles/g FW)
compared to those reared in the laboratory (mean = 159.08 (imoles/g FW: with mangrove mud substrate; mean = 45.18 fjimoles/g
FW without substrate), which were analyzed weekly until week 3. indicating that stored elemental sulfur is being utilized by the
bacteria in the absence of sulfide. Total sulfur content of mangrove mud in situ was higher than that used as substrate in the experiment;
where there were no significant differences from mitial to final readings. This shows that mangrove mud ;/; silii is linked to a steady
sulfur source.

KEY WORDS: Anndonlia edenmlu. lucinid. gills, elemental sulfur

INTRODUCTION

The mangrove mud clam Anodontia edentula (Linne, 1758) is
widely distributed in the Indo-West Pacific region (Poutiers 1998).
In the Philippines, it is one of the popular bivalves harve.sted from
sandy-muddy bottoms near mangrove areas (Poutiers 1998, Sotto
and von Cosel 1982 ). It is one of the most noteworthy species and
a highly prized delicacy in the coastal areas where it is abundant:
hence, an important source of food and livelihood. It grows to a
maximurn size of 8-9 cm shell length (SL), total weight of 180-
210 g and is a potential aquaculture species.

A. edentula belongs to order Veneroida, family Lucinidae
(Poutiers 1998), together with most eulamellibranchs containing
symbiotic bacteria (Schweimanns and Felbeck 1985).

Animal-bacteria symbioses in marine mollusks have been ob-
served in hydrogen sulfide-rich habitats, such as anoxic basins,
sewage outfalls (Felbeck et al. 1981), seagrass beds (Cavanaugh
198,3). mangrove swamps (Frenkiel et al. 1996. Vetter 1985). and
in organically rich sediments (Janssen 1992).

The presence of elemental sulfur in the gills of some clams as
energy source for clam-bacteria symbiosis has been reported by
Vetter (1985) for Lucinoma anmdata. Calyptogena elongata, and
Lucina floridana and Dando et al. (1985) for Myrtea spinifera. In
a review of Somero et al. (1989). the gills of the bivalve Calyp-
togena ponderosa have the highest elemental sulfur content
(2593.8 (jimole/g): whereas. Solemya reidi gills have the lowest
(15.8 |xmole/g).

Symbiotic bacteria in Philippine bivalves were first discussed
by Janssen (1992) after his observation of bacteria in the gills of
Codakia tigerina and A. edentula (using light microscopy), and
Fimbria fimbriata (using transmission electron microscopy). In the
present study, the observation of deep brown to deep purple, thick
and fleshy gills having a single demibranch on each side of the
gonad oi A. edentula agrees with the description of Allen (1958)
for several genera of lucinids, Dando et al. (1985) for M. spinifera
and Distel and Felbeck (1987) fori, aequizonata. L. anmdata and
L. floridana, all of which are known for endosymbionts. These

typical characteristics of lucinid gills possessed by A. edentula
may support the claims of Janssen (1992).

To support further the evidences mentioned above, this study
aimed to establish sulfur-oxidizing metabolism in A. edentula by
analyzing elemental sulfur content of its mantle, gill, and foot
tissues.

MATERIALS AND METHODS

Collection of Samples

A. edentula specimens were obtained from a narrow band ( 10-
15 m) of 15-20 ha mangrove area in Bgy. San Roque, Estancia,
lloilo in central Philippines (1 ri4'N, I23°8'E) (Fig. 1). The trees
are mostly secondary growths of Sonneratia and Avicennia spp.
During low tide of spring tide periods, a very wide tidal flat is
exposed, reaching 200-250 m seaward from the mangrove forest.
There is no freshwater input in the study area.

Clams were collected during the ebb of a spring tide at daytime
with the help of a clam collector who can recognize the opening of
the clams" anterior inhalant tube on the surface of the inud. A
specialized gear made of flattened iron bar attached to a wooden
handle was used in digging the substrate. With this, the tube that
led to the clam was carefully traced. Depth of each clam frotn the
surface was measured during collection.

Physicochemical parameters were monitored during clam col-
lection. Temperature was measured with a mercury thermometer;
salinity with an Atago refractometer; pH with WTW pH 192
meter; and dissolved oxygen with a YSI Model 5 IB DO meter.
Total sulfur, water, and organic matter content of the sediment
were determined as prescribed by Beaton et al. ( 1968) and Dando
etal. (1985).

Analyses of Sulfur Content

The presence of elemental sulfur in the tissues of A. edentula
was examined, because elemental sulfur is the by-product of sul-
fide oxidation carried out by symbiotic bacteria (Atlas 1995) and
is stored in the periplasmic space (Vetter 1985). Total sulfur in the

241

242

Lebata

Figure 1. Map of Bgv. San Roque, Estancia, Iloilo in central Pliilip-
pines showing location of collection site of Anodonlia edentula.

substrate was measured both in situ and during laboratory experi-
ments (weel<s 1, 2. and 3) to Icnow the levels in the clam's natural
environment and the changes under laboratory conditions.

Elemental sulfur content of the tissues (mantle, gill, and foot)
and total sulfur content of the substrate were determined initially
(from newly collected samples) and through time (1-3 weeks)
from laboratory-reared clams.

Clams and mangrove mud were collected from the study site
and transported to the laboratory of the Aquaculture Department of
Southeast Asian Fisheries Development Center (SEAFDEC/AQD)
in Tigbauan. Iloilo. Upon arrival, all clams were measured (shell
length, SL; shell width. SW; shell height, SH) using a caliper,
weighed (total weight, TW) using a Mctller AE163 analytical bal-
ance, and numbered individually.

Elemental Sulfur Analysis

Six clams were randomly selected and dissected tor their
mantle, gills, and foot. Gill color of each clam was noted and
described (Vetter 1985), and total wet weight and individual
weights of each tissue were measured using a Mettler AE163
analytical balance. Tissues were then dried to constant weight in an
oven at 60 C, then pulverized by mortar and peslle. Elemcnlal
sulfur was extracted from the dried tissues using acetone and pre-
cipitated with barium sulfate .seed suspension. Acidity of the me-
dium (for sulfur extraction) was maintained by the addition of
barium chloride (Beaton et al. I'X-iH). Extracts were then subjected
to lurbidimelry. Absorbance was measured in a Shimad/u IIV-
1601 spectrophotometer al 44(1 iiiii wavelength. I^lcnK-nlal sulfur

was computed and expressed as pimoles per g fresh weight
(|xmoles/g FW).

For comparison purposes, mantle, gill, and foot tissues of
newly collected Anadara antiquata — another mangrove-
associated bivalve inhabiting the same habitat as A. edentula —
were also analyzed for elemental sulfur.

Two treatments (with and without mud substrate) replicated
three times were prepared for sulfur analyses (elemental for the
tissues and total for the substrate) through time. For the treatment
with mud, three glass aquaria (0.75 x 0.44 x 0.42 m) were pro-
vided with 0. 15 m newly collected mangrove mud to provide a
substrate close to that found in their natural environment (Distel
and Felbeck 1987, Vetter 1985), 100-L seawater (salinity condi-
tions the same as in the field, 33-35 ppt), air stones, and 2-mm
mesh black net cover. For the other treatment, the same were
provided, except mud. Eight randomly selected clams were
stocked in each aquarium. Sampling was done weekly for 3 con-
secutive weeks starting 1 week after the initial analysis. (The ex-
periment could not be extended for a longer time, because 3 weeks
is the maximum period the clams can survive in seawater without
substrate.) Two clams were randomly chosen from each aquarium
and processed as in the initial analysis. Gill coloration was scored
(very light brown = 2; light brown = 4; brown = 6'. dark brown
= 8; and blackish brown = 10) and correlated with its elemental
sulfur content.

Temperature, salinity, pH, dissolved oxygen, water and organic
matter content, and total sulfur content of the mud were taken
during the initial and the weekly samplings.

Total Sulfur Analysis

For the total sulfur content analysis, sediment samples were air
dried and ashed in an Automatic Muffle Furnace MFD-200N at
700 °C for 40 min. Total sulfur was extracted using hydrochloric
acid, processed as in elemental sulfur and the absorbance measured
at 440 nm (Beaton et al. 1968). Total sulfur was expressed in
percentage. All analyses were conducted at the Centralized Ana-
lytical Laboratory of SEAFDEC/AQD. Elemental sulfur contents
were compared: ( 1 ) among tissues; (2) with time; and (3) between
treatments (with or without mud substrate!. Analysis of variance
(ANOVA) was used to determine significant differences between
means of elemental sulfur content of tissues at different times and
treatments (a = 0.05) (SAS 1988) and Duncan's Multiple Range
Test (DMRT) to isolate these differences (Gome/ and Gomez
1984). Weekly means of sulfur content of clams reared with and
without mud substrate were compared using ?-test (SAS 1988).
"Clams" in the text refer to A. edentula. unless otherwise stated as
A. aniiquiila.

RESULTS

During sample collection, substrate temperature ranged from
27.0-30.0 'C (mean = 28.70); salinity 33.0-36.0 ppt (mean =
35.0); pH 5.15-6.55 (mean = 5,63); D.O. 0.2-1.0 ppm (mean =
0.38); water content 49.4-56.0% (mean = 53.4); organic matter
5.08-6.58% (mean = 6.24); and total sulfur content 1.. 3-2.2%
(mean = 1.8). There was no rainfall during the entire collection
period.

/\. edentula were colloclcd at depths ranging from ().2S-()..'iO m
from the surface. Its fool was observed to extend several limes
longer than the clam's shell length. On the other hand, /\. anti-
quata. which were analyzed for comparison purposes, thrive al

Elemental Sulfur in Lucinid Clam A. edentula

243

shallower depths (surface-0.1 m). Its foot was hatchet-like and
does not extend longer like the foot of A. edentula.

During laboratory culture of the clams, water temperature
ranged from 27.0-28.5 °C (mean = 27.9): salinity 33.0-35.0 ppt
(mean = 34.0): pH 7.6-8.3 (mean = 8.05); and D.O. 3.4-6.0 ppm
(mean = 4.41 ). Total sulfur content of the substrate ranged from
0.3-0.7% (mean = 0.4): water content 49.0-66.8% (mean =
58.18). and organic matter content 5.15-6.58% (mean = 5.72).

The organic matter content of the newly collected substrate
(6.24 ± 0.29%) was not significantly different from those stocked
with clams (5.72 ± 0.57%) for 3 weeks. However, sulfur content of
mangrove mud in situ was higher (1.8 ± 0.15%) than the one used
as substrate in the experiment (0.4 ± 0.1%); the latter had no
significant difference from initial to final readings.

In newly collected clams, the gills were deep brown to deep
purple, thick and fleshy, as compared with the gills of individuals
kept for 3 weeks in the aquaria, which were thin and filamentous.
In the course of the 3-week sampling period, there was a slight
fading of color in the gills. Correlation analysis showed no rela-
tionship between gill coloration and elemental sulfur content.
However, gills of newly collected A. antiquata were reddish
brown, thin, and filamentous.

There was not much change in the total wet weight of the
clams. Newly collected clams were slightly lighter (36.01 ± 2.06 g)
compared with clams weighed weeks later (36.84 ± 2.05 g) during
laboratory experiment.

Elemental sulfur levels were significantly different (ANOVA.
P < 0.0001 ) in the gills (247.64 ± 63.28 p,moles/g FW) compared
with the mantle (0.84 ± 0.22 (j.moles/g FW). Elemental sulfur was
absent from the foot tissues (Fig. 2). Results also showed a sig-
nificant decrease (ANOVA, P < 0.01) in elemental sulfur between
the newly collected clams and those stocked in aquaria (both in
mud and without mud substrate) and analyzed 1-3 weeks later (Fig
3a). Minimal sulfur was detected in the mantle only at the start of
the experiment (Fig. 3b).

Results further showed that elemental sulfur in the gills of
clams stocked in mud substrate were significantly higher than
those stocked in seawater only for weeks 1 (r-test, P < 0.05) and 2
(/-test, P < 0.05). However, there was no significant difference
between these two treatments in week 3 (Fig. 4). For the mantle,
there was no significant difference between treatments. No el-
emental sulfur was detected in the mantle, gill, and foot tissues of
A. antiquata (data not shown).

a) Gills

rr^ Week2

I L M I [I \A/aolc 3

'' 1 b) Mantle

b b

With mud Without mud

Figure 3. Means ± SE of elemental sulfur content of Aiiodontia eden-
tula a) gills and b) mantle from initial to week 3 readings in clams
stocked in mangrove mud substrate and in seawater only. Means with
the same superscript are not significant. (ANOVA, a = 0.05)

DISCUSSION AND CONCLUSION

The major natural sources of sulfur in the soil are organic
matter and soil minerals. Most of these are bound, but over time,
microorganisms can simplify them into soluble inorganic forms
(Brady 1990). Hydrogen sulfide and other sulfides are produced
during mineralization of organic sulfur (proteins and other organic
combinations) under anaerobic conditions (Atlas 1995, Brady

5> 500

Figure 2. Means ± SE of elemental sulfur content of different tissues
of Anodontia edentula (ANOVA, P < 0.0001).

Week t Week2 Week3

Figure 4. Means ± SE of elemental sulfur content of Anodontia eden-
tula gills between treatments from week 1 (/-test, P < 0.05), week 2
(/-test, P < 0.05) week 3 (/-test, P > 0.05). Means with the same super-
scripts are not significant.

244

Lebata

1990). These sulfide ions will undergo hydrolysis to form gaseous
H-,S causing the rotten egg smell of swampy or marshy areas
(Brady 1990) including mangroves where A. edentida thrives.

In a clam-bacteria symbiotic relationship, the clam's anatomy
and its location in the substrate/mud play very vital roles. Accord-
ing to Distel and Felbeck (1987), clams should be strategically
situated in an interface between a sulfide-generating zone (anoxic)
and water with sufficient oxygen (oxic). The foot o{ A. edenlula.
which can extend up to several times longer than its length, gives
it the capacity to construct a ventilation burrow (Reid and Brand
1986) and allows it to draw water from the surface (Dando et al.
1985). The tip of the foot is specialized for the construction of this
inhalant tube. It is provided for with glands that lay down mucus
for the building of the tube (Allen 1958). The clam gains access to
oxygenated water through this inhalant tube. Its location (0.28-
0.50 m deep in mangrove mud) may allow it direct access to
sulfide. In cases where sulfide is limiting. Childress et al. (1991 )
have proved that the foot can dig deeper and is responsible for the
uptake of sulfide from deeper parts of the substrate. Sulfide is
taken up across the foot of the clam and into the blood that trans-
ports it to the gills for use of the bacterial symbiont (Ruppert and
Barnes 1994).

Sulfide is a highly reduced energy molecule and a variety of
biological systems have evolved to oxidize sulfide in orderly en-
zyme-regulated steps to harness the energy and avoid poisoning
(Bagarinao 1992). In a clam-bacteria symbiosis, bacteria are as-
sumed to provide the mollusk with chemosynthetically fixed car-
bon dioxide via aerobic oxidation of sulfide (Vetter 1985). Oxi-
dation of sulfides and other reduced sulfur compounds provides
energy to the bacteria to fix carbon dioxide into organic com-
pounds that become available to the host clam (Distel and Felbeck
1987, Kelly and Harrison 1989). The Calvin cycle is the main
metabolic pathway used by the bacteria to convert carbon dioxide
to organic carbohydrates powered by the energy (ATP) generated
from the oxidation of sulfides (Atlas 1995). In the process of
oxidation, sulfide is converted into elemental sulfur and stored for
future use (Childress and Mickel 1982, Vetter 1985). Using EDX
microanalysis, Reid and Brand (1986) found sulfur as the domi-
nant elemental inclusion of bacteria in a lucinid clam Parvihicina
teniiiscutpla.

In this study, elemental sulfur was present in highly significant
amounts in the gills (max: 1907.20 |xmoles/g FW from a newly
collected clam: min: 1.38 jjimoles/g FW from a clam reared with-
out substrate and analyzed at week 3) (Fig 3) and based on the
compilation of Somero et al. (1989). these values are within the
range of elemental sulfur content (0.22-2593.8 |j.moles/g FW) for
different species of bivalves containing symbiotic bacteria. The
very wide difference between the maximum and minimum values
may be attributable to the mobili/alion of elemental sulfur by the
bacteria in the absence of external sulfide (Vetter 1985) in aquaria
not provided with mud substrate. This is clearly exhibited by the
significant decrease in elemental sulfur content of the gills from
the initial sampling to week 3 (Figs. 3a. 4). The initial samples
showed to have significantly higher elemental sulfur content than
those stocked in mud and those in seawatcr (Fig. 3a). lurthcrmore.
clams .stocked in mud have significantly higher sulfur content at
week.s I and 2 than those stocked in seawatcr only. However, at
week 3, there was no significant difference between the two treat-
ments. In the first 2 weeks, bacteria in clams maintained in mud
may have utilized all a\ailablc sulfide in the mud; whereas, those
stocked in seawatcr only may have depcruleii on llicu clcnicntal

sulfur reserves. Moreover, around week 3. the clams stocked in
mud may have also utilized their sulfur reserves because of inad-
equate sulfide supply in the mud. In the absence of external sul-
fide, elemental sulfur represents a novel inorganic energy reserve
for the animal-bacterial symbiosis (Vetter 1985), which, according
to Powell and Somero (1985), is the most reduced nontoxic inor-
ganic form of sulfur.

If sulfur globules are confined in the periplasmic space of bac-
teria (Vetter 1985) then A. edenndu must have these endosymbi-
otic bacteria in their gills to store sulfur.

The presence of elemental sulfur in very minimal amounts in
the mantle may be attributable to the connection that links the
mantle and the gill, which may have served as a passage to some
bacteria containing elemental sulfur globules. There is an insertion
and fusion of the posterodorsal margins of the gills with the mus-
cular posterior mantle edge (Reid and Brand 1986). However, this
may also be attributable to contamination during dissection.

The deep brown to deep purple, thick and fleshy gills of A.
edentida were also observed by Felbeck et al. (1981) in bivalves
collected from sulfide-rich habitats containing sulfide oxidation
enzymes in contrast with the small, light-colored gills of those
lacking these enzymes. According to Distel and Felbeck (1987).
the dark coloration and thickness of the gills are attributable to the
presence of a thick layer of subfilamentar tissue perforated by
regular arrays of bacteriocyte channels formed by bacteriocyte
cylinders containing the bacterial symbionts.

The very slight, nonsignificant increase in wet weight of cul-
tured clams (3 weeks) compared to the newly collected ones used
for the initial analysis may be caused by the water trapped inside
the clams" body cavity. Newly collected clams were not weighed
immediately in the field but were transported to the laboratory and
weighed 8 h after collection. Water trapped inside the body cavity
was exuded. During laboratory experiment, clams were weighed
immediately upon harvest and so water trapped inside the body
cavity was released only during dissection and has, therefore,
added to the weight of the individual.

The absence of elemental sulfur in .4. anttqiiata simply shows
that only lucinid clams harboring endosymbiontic bacteria in their
gills are capable of sulfur-oxidizing metabolism and ha\e the ca-
pacity to store these sulfur globules.

The results of the experiment, therefore, support the presence
of endosymbiotic bacteria in A. edentida as exhibited by the pres-
ence in significantly higher amounts of elemental sulfur in the

ACKNOWLEDGMENT

I am grateful to the Philippine Council for Aquatic and Marine
Research and Development (PCAMRD) of the Department of Sci-
ence and Technology (DOST) for the study grant; SliAFDEC/
AQD for the use of laboratory facilities and field equipment: J. D.
Taylor of the Natural History Museum (London) for the identifi-
cation of A. edentida: J. H. Primavera for the invaluable help
throughout the study; J. A. Ingles, A. del Norte-Campos, N. B.
Armada. L. V. Laureta. T. U. Bagarinao, E. de .lesus, and N. Golez
for carefully reviewing the manuscript and for their suggestions
and recommendations: F. Harder. A. Asutilla, P. Bantilan, A. An-
drada, and M. B. Lavalle for the laboratory' assistance; V. Balinas
and D. Mcciano for the statistical analyses: E. Ledesma for the
figure; and the Bata-anon couple for their expertise in clam col-
lection.

Elemental Sulfur in Lucinid Clam A. edentvla

245

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animal tissue of the gutless clam Soleinya reidi. Biol. Bull. 169:164-
181.

Reid. R. G. B & D. G. Brand. 1986. Sulfide-oxidizing symbiosis in Luci-
naceans: implications for bivalve evolution. Veliger 29:3-24.

Ruppert. E. E. & R. D. Barnes. 1994. Invertebrate zoology, 6th ed. Saun-
ders College Publishing, FL. 1 106 pp.

SAS. 1988. SAS/STATTM user's guide, release 6.03 edition. SAS Institute,
Inc. Gary. NC. 1028 pp.

Schweimanns, M. & H. Felbeck. 1985. Significance of the occurrence of
chemoautotrophic bacterial endosymbionts in lucinid clams from Ber-
muda. Mar. Ecol. Prog. Ser. 24:1 13-120.

Somero, G. N., J. J. Childress & A. E. Anderson. 1989. Transport, me-
tabolism, and detoxification of hydrogen sulfide in animals from sul-
fide-rich marine environments. Crit. Rev. Aquat. Sci. 1:591-614.

Sotto. F. B. & R. von Cosel. 1982. Some commercial bivalves of Cebu.
Philippines. Philipp. Sci. 19:43-101.

Vetter. R. D. 1985. Elemental sulfur in the gills of three species of clams
containing chemoautotrophic symbiotic bacteria: a possible inorganic
energy storage compound. Mar. Biol. 88:33^2.

Journal of Shellfish Reseciirh. Vol. 19, No. 1. 247-250. 2000.

EVALUATION OF TAG TYPES AND ADHESIVES FOR MARKING FRESHWATER MUSSELS

(MOLLUSCA: UNIONIDAE)

DAVID P. LEMARIE, DAVID R. SMITH, RITA F. VILLELLA, AND
DAVID A. WELLER

U.S. Geological Survey.
Biological Resources Division,
Leetown Science Center,
Aquatic Ecology Laboratoiy,
Kearueysville. WV 25430

ABSTRACT Prior to initiating a long-term tagging program on freshwater mussels, we evaluated three varieties of tags (Northwest
Marine Technology Visual Implant Tag, Floy Fingerling Tag, and Hallprint Shellfish Tag) two types of adhesives (3M two-part epoxy
and Krazy Glue cyanoacrylate). and four bonding times before immersion in water (2, 5, 10. and 15 min). Tags were applied to empty
shells for two phases of testing. First, legibility was visually scored for each of the immersion times. The two-part epoxy became cloudy
at immersion times < 15 min. was easily abraded after curing, and was. therefore, eliminated from further testing. The visual implant
tag also was eliminated from further testing because the printing dissolved in the cyanoacrylate. In the second phase, the fingeriing tag
and the shellfish tag bonded with cyanoacrylate were tested for durability and retention under natural conditions in a shallow stream
and under highly abrasive conditions in a standard gem tumbler containing coarse metal shavings. Tag losses after 16 wk in the
instream test were 5.39^ for the shellfish tag and 2.69^ for the fingeriing tag. These tests revealed no loss of legibility after the removal
of material that accumulated on the tag surface. There was no appreciable wear of either tag type after 1 wk in the gem tumbler. Flexible
polyethylene shellfish tags were chosen for field application because they are thinner and available with a larger number of individual
codes than fingeriing tags. Tags bonded to the shells of live mussels with cyanoacrylate can be immersed in water in as little as 2 min
after application without affecting retention. This method was used to double-tag 1,372 mussels at a monitoring site on the Cacapon
River in West Virginia. After 2 y. the total tag loss in 325 recovered mussels was 0.46%.

KEY WORDS: Freshwater mussels, tag, adhesive, tag retention

INTRODUCTION

External identification of individual freshwater mussels is
highly desirable for tracking passive and active movements,
growth analyses, population studies, and laboratory experiments.
The marking of freshwater mussels is particularly problematic be-
cause these are long-lived species, and programs to collect popu-
lation information could potentially last for many years, requiring
excellent long-term legibility and retention. Additionally, marks
on freshwater mussels are subjected to abrasion from animal
movement through mineral substratum and water-borne materials
in fast-tlowing water. Any marking method must have minimal
effects on the survival, growth, and behavior of the tagged animal.
The ideal marks should be easy to apply, inexpensive, and readily
available, especially when needed for large-scale marking pro-
grams. In addition to high legibility, the tags should be small and
of subdued colors to avoid the potential for increased susceptibility
to predation in field studies (although bright colors for easy vis-
ibility in laboratory studies may be desirable) and should be com-
mercially available with a large number of codes.

Freshwater mussels have been individually marked by scratch-
ing numerals (Isley 1914, Couilliard et al. 1995), drilling codes in
the periostracum (Thoma et al. 1959), attaching pendant tags with
wire passed through a hole in the shell (Isley 1914), and attaching
plastic labels with adhesive (Neves and Moyer 1988). Sequentially
numbered plastic tags have been obtained from commercial ven-
dors (Neves and Moyer 1988) or created with Dymo label makers
(Balfour and Smock 1995). Adhesives used for plastic tags include
dental cement (Downing and Downing 1993). wet surface repair
putty (Balfour and Smock 1995), and cyanoacrylate (Neves and

Reference to trade names does not constitute U.S. Government endorse-
ment of commercial products.

Moyer 1988). Cyanoacrylate also has been widely used to attach
plastic tags to a variety of marine shellfish, including abalone
(McShane 1989), scallops (Heald 1978, Williams and Dredge
1981, Gwyther 1989), and limpets (Treble et al. 1993). Neves and
Moyer (1988) used this combination for freshwater mussels and
found no apparent effects on growth or survival up to 4 y later.
While long-term tag retention and legibility have been documented
for freshwater mussels, they have not been quantitatively evalu-
ated.

Because we desired to establish retention and legibility infor-
mation before initiating long-term tagging programs, we con-
ducted several short-term tests to select tags and adhesives. We
elected not to scratch codes into the periostracum for fear that this
action could cause increased shell erosion and the high-contrast
marks could make the animals more visible to predators. Two-year
retention and legibility results from a field monitoring study, ini-
tiated during the summer of 1996. also are presented here.

MATERIALS AND METHODS

The fingeriing tag and the visible implant tag (so named for its
intended use in clear tissue on fish) were initially selected and
were used in adhesive immersion and legibility tests (Table 1 ).
Shellfish tags were acquired after the adhesive tests were complete
and were used in all subsequent evaluations. Shellfish tags are
similar in size to the fingeriing tags but are significantly thinner
and carry larger characters and a greater number of characters.
Both the fingeriing tag and shellfish tag were previously used for
inarking freshwater mussels (R. Neves pers. comm.).

We selected adhesives that were readily available at reasonable
cost, easily dispensed, fast curing, and known to be durable while
immersed in water. A fast-curing adhesive was particulariy desir-
able to minimize emersion time, thus reducing stress to the mussels

247

248

Lemarie et al.

TABLE 1.
Tags used in evaluations.

Characteristics

Fingerling Tag

Visible Implant Tag

Shellfish Tag

Manufacturer's model no.
Manufacturer

Tag size (mm)
Tag thickness (mm)
No. of characters
Character height (mm)

FTF-69

Floy Tag and Manufacturing. Inc.

Seattle, WA
3.2 X 4.8
0.65

3 numeric
1,3

None

Northwest Marine Technology.

Shaw Island, WA
1.0 X 2.5
0.09

1 alpha + 2 numeric
0.7

FPN

Hallprint Pty. Ltd.. Holden Hill.

SA, Australia
4x8
0.15

1 alpha + 3 numeric
l.S

and providing a more efficient tagging process. Because tags will
be exposed to highly abrasive conditions as animals move through
the mineral substrate, our initial assumption was that it would be
necessary to cover the lags with clear adhesive to protect the
printed codes, especially in the case of the visible implant tags,
which are very small and delicate. A two-part epoxy (DP-IOO. 3M,
St. Paul, MN) and a cyanoacrylate (Krazy Glue, Borden, Inc.,
Columbus, OH) fit all of these considerations, and were selected
for the initial round of tests.

The tag and adhesive combinations were tested in a two-phase
process. First, we tested the ability of the adhesive to form a secure
bond and to remain clear when immersed in fresh water shortly
after application. Second, we evaluated the durability of the se-
lected adhesive and tag types when exposed to natural stream
conditions and a highly abrasive environment. Tags were applied
to empty shells collected from local streams. To prevent exfolia-
tion of the periostracum due to drying, all shells were stored in
water before and after tagging. Unsatisfactory adhesives and tags
were eliminated at each step. The tag and adhesive combination
that was found to perform best in these initial tests then was used
to double-tag live mussels for monitoring in a multiyear field
study. Double-tagging assumes that both tags are retained inde-
pendently and can be used to estimate tag loss (Arnason and Mills
1981).

Evaluation of Adhesives

Adhesives were evaluated initially by attaching tags to the ex-
terior surfaces of empty shells and allowing them to air dry for 2,
5, 10, and 15 min prior lo immersion in water. A maximum du-
ration of 15 min was chosen to minimize the potential stress to live
animals and to maintain efficient tagging rates in field applica-
tions. Ten replicates of each treatment (tag type, adhesive, time to
immersion) were prepared. All tags were completely covered with
the adhesive. Tagged shells were stored in 1-L containers filled
with spring water. Weekly observations were conducted through 4
wk to evaluate legibility and the retention of the tags. Shells were
removed from the water to conduct the observations, then imme-
diately were replaced. Legibility was scored by a single observer
(DPL) as easy to read (i.e., it could be read immediately), difficult
to read (i.e., it required close scrutiny or scraping of encrusting
glue), barely legible (i.e., despite the aforementioned efforts, an
"educated guess' was still necessary to make oul the numbers), or
illegible. Numeric scores of .3 through 0 were used to calculate
mean legibility scores for each trealmenl.

Evaluation of Tags

Based on the results of the initial tests, only cyanoacrylate was
used lor further testing. In addition, thie to legibllit> problems to

be discussed later, the visible implant tags were replaced with
shellfish tags for subsequent evaluations (Table I).

Instream Immersion Test

As in the adhesive test, shells were immersed 2, 5, 10, and 15
min after the application of tags. After remaining in water for I
wk, one shell from each treatment was imbedded in a natural
position in a block of concrete. Ten replicate blocks were prepared.
Blocks were placed perpendicular to the current in a section of
Hopewell Run, a second-order stream in Jefferson County, WV,
with fairly homogeneous water depth and velocity. The blocks
were randomly rearranged weekly to ensure equal exposure to
variations in tlow conditions. Tags were examined after 16 wk to
evaluate legibility and retention.

Tumbler Test

A minimum of four examples of both tag types were applied to
mussel shells using cyanoacrylate, which was allowed to cure for
2-3 min then was immersed in water. To fully test the durability
of the lag material and printing, care was taken to avoid placing
adhesive on the surface of the tags. After remaining in water for
approximately 24 h, individual shells were placed in a rock tum-
bler (Natural Science Industries, Far Rockaway, NY) containing
38 g of coarse metal shavings. The tumbler was operated for I wk
with periodic examinations.

Field Tests

Shellfish tags were attached to live mussels with cyanoacrylate
and were monitored at a site on the Cacapon River in West Vir-
ginia. The site was 200 m long and was divided into 10 20-m long
sections. Each section was .searched for 30 min using viewing
buckets, and all mussels encountered were remoxed from the sub-
strate for identification, measurement, and tagging, then they were
returned to the substrate in the same section where they were
found. Each animal was double-tagged with one tag placed on the
posterior slope of each \al\e. The area of tag application was
cleaned with a medium grit sandpaper or scrub pad, then was
rinsed and blotted dry. A sinall amount of cyanoacrylate was ap-
plied to the shell (i.e., a volume sufficient to completely attach the
tag with minimal excess glue), then, using forceps, the tag was
placed on the adhesive and pressed gently.

The adhesive was allowed to air dry for at least 2 min before
returning the animal to the water. The initial survey and tagging
were conducted in July 1996, with subsequent surveys conducted
in Januaiy, April, June, and October 1997, and in June 1998.
Additional animals were lagged at each interval. Water tempera-
ture anil iurbiilii\ were measured during each sur\cy.

Evaluation of Tags and Adhesives

249

RESULTS

Evaluation of Adhesives

The characters on the tlngerling tag were normally readable
without magnification. The characters on the visible implant tags,
however, are approximately 0.7 mm in height and were most easily
read with the aid of a magnifying lens. Regardless of taa type (Fio.
1). the epo.xy did not perform well, becoming cloudy when im-
mersed for times < 15 min. Legibility in the 15-min group was
impaired by the roughness of the surface of the adhesive. Addi-
tionally, the epoxy remained soft and was easily abraded during
examination.

The printing on the visible implant tags dissolved in cyanoacry-
late. resulting in complete loss of legibility in all tags in this
treatment. Cyanoacrylate provided good visibility with the finger-
ling tags regardless of time to immersion, although minor surface
rippling was noted in the groups immersed 2 and 5 min after
application.

Evaluation of Tags
Instream Immersion Test

The adhesive covering the tags acted as a substrate for the
attachment of silt and periphyton, somewhat impairing visibility. It
was frequently necessary to scrape the surface of the glue to re-
mo\'e attached material. In some cases, it was necessary to remove
the glue from the surface of the tag by gently scraping with a knife.
There was no loss of legibility after surface materials had been
removed. Tag losses for shellfish tags and fingerling tags were
539c and 2.6%. respectively, with no apparent relationship to the
tour drying times prior to immersion.

Tumbler Test

Neither tag type showed significant wear when compared to
new tags, and no tags were dislodged during the tumbling process.
As an extreme test, a shell with a shellfish tag was tumbled for an
additional 3 wk with metal shavings, plus 4 wk with up to 100 g
of sand in the chamber, and still did not show any appreciable
wear.

Field Tests

Between June 1996 and October 1997. 1,372 mussels were
tagged at the Cacapon River site. Species consisted of predomi-
nantly Elliptio compkmata (Lightfoot, 1786) (85.6%). EUiptio
fisheriana (Lea. 1838) (7.7%), and Lampsilis cariosa (Say, 1817)

Easy 10 read (3)

Difficult to read (2)

Barely legible ( 1 )

Illegible (0)

Fingcrtiog Tag/Cyanoaciylatc
Fingerling Tag/Epoxy
Visible Implant Tag/Epoxy

Time to Immersion (min.)

Figure, 1. Mean legibility of tag and adhesive combinations (numeric
score) after immersion in water.

(5.9%) and a few individuals of Alasmidonta varicosa (Lamarck.
1819), Lasmigona siibviridis (Conrad. 1835). and Strophitiis un-
dulatus (Say, 1817) (total < 0.9%). During periodic surveys
through June 1998. 325 tagged mussels were recaptured. The time
at large for recaptures ranged from 69 to 722 days. Survey tem-
peratures ranged from < 0 to 24°C, and turbidity ranged from 1 4
to 14.3 NTUs.

Only three tags were lost (0.46% of all tags recovered); two
were missing when recaptured after 70 and 378 day in silii. and one
was rubbed off while removing an attached caddisfly case after
447 day in situ. Loss of legibility was observed only once, after 69
days in situ (0.15%) and may be attributed to a printing error that
was not recorded at the time of tagging. Additionally, six tags
(0.92%) were reported to be loose when examined 69-343 days
after tagging. Typically, one end of the tag was not well-adhered
to the shell surface.

DISCUSSION

The results of this study indicate that the shellfish tag adhered
with cyanoacrylate provides a good long-term marking method for
freshwater mussels. While any tag loss is undesirable, the minimal
losses observed in the field tests would not seriously affect popu-
lation estimates based on these surveys. Conducting tests in the
laboratory eliminated the need for trial and error exercises in the
field and allowed the establishment of protocols before project
initiation. This testing process eliminated an adhesive with unde-
sirable characteristics, avoiding tag losses and illegibility that may
have impacted long-term studies.

Although the two-part epoxy formed a clear, hard bond after a
15-min drying time, we felt that this was too long for safe handling
of the mussels and would adversely affect the efficiency of field
survey operations. Cyanoacrylate bonded quickly and was prefer-
able because it did not require mixing. We also found that it was
not necessary to cover the tag with adhesive, as the tag surface and
printing are highly durable. Tumbler tests indicated that the print-
ing on both tag types is of sufficient durability that complete
coverage of the tag with adhesive is unnecessary. Clear adhesive,
however, is still desirable in the event that glue gets on the tag
surface.

Tag retention was greater in the field tests than in the instream
immersion tests. Because old shells collected in previous surveys
were used, higher losses may have been associated with a decrease
in the integrity of the shell surface, even though the shells had been
stored in water prior to tagging. In addition, tags on empty shells
were more exposed to flowing water than those on live animals
that could burrow into the substrate. Although water conditions
were measured in both the stream immersion test and field tests,
they were measured only at the observation intervals and may not
reflect the full range of conditions. The evaluation of adhesive
durability under different water conditions (e.g., temperature
range, hardness, and alkalinity) should be undertaken in the future.
For our freshwater mussel surveys, shellfish tags are preferred
over the fingerling tags for several reasons. Primarily, they are
much thinner, reducing the opportunity for objects to snag on the
tag edge. Treble et al. (1993) suspected that losses of tags were
caused by abrasion and snagging as limpets moved through narrow
crevices. Shellfish tags are also available in a wider range of
colors, including subdued natural colors that may reduce visibility
to predators (e.g., gray and beige). In addition, they use a combi-
nation of letters and numerals, which provides a large number of

250

Lemarie et al.

individual codes for each tag color. Finally, the shellfish tags come
on a roll, rather than loose, making them easier to handle in the
field.

While costly in terms of the number of tags and the time to
mark, the use of double tags will benefit large-scale, long-term
tagging programs. It will allow a continuous evaluation of tag
losses, the rate of which may change overtime (Treble et al. 1993).
Establishing rates of tag loss will provide correction factors for
population estimates based on recapture of tagged individuals (Se-
ber 1982. p. 94). Because entire tags can become obscured by
algae, insect cases, and other materials, the use of two tags also
may increase the possibility that a tagged animal is recognized.
Although these tests were physically rigorous, it is uncertain how
long the adhesive will be effective. Differences in tag retention

among species due to morphology (e.g., periostracum texture or
animal size) and behavior (e.g., substrate preference or burrowing
depth) are also unknown. Long-term monitoring and reporting to
the research community will aid in the development of future
tagging programs.

ACKNOWLEDGMENTS

We thank W. Bartles, A. Dunn, C. Horton, J. Lambert-
Newman, M. Morton. P. Pooler, N. Thomsen. K. Voges, and P.
Young for their assistance in the field. We also thank the tag and
adhesive vendors who provided valuable technical information on
their products, and in some cases, provided free samples for test-
ing. Reviews of the manuscript by J. Layzer, W. Leilis, E. Pendle-
ton, and K. Weike are greatly appreciated.

LITERATURE CITED

Arnason, A. N. & K. H. Mills. 1981. Bias and loss of precision due to tag
loss in Jolly-Seber estimates for mark-recapture experiments. Can. J.
Fish. Aquat. Sci. 38:1077-1095.

Balfour, D. L. & L. A. Smock. 1995. Distribution, age structure, and
movements of the freshwater mussel ElUptio comptanata (Mollusca:
Unionidae) in a headwater stream. / Freshwat. Ecol. 10:255-268.

Couilliard, Y., P. G. C. Campbell. A. Tessier, J. Pellerin-Massicotte & J. C.
Auclair. 1995. Field transplantation of a freshwater bivalve. Pygunodon
grandis. across a metal contamination gradient: 1. Temporal changes in
metallothionein concentrations in soft tissues. Can. J. Fish. Aquat. Sci.
52:690-702.

Downing, W. L. & J. A. Downing. 199.^. Moliu.scan shell growth and loss.
Nature. 362:506.

Gwyther, D. 1989. Scallop tagging. In: D. A. Hancock (ed.). Tagging—
Solution or Problem? Australian Society for Fish Biology Tagging
Workshop, Proceedings No. 5. p. 104.

Heald, D. 1978. A successful marking method for the saucer scallop Ainii-
siiim balloti (Bernardi). Aii.st. J. Mar. Frcwimater Re.s. 29:845-851.

Isely, F. B. 1914. Experimental study of growth and migration of fresh-

water mussels. In: Report of the U.S. Commissioner of Fisheries for the
fiscal year 1913. Doc. 792. U.S. Bureau of Fisheries. Government
Printing Office. Washington. D.C. pp 1-24.

McShane, P. E. 1989. Tagging abalone. In: D. A. Hancock, (ed.). Tag-
ging— Solution or Problem? Australian Society for Fish Biology Tag-
ging Workshop. Proceedings No. 5. pp 65-67.

Neves, R. J. & S. A. Meyer. 1988. Evaluation of techniques for age
determination of freshwater mussels (Unionidae). Anier. Malacol. Bull.
6:17y-188.

Seber. G. A. F. 1982. The Estimation of Animal Abundance and Related
Parameters, 2nd ed. Griffin & Company. London.

Thoma. B.. G. Swanson & V. C. Dowell. 1959. A new method of marking
fresh-water mussels for field study. Proc. Iowa Acad. Sci. 66:455-457.

Treble. R. J., R. W, Day & T. J. Quinn IL 1993. Detection and effects on
mortality estimates of changes in tag loss. Can. J. Fish. Aquat. Sci.
50:1435-1441.

Williams. M. J. & M. C. L. Dredge. 1981. Growth of the saucer scallop.
Amusium japonicmn balloti Hahe in central eastern Queensland. .Ausf.
J. Mar. Frcslnmt. Res. 32:657-666.

Journal of Shellfish Research. Vol. 19, No. 1. 251-25S. 2000.

BACTERIAL PATHOGEN CONTAGION STUDIES AMONG FRESHWATER BIVALVES AND

SALMONID FISHES

CLIFFORD E. STARLIPER' AND PATRICK MORRISON^

'USGS-BRD Leelown Science Center

National Fish Health Research Laboratory

KearneysviUe. West Virginia 25430
'U. S. Fish and Wildlife Senice

Ohio River Islands National Wildlife Refuge

Parkersburg, West Virginia 26101

ABSTRACT A part of the conservation efforts of native freshwater bivalves is a relocation program whereby animals are collected
and moved to a safe refuge for maintenance and propagation. With the rearing of two different hosts, mussels and fish, on the same
facility there is a question of the possibility for contagion of pathogens. The studies presented here are part of a continuing effort to
address the concerns of contagion. Freshwater bivalves collected throughout the 1997 season were cultured for fish pathogens. Counts
of total bacteria on cytophaga medium ranged between 1.07 x 10' and 4.99 x 10' cfu/g of mussel soft tissues. The predominate groups
of bacteria were motile Aeromonas spp. and Pseudomonas spp., both of which include members that are opportunistic pathogens to
salmonid fishes. No primary fish pathogens were cultured; however, cells with correct morphology for Renibacleriuin salmoniitanim.
cause of bacterial kidney disease, were detected from mussel soft tissues in all six trials using the direct fluorescent antibody test.
Groups of mussels were subjected to 24-h waterbome challenges using bacteria cultured from healthy fish; no mortality occurred to
any of the animals. Another group exposed to the fish pathogen Aeromonas salmonicida also showed no effects of the challenge;
however, susceptible fish became infected and died after the fish were added to cohabit with this group of mussels.

KEY WORDS: Freshwater bivalves, salmonid. pathogen, disease, contagion, bacteria, cohabitation

INTRODUCTION

Over 70% of the approximately 300 species and subspecies of
freshwater bivalves (Unionidae) native to North America are cat-
egorized as endangered, threatened, or of special concern (Will-
iams et al. 1993). Bivalves are particularly susceptible to impacts
from environmental changes, not only because they are sessile,
filter-feeding animals, and adverse effects placed on their inter-
mediate fish hosts that are necessary for glochidia development
also may indirectly affect them.

A number of factors have been documented as contributors to
the decline in numbers of freshwater bivalves. Human disturbance
in the terrestrial environment, such as agriculture and develop-
ment, result in siltation that may impair growth and respiration and
lead to suffocation (Ellis 1936. Kat 1982). Also, habitat alteration
from dredging or dam construction can result in changes in fiow or
temperature and also affect movement of the intermediate fish host
(Fuller 1974; Keller and Zam 1990). In recent years, the zebra
mussel (Dreissena polymorpha) has become a major threat to na-
tive bivalve populations in large river systems (Herbert et al. 1991 ;
Nalepa 1994). Zebra mussels are able to proliferate and compete
very favorably against native animals (Gillis and Mackie 1994).
Their high spatial tolerance allows them to colonize in great num-
bers, leading to mortality of native bivalves by impeding feeding
and respiration (Griffiths et al. 1991. Haag et al. 1993. Leach 1993.
Mackie 1991), In 1996. densities of zebra mussels in the lower
Ohio River (near river mile 814) exceeded 14.000 per square
meter, and mortality to the native populations exceeded 30% (P.
Morrison, Ohio River Islands National Wildlife Refuge. U,S, Fish
and Wildlife Service, pers. comm.).

In the mid 1990s, the U.S. Fish and Wildlife Service (USFWS)
along with other federal, state, and private partners initiated con-
servation efforts. One such program was to isolate native animals
from impending zebra mussel infestation from selected large riv-
ers. Under this program, individuals are collected and relocated to

safe refugia for maintenance and propagation with the hope of
future successful reintroduction. These refugia. which are free of
zebra mussels, include sahnonid fish-rearing hatcheries, which
may culture such species as rainbow {Oncorhynchus mykiss) and
brook (Salvelimis fontinalis) trout. With the rearing of these two
hosts (mussels and fish) on the same facility, there exists the
possibility for contagion of pathogens either by shared water or via
contaminated equipment such as boots, nets, and buckets. The
question of contagion is particularly relevant, because one of the
two hosts (freshwater mussels) is originating from a natural envi-
ronment and might be exposed to pathogens of both mussels and
fish that could be introduced to a hatchery along with their relo-
cation. All of the recognized primary bacterial pathogens of salmo-
nids involve, to varying degrees of significance, horizontal trans-
mission, and. hence, there is a potential to develop disease in fish
that might be exposed to pathogens via contamination or by some
vector. It can be speculated that bivalves may serve as a pathogen
vector. On the other hand, it is not known if certain microbial flora
of healthy resident fish might pose a disease threat to mussels once
they are exposed to the pathogen, and they are reared in intensive
culture situations. There are no single pathogens described that
produce disease in both salmonid fishes and freshwater bivalves.
There is a wealth of knowledge on diseases to salmonids, but there
are few reports of diseases and epizootics that occur in native
freshwater bivalves. However, it has been demonstrated that fresh-
water mussels may serve as a vector for fish pathogens, because
Flavobacterium cohunnare. the cause of columnaris disease, was
isolated from a single Amhlema plicala that was collected from the
Ohio river (Starliper et al. 1998).

Presented here are studies that are part of a continuing effort to
evaluate the potential for contagion of bacterial pathogens. We
report on efforts to isolate salmonid bacterial pathogens from
freshwater bivalves that were collected from their natural environ-
ment (without quarantine), and to produce disease or mortality

251

252

Starliper and Morrison

experimenially in bivalves using the representative bacterial flora
from healthy fish and two fish bacterial pathogens.

MATERIALS AND METHODS

Freshwater Bivalves and Fish

Ail bivalves were collected by brailing between August and
November 1997, and this duration was representative of that year's
sampling season. Duration of sampling seasons varies from one
year to another, depending upon water temperature and when ani-
mals emerge from the river bottom. The collection site was be-
tween river miles 175 and 177 at Muskingum Island of the Ohio
River, which is near Boaz. Wood County. West Virginia. This
region of the river is not easily accessible by land, and the only
public use is pleasure boat traffic. The site supports 28 species of
freshwater mussels, including two federally listed species (Lainp-
siUis abrupta and Cyprogeniii stegaria). The island and its under-
water acreage, including the mussel beds, are protected from de-
velopment, because it is within the Ohio River Islands National
Wildlife Refuge. No harvesting or collecting of mussels is allowed
on the refuge except for scientific or management purposes.

Bivalves used to evaluate the presence of fish pathogenic bac-
teria were collected at 2 to 3 week intervals during this season.
Twenty animals were collected on each of six dates (trials 1-6.
respectively), except for trial 6, when only eight were collected
because of low water temperature. Upon collection, animals were
kept cool and moist and shipped overnight by commercial carrier
to our laboratory for bacteriological analysis the following day.
They were not placed in other water in the interim between col-
lection and analysis. Species and physical data for the bivalves
represented in this study are presented in Table I. Also, the dis-
tribution data for Amblema plicala, which was the species most
frequently collected during the season, are given in Table 2. Ohio
River surface water temperatures (°C) were recorded at the time
and collection location for each trial.

Two hundred animals, representing six species, were used as
subjects for bacterial challenges with flora isolated from healthy
fish: A. plicata (123). Qiiadnda metanevra (33), Q. qiiadnila (21 ).
Ohliquaiia reflexa ( 10). Q. pustiilosa (9). and Pleurohema corda-
liiiii (4). Physical data of the animals used in the challenge studies
are not presented: however, mean values were vei7 similar to those
animals used for fish pathogen isolation, which are presented in
Tables 1 and 2. These animals were quarantined for 30 days at the
Ohio River Islands National Wildlife Refuge (Parkersburg. WV) to
eliminate any zebra mussels (Gatcnby et al. I99S). They were then
transported to the Leetown Science Center, where they were ac-

climated to (over 2-3 hours) and maintained in pathogen-free
spring water (12 °C) delivered via a flow through system.

The fish added to the tanks to cohabit with mussels following
their (mussels) challenge were the Nashua strain of brook trout
(Salveliims fontincdis). about 50 g each, and the Shasta strain of
rainbow trout (Oncorhynchus mykiss), also about 50 g each. The
fish were certified as fish pathogen-free through biyearly fish
health inspections by the U.S. Fish and Wildlife Service's Fish
Health Unit. Lamar. Pennsylvania. The fish were maintained in the
same water as described for the mussels; all in vivo studies were
also done using the same water source.

Collection and Processing of Tissues from Bivalves

Tissues were collected and prepared using a procedure similar
to that developed by Starliper et al. (1998). The external shell
surface of each animal was cleaned by brushing with 200-ppm
chlorine, rinsed in deionized water, and allowed to dry. An oyster
knife was used to pry open the shell valves, and the adductor
muscles were cut. All soft tissues were excised from the shells and
were separated into two samples. One consisted primarily of di-
gestive tract tissues, denoted "gut" (e.g.. stomach, intestine), while
all remaining soft tissues, denoted "OT" or other tissues, (e.g.,
mantle, gill. foot, lymph) comprised the second. The gut sample
was removed first, and effort was made not to contaminate the
remaining tissues with bacterial contents from the gut. Soft tissue
samples were placed in preweighed. sterile stomacher bags. The
bags with tissues were weighed, and the difference of the two
weights was the weight of the tissue sample.

Isolation and Growth of Bacteria

For bacterial isolation, the tissue samples were diluted in sterile
0.1% peptone- 0.05% yeast extract (PEP-YE). To each sample, a
volume (mL) of PEP-YE equal to the tissue weight (g) was added
yielding a 1:2 dilution of tissues. This was homogenized for 120
seconds using a Model 80 stomacher (Seward Medical. London
SEl IPP. UK). A portion of the supernatant from each homoge-
nate was transferred to a sterile tube for ease of handling and three
serial tenfold dilutions were prepared from this, also in PEP-YE.
Four drops (0.025 mL each) from all dilutions were applied to the
surface of each bacteriological medium. After the drops had been
adsorbed, plates were incubated at the appropriate temperature and
duration specified in the appropriate reference materials (see Me-
dia employed). Following incubation, colonies were enumerated
by counting the lowest dilution with single colonies, and this was
converted to a standard colony forming units per g of tissue (cfu/
g). For the selective and/or differential media used for specific

TABLE L

Mean value.s for physical data on freshwater bivalves from the Ohio River assayed for presence of bacterial fish pathogens. Animals were
collected al six different times (trials 1-6) durin)> the I***)? collection sea.son.

Species

No.

Length (mm)

Width (mm)

VVeijjht (n)

'7f Soft Tissue"

Gut/OT %"

Amhlemu pliculu

88

loo.y

74.4

238.5

12.5

61/.^9

Qtuulrula quadruUi

7

74.1

5X.4

123.0

10.5

62/38

Ohliqiiariii reflexa

.S

58.8

4(1.8

75.2

11.2

64/36

PU'i<n)l>cmii cordaliim

3

77.0

65.3

142.9

11.7

57/43

Qiuulnilii mcianevni

3

67.3

57.0

107.6

12.0

61/39

QuculniUt ptt.\Uil()s(i

1

.57.0

54.0

63. 1

15.2

53/47

lillipsaria lincoUilii

1

45.0

34.0

25.7

10.9

56/44

" % Soft tissue = the percentage of the total weight that is comprised of soft tissue.
'" Ciut/OT '* = Ihe percentage cif the total weight iif solt lissue in gut arul OT ^amples

Disease Concerns of Mussels and Fish

253

TABLE 2.
Mean values of physical data for Amblema plicala collected during the 1997 sampling season.

Trial

No. Animals

Length (mm)

Width (mm)

Weight (g)

Soft Tissue ( % f

Gut/OT Ratio''

1

13

105.8

76.9

269.9

11.9

59/41

2

18

102.4

76.5

247.6

11.6

59/41

3

18

I0I.9

76.9

252.4

12.1

62/38

4

20

87.9

64.3

153.7

12.7

61/39

5

17

108.6

78.7

283.2

13.9

64/36

6

2

II2.5

80.5

294.7

13.1

63/37

" Percentage of the total weight of the bivalves that were soft tissues used for isolation of bacteria.
'' Percentage of the total soft tissues used for: Gut (/) and OT samples for isolation of bacteria.

isolation of pathogens, suspect colonies that were picked to fresh
media had their identity confirmed as to that particular pathogen,
or not. Biochemical tests employed for bacterial characterizations
were described in the appropriate reference papers or with standard
biochemical characterization (Koneman et al. 1988; MacFaddin
1980). The sets of 1:2 dilutions were used to prepare smears on
microscopic slides for detection of Renibacterimn salmoninanuu
cells using the direct fluorescent antibody test (FAT; Bullock et al.
1980) and commercially available FITC-conjugated antiserum pre-
pared in goats to the whole cells (Kirkegaard and Peny Labora-
tories, Inc., Gaithersburg, MD). One hundred microscopic fields of
view were observed from each stained homogenate at 1 ,000x mag-
nification using a Reichert Diastar Model 420 microscope with a
halogen light source (Cambridge Instruments Inc.. Buffalo. NY).
An R. salmoninaniin-posh\\'e kidney tissue from a diseased
salmon served as a control slide for reference to the correct cell
morphology.

Isolation of A. sahnonicida from fish that died as a result of
cohabitation with A. plicata that were previously challenged with
the bacterium was done using a sterile inoculation loop to collect
kidney tissue, and this was used to inoculate primary isolation
plates. The medium for isolation oi A. salmonicida was tryptic soy
agar (TSA; Difco Laboratories. Detroit. MI) supplemented with
0.01 % coomassie brilliant blue (CBB; Cipriano and Bertolini
1988). The identities of suspect, blue colonies were confirmed as
A. salmonicida with standard biochemical characterization tests.

Bacteria used for the waterbome challenges of bivalves were
isolated from rainbow trout at the White Sulphur Springs National
Fish Hatchery. White Sulphur Springs. WV. This facility was se-
lected, because mussels are currently being held there, and the
resident populations offish are healthy and well maintained; there-
fore, they would be representative hosts of normal tlora bacteria
that relocated mussels encounter, either via the fish as a source or
from their common water supply. Mucus and kidneys from 100
randomly selected rainbow trout were used to inoculate BHIA and
CYTO plate media (described in Media employed). The resulting
bacterial growth on the primary isolation media was observed, and
the most frequent colony types, on the basis of morphology, were
noted. Subcultures of the predominate bacterial types were estab-
lished by transferring representative single colonies onto fresh
plates. These isolates were used to challenge the groups of fresh-
water bivalves.

Media Employed

Thirteen media were employed to isolate bacteria, including
fish pathogens from mussels. These media are routinely used by
fish health personnel to culture environmental bacteria commonly
found on fish or in aquatic environments and for isolation of spe-

cific pathogens. Two were used to determine total bacterial counts,
brain heart infusion agar (BHIA; Difco Laboratories, Detroit, MI)
and Cytoplmga agar (CYTO). a medium of reduced nutrient con-
centration (Anacker and Ordal 1959); plates were incubated at 22
°C for 48 hours. An additional set of CYTO plates were incubated
at 15 °C for culture of Flavobacteriiiin psychrophihim, cause of
bacterial coldwater disease of salmonid fishes (Bemardet et al.
1996). The cooler incubation temperature is favorable for growth
of F. psycliropliiluin. and it also retards the growth of other, en-
vironmental bacteria that facilitate isolation. Three media were
used for Gram-positive bacteria. Rogosa SL (ROGO; Difco Labo-
ratories, Detroit, MI) was used for Lactobacillus and related gen-
era. Azide blood agar base (ABA; Difco Laboratories. Detroit, MI)
containing 5% sheep erythrocytes (Bio-Whittaker. Walkersville,
MD) was used for other species, including Streptococcus spp. and
Staphylococcus spp. Incubation of ROGO and ABA plates was at
28 °C for 3 days. A third, the selective medium (SKDM) described
by Austin et al. ( 1983), was used for isolation oi R. salmoninarum.
cause of bacterial kidney disease (Bullock and Herman 1988). To
enhance growth of this bacterium, the sterilized SKDM was cooled
and supplemented with 17r filter sterilized culture metabolite
(Evelyn et al. 1990) before pouring plates. Following incubation at
15 °C for up to 4 weeks, suspect R. salmoninarum colonies (Sand-
ers and Fryer 1980) were suspended in 0.5 mL PEP-YE; 100 jil of
this was placed on a microscopic slide and air dried. The dried
smears were subjected to the direct fluorescent antibody test, as
previously described. Other media used were for isolation and
enumeration of Gram-negative fish pathogenic bacteria and unless
indicated, were incubated at 28 ^C for 2 to 3 days. Two were for
isolation of Yersinia ruclieri. cause of enteric redmouth disease.
They were the differential medium described by Waltman and
Shotts (SW; 1984) and the differential and selective medium of
Rodgers (ROD; 1992). Suspect colonies were transferred to TSA.
Two media were for Aeromonas spp. One was CBB for A. salmo-
nicida, cause offish furunculosis; these plates were incubated at 22
°C; suspect blue colonies were transfened to TSA. The other me-
dium (SGAP-IOC) was for growth and enumeration of motile
Aeromonas spp. (Huguet and Ribas 1991, Jenkins and Taylor
1995). Although other organisms may grow on SGAP-IOC, such
as Pseudomonas fluorescens, they are easily distinguished from
Aeromonas spp. by colony characteristics and additional biochemi-
cal tests, such as fermentation of glucose. Pseudomonas isolation
agar (PIA; Difco Laboratories, Detroit, MI) was for growth of
Pseudomonas spp., of which many members of this or related
genera are commonly found in aquatic environments or as part of
the flora of healthy fish. Eduardsiella isolation medium (ElM;
Shotts and Waltman 1990) is both differential and selective and
was used for Edwardsiella ictahtri. cause of enteric septicemia of

254

Starliper and Morrison

catfish, and E. tarda, a potential pathogen often found in warm-
and cold-blooded animals. Selective Cytophaga agar (SCA;
Hawke and Thune 1992) was employed as a selective medium for
Flavobacterium colitinnare, these plates were incubated at 37 °C
for additional selection.

Challenge Procedures: Mussels and Fish

For the bacterial challenges of mussels. 20 tanks (27-L each)
were used, each containing 10 mussels. The 200 animals, listed
previously, were distributed equally by species among the 20
groups. The animals were allowed to acclimate in the tanks for 2
d before challenge. Sixteen of the tanks received bacteria: this
included 14 that were of the predominate normal flora from rain-
bow trout from the White Sulphur Springs Hatchery, seven that
were originally isolated on BHIA, and seven from CYTO. Another
two tanks were challenged with the known fish pathogens A.
salmonicida and R. salmoniimnim. and there were four control
groups exposed, one each for the four sterile media used to grow
the challenge bacteria. The challenge strain oi A. sabmmicida orig-
inated from a furunculosis-diseased Atlantic salmon {Salmo salar)
from Vermont, and the R. salmoninantm isolate (ATCC33209)
was from a Chinook salmon {Oncorhynclms tshawytscha) from
Oregon. The normal flora bacteria were each grown in a 20()-mL
quantity of either BHI broth or CYTO broth, the same medium that
was used for original isolation. Aeromonas salmonicida was grown
in 200 mL of TS broth, and R. sabnoninanim was grown in 200
mL of KDM2 broth. Cultures were grown for 48 hours, except for
R. salmoninanim, which was grown for 14 days. A viable cell
count was done for each culture, and this was determined by
preparing sets of serial tenfold dilutions in the homologous broth
medium, and drop inoculating known quantities on plate media.
Colonies were quantified, and the number of cfu/mL of tank water
at the start of the challenges was calculated. To perform a chal-

lenge, the water supply to the tank was turned off, the culture was
poured and mixed in, and exposure was for 24 hours. Then, the
water was turned on and allowed to rinse for 24 hours; then to each
tank, 10 fish were added: five rainbow and five brook trout. The
mussels and fish were then observed for a period of 21 days for
mortality and development of abnormal signs or pathology. Kid-
neys of fish that succumbed were cultured onto the appropriate
medium, and subsequent bacterial growth was identified to con-
firm re-isolation of the bacterium used to challenge the mussels.

RESULTS

Physical data on the animals collected for isolation of bacteria
are presented in Tables 1 and 2. The predominate bivalve species
collected for the six trials was A. plicata. accounting for 81.5 % of
the total. The average weight of the animals varied, depending on
species, and ranged from 25.7 g for the single E. lineolata to 294.7
g for the A. plicata. However, regardless of over-all size, the
percentage of the total weight comprised of soft tissues ranged
from 10.5 to 15.2 %. Of the total soft tissues, the ratios of our
portioned gut to OT samples were also similar among the hosts.
The percentage of soft tissues collected as gut samples ranged
between 53 to 64 %. with the paired OT samples comprising the
balance of the total soft tissues. With exception of trial 4, the
distribution of A. plicata physical data remained relatively similar
throughout the season (Table 2). In five of the trials, the mean
weights of the A. plicata ranged from 247.6 to 294. 7g: whereas,
with trial 4, the average weight was less, 153.7 g.

Results of the bacteriological analyses including enumeration
of bacterial flora, numbers and qualitative characterization of sus-
pect colonies, DFAT staining for R. salmoninantm in tissue ho-
mogenates and Ohio River surface water temperatures at the time
and location of collection for the six trials are given in Table 3.
Total bacteria counts using the routine growth media BHIA and

TABLE i.

Mean bacterial counts (cfu/g), colonies selected and characterization results of suspect fish pathogenic bacteria isolated from freshwater
bivalves sampled six times (Trials 1-6) during the 1997 collection season. The data are mean or summary values for the number of animals

per trial. Trials 1-5 had 2U animals each, trial 6 had eight.

Medium"

Trial 1 (29 C")

Trial 2 (27 C)

Trial 3 (24 C)

Trial 4 (21 X)

Trial 5 (20 C)

Trial 6 (ICC)

BHIA

1.07 X 10"

2.90 X 10'

2.31 X 10'

2.10 X 10'

2.85 X 10'

2.01 X K)-"

CYTO at RniT

1.81 X 10'

4.99 X 10'

2.91 X 10'

4.29 X 10'

2.73 X 10'

1.07 X 10'

SGAP-IOC

3.81 X I0-*

8.39 X lO-*

3.77 X K)-*

2.03 X lO-*

8.07 X IC

2.95 X 10'

PIA

8.37 X 10'

5.41 X lO'*

7.61 X 10'

6.69 X 10'

2.01 X K)-*

3..16X 10'

CYTO at 15 °C

NS'

NS

1 (())''

14(0)

NS

NS

.SW/ROD

18(0)

12(0)

21(0)

21 (0)

21(0)

10(0)

CBB

3 (Ol

12(0)

11(0)

13(0)

12(0)

7(0)

EIM

fi(0)

10(0)

7(0)

8(0)

7(0)

8(0)

SCA

N.S

NS

NS

NS

NS

NS

ROGO

NG

2. I.2x lO^"--

NG

NG

2,8.0x 10'

I.S.Ox 10'

ABA

3. 5.90 X 10'

I.3.20X IO-

NG

NG

NG

NG

SKDM

9(0)

NS

9(0)

14(0)

12(0)

12(0)

DFAT

3:3, 1.40'

2: 1. 1

2: 1.5

3:2. 1. 1

8: 2. 1. 1. 1. 1. 1. 1. 1

1: 1

" Media for total counts: BHIA, brain heart Inlusion agar; CYTO, Cytophut^a agar: SGAP-IOC, for niotilc Aeromonas spp.: ?\.\. Psi-iuloinonns isolation

agar. Grain-negati\'e media: CYTOCn'15 'C. for i'Uivohacterium psychrophiliim: SW/ROD. two media for Yersinia nickeri: CBB. for .4. salmonicida:

bIM. Edwardsiella isolation mcdiimi: SCA, for F. columnare. Gram-positive media: ROGO. primarily for UulobaeiUns spp.: ABA. a/ide hlood agar:

SKDM. for Kenilhii leriiim salmoninanim: DFAT. direct fluorescein antihody test for R. salmoninarum of 1:2 dilution of each tissue.

'' Temperalure of Ohio River surface water at time and location of collection.

••' NS = bacterial growth, but no characteristic colonies present. NG = no growth.

'' Number of suspect colonies picked for biochemical characterization or for SKDM, DFAT for ft. salnioninionni (number positive).

■•' Number of samples with growth, mean cfu/g of those with growth.

' Number of tissues positive: number of positive cells per 100 micro.scopic fields of each positive sample.

Disease Concerns of Mussels and Fish

255

CYTO remained quite similar througliout the sampling season,
with exception of trial 6 for which counts were less, presumably
because of the colder water temperature. The average bacterial
counts on BHIA ranged from 2.01 x 10"* to 2.90 x 10^ cfu/g of soft
tissue and for CYTO. the range was 1 .07 x 1 0' to 4.99 x 1 0^ cfu/g.
The range for Aeronwnas spp. isolated on SGAP-IOC was 2.95 x
10' to 8.39 X lO'* cfu/g; whereas, for Pseudomonas spp.. counts on
PIA were between 3.36 x lO"* and 5.41 x 10"* cfu/g of soft tissue.
All of the Aeromonas spp. were presumptively identified as being
of the motile Aeromonas spp. group, because the only member of
this group that is nonmotile and considered significant with regard
to fish disease is A. salmonicida and none of the suspect blue
colonies transferred and characterized off CBB were nonmotile.
The sum of bacterial counts off SGAP-IOC and PIA accounted for
a large portion of the total bacteria. The water temperature was
lowest when animals were collected for trial 6. not only had the
animals burrowed beneath the surface of the river bottom and
limited the number collected but also resulted in the lowest counts
for total bacteria, motile Aeromonas and Pseudomonas. Con-
versely, the highest cfu/g of tissue for these three groups of bac-
teria were all recorded on trial 2, when the water temperature was
near the highest at 27 °C. The proportion of the total bacteria that
was comprised of motile Aeromonas and Pseudomonas was also
highest from trial 2.

A number of Gram-negative colonies suspected of being patho-
genic for fish were selected off the media CYTO at 1 5 "C, SW/
ROD, CBB, and EIM (Table 3). Suspect colonies were identified
based on meeting criteria set forth in the specific references. After
transfer and biochemical characterization, none was confirmed as
being a fish pathogen. There were no characteristic F. cotumnare
colonies cultured from any tissues of any of the trials. In a previous
year this bacterium was isolated from an A. plicata that came from
the same location in the Ohio River (Stariiper et al. 1998). The
selective Gram-positive isolation media, ROGO and ABA, sup-
ported very minimal growth relative to that of the media used for
Gram-negatives. Bacterial growth was present on ROGO medium
in three of six trials, but from only five of the 96 tissue samples
assayed. The counts of the tissues with growth ranged between
8.0x 10' and 1.2x 10" cfu/g. Tissues from two of the trials showed
growth using ABA medium and these were from only four of 80
samples with averages of 5.90 x lO"* and 3.20 x 10~ cfu/g for trials
1 and 2, respectively. Isolates selected from the ABA culture plates
were also identified as a-hemolytic Lactobacillus and were cata-
lase negative, and. therefore, differed from Carnohacierium pisci-
cola (formerly Lactohucilhts piscicola) that has been previously
noted to cause disease and low mortality primarily in postspawn-
ing rainbow trout (Stariiper et al. 1992). Only in trial 2 was there
any bacterial growth of any tissue samples on both ROGO and
ABA. Serological based observation of the 1 :2 tissue homogenates
for R. salmoninunini using the DFAT showed positive fluorescent
cells of correct morphology from every trial. Trial 5 had the largest
number of tissues (8 of 40) with at least one cell detected within
100 microscopic fields of view. The number of cells detected in
those positive tissue samples was either one or two for most, but
40 cells were seen in 100 fields from one Q. quadrula OT sample.
However, no bacterial colonies yielding fluorescent cells having
correct morphology for R. salmoninarum were noted on SKDM
primary isolation medium from these or any other tissue homoge-
nates. One suspect bacterial colony from SKDM growth plates did
yield excellent positive fluorescence, but the individual cells were

too large to be considered of correct R. salmoninarum cell mor-
phology; this was from a Q. metanevra OT homogenate.

At the start of the 24-hour bacterial challenges, the average
number of viable bacteria was 5.31 x lO'' cfu/mL of tank water for
the seven groups of freshwater bivalves exposed to the selected
normal flora bacteria isolated from fish and grown in CYTO me-
dia. There was an average of 4.89 x 10'" cfu/mL of challenge tank
water in the seven groups of animals exposed to the normal flora
bacteria isolated and grown in BHI medium. There was 1.98 x 10**
cfu/mL of tank water in the group exposed to A. salmonicida and
for R. salmoninarum, there was 3.53 x 10'^ cfu/mL. During the
actual 24-hour waterbome exposures, there was no mortality ex-
perienced in the bivalves. The only bivalve death in the duration of
the study, a Q. quadrula. occuired on day 8 of the 21 -day cohabi-
tation with fish and was in the group exposed to the TSB medium
control. There was no mortality in the brook or rainbow trout in
any groups that were placed to cohabit with mussels previously
challenged with the 14 bacterial types cultured from the White
Sulphur Springs National Fish Hatchery. Also, there were no
deaths of fish in the R. salmoninarum group or the four control
groups exposed to bacteriological media only. However, in the
group of animals challenged with A. salmonicida, mortality in
brook trout began on day 8 of cohabitation, two more died on day
15, and the remaining one on day 21. One rainbow trout died on
day 21. Of these dead fish, there were no external lesions pro-
duced, but there was extensive internal pathology indicative of a
systemic. Gram-negative bacterial infection. There were elevated
amounts of red, ascitic fluid, hemoiThaging of internal organs, and
the hind gut was filled with yellow, pus-like material. Kidney
tissues of dead fish inoculated onto CBB plates resulted in heavy
growth with presumptive blue A. salmonicida. Single colonies
were picked, and their identity was characterized as A. salmonicida
with the following criteria, in addition to blue on CBB: production
of brown, water soluble pigment on TSA, K/A on triple sugar iron
agar, oxidase positive, nonmotile by the hanging drop method,
gelatin liquefaction positive, and a negative ornithine decarboxyl-
ase. The greater mortality in brook trout was expected, because
they are known to be more susceptible to A. salmonicida than are
rainbow trout (Cipriano 1982). After the 21 -day observation, kid-
ney tissues of the surviving rainbow trout were streak plated onto
CBB, and A. salnwnicida was i.solated and the identity confirmed,
as previously, from two of the fish. The 10 mussels in this group
were comprised of seven A. plicata and one each of Q. metanevra.
P. cordatum and Q. quadrula. After the 21 days of cohabitation,
the gut and OT soft tissue samples of each of these animals was
excised, homogenized, and diluted as previously described with
drop inoculation onto CBB plates for isolation of A. salmonicida.
From these, no suspect colonies were cultured from any of the 20
soft tissue homogenates; therefore, A. salmonicida was not re-
isolated.

The 10 freshwater bivalves challenged with R. salmoninarum
included seven A. plicata, two O. reflexa, and one Q. pustulosa. In
contrast with the group challenged with A. salmonicida, no mor-
tality in fish occurred. After the observation period, the fish kidney
tissues of surviving fish were used to prepare smears on micro-
scopic slides for evaluation by DFAT. No fluorescent R. salmoni-
narum cells were detected. At this time, the same was done for the
gut and OT tissues of the ten mussels, and one A. plicata was
positive with two cells of correct morphology for R. salmoninarum
being detected in 100 microscopic fields viewed. However, it is
not known if these cells were alive, beecause the DFAT stains both

256

Starliper and Morrison

live and dead cells. Upon observation of the slides prepared from
the mussel tissues, there was a significant amount of small ( 1(jl in
diameter) fluorescent particles present that did not have uniform
shape and were not confused with intact cells. There were too
many, and each was too small to be quantified accurately. It is not
known if these entities in some way originated from the R. salmo-
niiianmi cells used for challenge or if they were artifactual stain-
ing. In either case, this is unique, because this is not typically noted
on stained slides prepared from fish kidneys, whether the tissue is
positive or not for the bacterium.

DISCUSSION

With intensive fish culture and fish health management, the
best defense against pathogenic diseases is prevention (Piper et al.
1982). All of the major bacterial pathogens of salmonid fishes may
involve horizontal transmission. This might occur among resident
individuals within a facility or could result from introduction of a
pathogen to resident fish by contamination with a new lot of fish
placed into the facility. Relocated fish could be carriers of a patho-
gen and because of their past exposure may have some innate
immunity that would allow them to harbor an organism and not
display any obvious signs of disease. Then, when naive fish are
exposed to bacteria shed by the carriers, an epizootic may ensue.
Because of this potential, it is imperative that fish to be relocated
undergo a health inspection to identify pathogens, including those
that are not obvious because of a lack of clinical signs. The po-
tential for introduction of pathogens via relocated freshwater bi-
valves is also of concern. Animals may be originating from open
and uncontrolled environments, such as the case with those from
the Ohio River, where they might be expo.sed to wild fish that
could be diseased. Bacteria are shed into the environment and the
bivalves could uptake bacteria either as a food source or by simply
filtering contaminated water. The primary pathogens of salmonid
fishes are not known to cause diseases in freshwater bivalves;
therefore, animals would be unlikely to become carriers of a patho-
gen in the sense that susceptible fish can following their survival
of an epizootic. Once freshwater bivalves are removed from the
source (shedding) of a bacterial pathogen and relocated to a patho-
gen-free water supply, such as could be the case of quarantine for
zebra mussels, the length of time the pathogen remains present and
viable in the tissues and could still be infective to fish is not
known. This is especially important considering the results of the
cohabitation study where mussels were exposed to A. salmoiiicida.
In a study by Plusquellec ct al. (1994) with two marine bivalves,
the mussel Mytilus ethilis and the oyster Crassostrea gigas. reten-
tion of the enteric human pathogenic Salmnnetla following artifi-
cial exposure and air drying showed retention of viable cells of at
least .S days al 12 to 15 C and as long as 20 days at 10 C. However,
when the clam Mcneiuirid nienciuirin was artificially exposed to
Escherichia coli or S. lypliiniiiiiinn. and the infected animals were
moved to flowing, pathogen-free seawaler. they are able to reduce
viable cell loads in tissue homogcnates within 24 hours by factors
of 10'' and 10''. respectively, from a starting tissue load of between
1-2 X 10*^ cfu/g (Timoney and Abston 1984). A change in bacterial
flora was similarly noted within 24 hours in freshwater bivalves
that were not initially exposed artificially to bacteria, rather these
animals were simply relocated from one water supply to another
(,Starliper et al. 1998). In this study, the total bacterial count per
gram of soft tissue remained quite stable at both water sources, but
the most notable change was a relative increase of nonfermenting
bacterial types, coinciding with a decrease in other bacterial types
alter 24 hours of being in the different water supply.

The bivalves that were collected in trial 4 were smaller than
those collected in the other five trials (Table 2). The smaller ani-
mals were several years younger than the larger ones. When col-
lecting animals from a large environment, such as the case with the
Ohio River, a group with size characteristics unique to other
groups collected is not unusual. Bivalves are not evenly deposited
on the river bottom, rather, they often occur clustered as to age and
species, which has to do with the movement of the host and where
the juveniles drop from the host. This is particularly relevant with
A. plicata. because of the variety of fishes that may act as hosts and
include many species of the family Centnircliidae. sauger (5;/-
zostedion vitreum). and the flathead cattish iPylodictis olivaris).

In the study by Starliper et al. (1998), Flavobacteriitm colum-
nare. cause of columnaris disease in many cool and warm water
fishes, was isolated from an -4. plicata. This animal was assayed
directly after being removed from the Ohio River and had not been
placed into pathogen-free water for any time prior to analysis. The
pathogen was not isolated from animals that were collected at the
same time, and location but had been in pathogen-free, tlow-
through water for 24 hours. Alone, information on the isolation of
a fish pathogen from a bivalve that could be intended for relocation
to a salmonid rearing facility is discouraging. However, before
relocation, if they undergo quarantine according to a protocol such
as that of Gatenby et al. ( 1998) to ensure that zebra mussels are not
inadvertently spread and if the animals are maintained in patho-
gen-free water, there is the chance that pathogens may be depu-
rated during the quarantine. This is surmised from the demon-
strated rapid change in fiora after being moved to a different water
supply and because F. coliininare was not isolated from animals
after having been relocated for 24 hours.

The detection of cells of correct morphology for R. satmoni-
narum from the DFAT of mussel tissue homogenates could be-
come a concern for fish health managers of salmonid rearing fa-
cilities. This bacterium and the disease it causes, when in a popu-
lation of fish, presents significant health management problems,
because a primary means of pathogen transmission is vertical, in
addition to the fact that it is horizontally transmitted. Therefore,
presence of any fluorescent bacterial cells of correct morphology
noted in fish kidney tissues or ovarian fluids of spawned fish is not
good, particularly at those facilities that are involved with egg
production and shipment. The nature of the bacterium's being very
slow growing and difficult to isolate, especially in low numbers,
only adds to the dilemma of fish health managers faced with the
situation. When fish health inspections yield only minimal num-
bers of positive cells using the DFAT or minimal positive results
from other serology-based assays in the absence of bacterial iso-
lation or clinical disease signs in fish, management personnel are
faced with difficult decisions on the health status iif the population.
Therefore, if relocated mussels might be dctennined to be a source
of a bacterium thai could result in positive cells by DFAT, regard-
less of a lack of culture isolation, a significant concern is realized.
An important topic for further study would be lo evaluate if a
.^0-day quarantine in a clean or different water supply would result
in depuration of cells that may yield fiuorescence.

In the six trials of the present study, the results using the DFAT
and culture on .SKDM for detection of R. saliiuminanim from
bivalve tissues were not in agreement; no cells were noted from
culture; whereas, there were using the DFAT for tissues. Discrep-
ancies of this sort are not uncommon using various methodologies
for detection of this bacterium in fish (Cipriano et al. 1985; Teska
ct al. 1995). A number of factors contribute to this and include the
slow and difficult nature of isolation and growth of R. salinoni-

Disease Concerns of Mussels and Fish

257

nartim. specificity and sensitivity of antisera. the host, and the
extent of infection within a population of tlsh. Fuilhermore, the
DFAT was originally developed as a method for quick, presump-
tive diagnosis of clinical bacterial kidney disease (Bullock et al.
1980) that would later be confirmed by bacterial culture. The
ad\ antage was that a DFAT could be done in hours, as compared
to weeks for primary isolation. This affords quick intervention for
fish health managers to control the disease and prevent further
spread. With heavily infected fish, the two methodologies correlate
well, but when used for relatively healthy, pathogen carriers, the
agreement may decrease.

In the cohabitation group with mussels previously exposed to
A. salmoiiicida and brook and rainbow trout, it is noteworthy that
after 21 days, the bacterium was not re-isolated from the mussels"
soft tissues. This is interesting, because there were infected and
dying fish present in that tank water, and clinically diseased and
carrier fish are known to shed viable A. salmonicida cells. How-
ever, with only four rainbow left at the end, the quantity of bacteria
shed might have been too few to maintain bacterial presence
within the A. plicaui. ALso. the normal flora of the mussels may
have displaced and/or prevented further infection, because to mus-
sels, it is assumed that A. salmonicida is merely an environmental
organism and not pathogenic.

With the A. salmonicida challenge experiment, it is encourag-
ing that after being exposed to a load of viable bacteria in the water
that is greater than would occur naturally, the bacterium was not
isolated from animals after the challenge's observation period.
Again, it would be important to evaluate if the quarantine for 30
days to eliminate zebra mussels is sufficient for native bivalves to
concurrently depurate fish pathogens. Additional topics for re-
search are to extend beyond 24 hours the duration between when
mussels are removed from bacterial exposure and when suscep-
tible fish are introduced. Furthermore, the bivalves can be exposed
to lesser bacterial cell concentrations and a more natural challenge
method, those more analogous to what might be encountered in
nature and then evaluate contagion. Also, a determination should
be made on whether the bacterium enters the soft tissues or merely
is contained in the fiuid portion outside the soft tissues, but within
the shell.

Following the challenge and cohabitation involvins R. salmo-

ninarum and when fish and mussels tissues were evaluated by
DFAT. no cells were detected in fish, and only two were detected
from one A. plicata. These results were not surprising for this
bacterium. Reproduction of experimental bacterial kidney disease
in a laboratory by contact exposure is very difficult and requires
significant effort and time. Wolf and Dunbar ( 1959) were able to
produce mortality in brook trout by a noninjectable challenge with
R. salmoninarum only after fish were maintained in tanks with
bricks in place as a means for abrasion, then the water level was
dropped daily and viable cells were added. Still, it took 96 days for
the first death. In another study. IP injection of brook trout with a
number of viable cells similar to that per mL of tank water used in
the present study, the mean days to death for the group of fish was
about 28 days, and the first occurred on about day 25 (Starliper et
al. 1997). Because horizontal or contamination infection to fish is
difficult and because this bacterium has a limited host susceptibil-
ity range that is known to include primarily salmonid fishes, per-
haps the chance for mussels to act as vectors is remote. Particu-
larly, if mussels are able to rid the bacterium as noted in the present
study when exposed to R. salmoninanim. that after 21 days in
clean water only two cells were detected by DFAT of the soft
tissue homogenates. Furthermore, the high frequency of fluores-
cent particles in the tissues might be indicative of cellular debris
resulting from the bacterial cells being digested by the mussels.

All of the freshwater bivalves in these studies were used, be-
cause they are readily available, and they exist in high numbers;
however, they may not be species that will be collected as part of
the relocation program. Once techniques are developed using com-
mon animals and are available, the appropriate studies may then be
repeated using minimal numbers of surrogates selected to repre-
sent those animals that do fit the criteria for relocation.

ACKNOWLEDGMENTS

Appreciation is extended to those facilities that provided patho-
gen-free fish: The Conservation Fund, Freshwater Institute, Shep-
herdstown, WV; Paint Bank State Fish Hatchery. Paint Bank. VA;
and White Sulphur Springs National Fish Hatchery. White Sulphur
Springs, WV. Also, thanks to Ms. Rita Villella and Dr. G. L.
Bullock for their critical reviews.

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Joiiriwl of Shellfish Research. Vol. 19, No. 1. 259-263. 2000.

DEVELOPMENT OF ARTIFICIAL LOBSTER BAITS USING FISH SILAGE FROM

TUNA BY-PRODUCTS

LAURA CHANES-MIRANDA' AND MARIA TERESA VIANA^

Facultad de Ciencias Marinas
Universidad Aitlonoma de Bcija California
22 800 Ensenada. BC Mexico
^Institiito de Investigaciones Oceanologicas
Universidad Aittonoma de Baja California
22 800 Ensenada. BC Mexico

ABSTRACT The present work shows that an artificial bait can replace the traditional baits (raw fish) using tuna by-products
preserved with citric and phosphoric acids to produce an acid fish silage. After 2 months preservation by mixing minced viscera from
the tuna industry with 2.6% each of citric and phosphoric acids, the silage was ready to be blended in two different mixtures: acid, or
non-neutralized, and neutralized with 5% sodium carbonate to reach a pH close to 6. The baits were made by mixing both types of
silage (non-neutralized and neutralized) with starch, fishmeul. and fish oil to prepare a sausage-like bait weighing 150 g each. A
cellulose membrane was used to cover the bait and a cotton string to close them at both ends. After measuring the soluble protein loss
at different times throughout 48 h, it was shown that baits leach out up to 0.88 mg protein per g bait per hour. Both types of bait did
not show any significant difference compared to the natural bait (raw fish) when tested under commercial fishery conditions. The baits
were also tested for dehydration by hanging until a \49c water content was reached. However, the dry baits showed a lower leaching
rate when they were exposed to water. More experiments should be performed in order to conclude if dried baits are able to attract
lobsters in a way similar to the moist baits.

KEY WORDS: Pamilinis. lobster, artificial baits, baits

INTRODUCTION

The lobster is an economically important resource of Mexico,
especially in Baja California, their principal area of capture (Bri-
ones and Lozano 1994). According to the Mexican foreign trade
bank (Bancomext). 2.552 tons of lobster was captured in 1997 in
Mexico, where the spiny lobster (Panulirus interruptus) was the
most important species, which valued 22 million dollars. This
placed lobster sixth in economic importance of Mexican export
fishery products of 1997.

The fishery of lobster in Baja California it is one of the oldest
fisheries in the region. In 1959, the fishery was given in concession
to 19 cooperatives along the coast. In 1992, the private sector was
allowed to catch lobster with a governmental permission or con-
cession (Diario Oficial de la Federacion 1992), but the lobster
production still remained mainly cooperatives-controlled. Lobsters
are caught from September to February, depending on the repro-
ductive status of the animals. The lobsters are taken in traps, which
usually contain two entrances and one coinpartment for the bait.
Lobsters are nocturnal; therefore, traps are checked daily in the
early morning during open season, at which time, the lobsters are
removed, and the bait is replaced.

Bait is very important for this activity, because the fisherinen
depend upon a regular and quality supply. Bait generally consist of
fresh or frozen fish, which is obtained either by the head office in
the cooperative or the by fishermen, where sometimes a whole day
is devoted to obtaining sufficient bait for the following day. They
act as attractants by the leaching from the hydrolyzed protein or
fluids from fish (Ache et al. 1978), which is sometimes increased
by bacterial decomposition (Zimmer-Faust 1987; Zimmer-Faust
and Case 1983; Zimmer-Faust et al. 1996). The fishermen choose
their type of bait according to their prior experience with different
fish or invertebrate species or size of the bait, which often results
in longer periods of time to get their favorite bait. Cans with fish

in tomato sauce are also sometimes used, where small holes at both
sides of the can are made leaving the can for one or two days.

Baits are one of the main problems faced by this fishery since
large quantities of fish must be stored in short term refrigeration
facilities, resulting in large expenses for the co-operatives. One of
the main co-operatives of the region estimate that up to $50,000
U.S. are used for baits during the season (Jacobo Castro, Coop-
erative head officer, pers.coin.). Moreover, good quality fish suit-
able for human consumption are often used as bait, which has other
implications. According to FAO demand for fish products is in-
creasing and the use for certain products should be destined only
to humans (FAO 1997). Besides the problems above mentioned the
handling of fresh or frozen fish has considerable energy demands
and also requires that fishermen collect their bait daily from the
co-operative office. Fishery camps are sometimes far from the
storage rooms from the head office, which makes this type of
handling difficult.

Several artificial baits have been tried in order to substitute the
fresh and frozen fish (Mackie et al. 1980; Carr 1986; Daniel and
Bayer 1987; Miller 1990 and Mohan-Rajan and Shahul 1995), but
for some reasons non-of them are commercially available. In the
present work a new concept of bait is introduced, which uses tuna
by-products preserved as acid silage extruded in a cellulose mem-
brane.

MATERIAL AND METHODS

Baits Preparation

Tuna by-products were ensiled as described by Viana et al.
(1993). In summary, chopped tuna by-products were mixed with
2.6% phosphoric acid, 2.6% citric acid and 0.1% sodium benzoate
was added as a preservative. The mixture was blended to obtain a
homogenate and left for 60 days in plastic buckets, while adjusting
the pH lower to than 3.5. Further, the silage was separated in two

259

260

Chanes-Miranda and Viana

groups and one of them was neutralized using 5% sodium carbon-
ate as described by Raa and Gildberg ( 1982) prior to cold mixing
with the other ingredients as described in Table 1 . The sausages
were extruded in a pasta Machine (Rosito Bisani TRl 10) using a
funnel and cellulose membrane. The sausages were tied with cot-
ton string at both ends every 15 cm long, and were hang or frozen
until used.

Lab analysis

TABLE 2.

Proximate composition of the artificial baits tested in this study,
given as percentage.

Ingredients

Tuna By-Product Silage (%)

Protein (g/lOOg)
Total lipids (g/lOOg)
Total solids (g/lOOg)

27.4
11.9
46.1

Protein leaching

The baits were tested for protein leaching at 16 °C by immers-
ing 30g baits in triplicate in 250 niL beakers containing 100 mL
seawater at constant turbulence created by a horizontal lab mixer.
A I mL sample of seawater was taken at 0, 6, 12, 24. 30, and 48
h and soluble protein content was determined. Soluble protein was
estimated following the Lowry method (Lowry et al. 1951), using
bovine serum albumin (BSA) for the calibration curve. The
amount of soluble protein reported is expres.sed as mg protein
equivalent to bovine serum albumin (BSA) per g bait.

Dry matter loss

The remaining dry matter was measured al constant dry weight
by recovering the baits after 0. 6. 12, 24. 30. and 48 h immersion
in seawater. Percentage dry weight was calculated as the dried
residue weight of triplicate sainples of each bait after drying to
constant weight at 100 ^'C during 24 h.

Total protein

Total nitrogen was determined using triplicates samples ana-
lyzed by the Kjeldahl method (AOAC 1995). Crude protein was
calculated as % N x 6.25.

Microbiological analysis

Baits were tested for bacterial content under storage at days 0,
8. 16 and 183. Bails samples were stored in plastic bags at 3—1- °C
and total bacterial content was determined (Aerobic plate count
APC) following the procedure described by the FDA-AOAC
(1992).

Dehydration / Re-hydration tests

In order to determine the behavior of baits under storage, the
baits were dehydrated when hanged at 3^ "C until I59f humidity
was reached. The dry baits were tested for time of re-hydralion and
tested for protein leaching as described before.

TABLE 1.

Ingredient composition of the arlificial l)alls tested in this study,
given as percentage of inclusion either wet (silage) or dry matter.

Ingredients

Tuna By-ProducI .Silage

Tuna by-priiducis silage''
Fishmeal''

Modified corn slarcli
Fish oil
Sodium bcn/oate

70.9
15.0
10.0
4.0

0.1

Fishing experiment

Both neutralized and non-neutralized fish silage baits were
tested in the Co-operative of Emancipacion. BCS, Mexico during
6 days in January. 1996. For this. 150 neutralized and 60 non-
neutralized baits were prepared while only 41 neutralized and 12
acid baits were tested in pairs together with a traditional bait (fish
parts). Each bait (artificial or traditional) was introduced separately
to traps in the early morning (5:00 AM) and 24 h later the number
and size of lobster were registered.

Statistical procedures

A one way ANOVA (Zar 1999) was used to compare between
baits, non-neutralized vs. neutralized tuna-fish silage. To test pos-
sible differences between treatments on leaching and stability at
different levels of time an analysis of covariance (ANCOVA) was
used according to Sokal and Rohlf ( 1981 ). To test the baits func-
tionality in the lobster traps, a one way analysis of variance
(ANOVA) was used to compare between non-neutralized and neu-
tralized fish silages and traditional bait. The computer package of
Sigma-Stat for windows, version 1.1 (Jandel 1994) was used in
these statistical analyses.

RESULTS

The lobster baits showed a proximate composition of 27.4%
crude protein, 1 1.9% total lipids and 46.1% total solids (Table 2).
When the neutralized and non-neutralized baits were tested for
leaching of protein no significant differences were observed be-

TABLE 3.
Leaching of soluble protein.

" Made from Ihc cannery industry, kindly provided by Rowen SA. Mexico.
'' Kindly provided by Procsa Hnsenada. Mexico.

Neutralized Tun

a

Non-neutralized

Leaching

Bv -Product

Tuna Bv-Product

P

Time (h)

Silage (mg BSA/g)

Silage (mg BSA/g)

\NOVA

6

7.7 ±0.77

12.0+ 1.66

0.114

12

1 1 .4 ± 0.46

16.5 + 3.97

0.200

24

28.8 ± 2.02

23.6 ± 1.03

0.061

36

36.6 ± 2.03

31.8 ±3.33

0.25')

4S

39.4 ± 2.54

36..<i + 4.41

0.583

Leaching rale

(mg/g/hl

0.84

0.88

Covariance Table

n = 4() K =

= ().X95 K- = (1.802

Source of variation

SS

dt

MS Fs

P

within groups

S.tJO.-S

1

5.005 0.16

0.691

Time (X)

4667..';79

1

4667.579 149.587 0.000

Krror

ll.'i4..S16

37

31.203

Mean values obtained in nculrali/ed and non-neutrali/ed luna by-products
bails. Covariance analysis is indicated below.

Artificial Lobster Baits

261

TABLE 4.
Dry matter loss.

Time (h)

Dry Matter Loss of
Nonneutralized ( % )

Remnant Total
Protein After Baits
Were Immersed in

Seawater at
Different Times ( % )

0

6
12
24
36
48

Rate loss (g/lOOg/hr)

0

ND'

2.05 ± 0.682

5.67 ± 1.07

8.25 + 0.307

14.88 ±0.827

0.14

27.46 ± 0.23
27.08 ±0.1 2
26.03 + 0.04
24.24 ± 0.24
23.85 ± 0.37
22.24 ±2.70

0.11

ND. not determined.
Mean \alues of baits immersed in water at different times.

tween treatments at any time up to 48 h. as shown in Table 3. By
a coxariance analysis it was also corroborated that no differences
were found in the rate of leaching being 0.84 and 0.88 mg/g/h.
respectively.

The dry matter loss on non-neutralized baits showed a rate loss
of 0.14 grams per 100 grams per hour up to 48 h. The loss for the
first 24 h was below 59!- (Table 4). Moreover, by measuring the
remnant total protein it was shown a decrease in protein content
over time, from 27.5% to 22.2% after 48 h with a rate loss of 0. 1 1
g protein per 100 g per hour (Table 4).

The dehydration of baits to a constant 1 4.3% water content was
reached after 40 days from the original of 54. 1 % (Table 5 ). More-
over, those dehydrated baits showed a significantly lower leaching
of protein after being re-hydrated compared to the fresh bait (Table
6) with a leaching rate of 0.55 mg protein per gram bait per hour,
compared to 0.88 observed in the fresh bait. Non-neutralized baits
showed no significant growth of undesirable bacteria with a
growth of less that 25 colonies per gram even after 183 days.

The Table 8 shows the results obtained in the field with tradi-
tional baits (fresh mackerel) compared to the non-neutralized and
neutralized fish silage baits. No differences were detected between
all three different groups with the lobster at commercial size. How-
ever in the small lobsters group (within the illegal size) differences
were observed where the non-neutralized bait showed less attrac-
tant activity followed by the other two treatments, the non-
neutralized and the traditional baits.

DISCUSSION

Tuna by-products are rarely used in Mexico, resulting in large
amounts of products, which are often discharged, causing envi-

TABLE 5.
Mean values for water content in baits left at 4 °C until 40 days.

Time (Days)

Tuna Baits (Humidity %)

0
4
10

17
27
35
40

54. 1 1
.34.29
27.31
18.61
16.72
15.07
14.31

TABLE 6.

Mean values of protein leaching obtained in dehydrated baits either

neutralized and non-neutralized. Coyariance analysis is

indicated below.

Time (h)

Fresh Bait

(mg BSA/g)

Dehydrated Bait

(mg BSA/g)

6
12
24
36
48

Leaching rate
(mg/g/h)

12.0+ 1.66

16.5 ± 3.97

23.6 ±1.03
31.8 + 3.33
.36.5 ±4.4!

0.88

4.71 + 1.06
11.49 ±0.36
14.53 ± 1.50
19.59 + 2.19
21.53 ±0.47

0.55

0.00
0.20
0.00
0.02
0.01

Coyariance Table n = 40 R = 0.884 R" = 0.781

Source of variation

SS

df

MS

Fs

P

within groups

941.719

1

941.719

39.495

0.000

Time (X)

2203.992

1

2203.992

92.434

0.000

Error

882.223

37

23.884

ronmental problems in nearby cities. However, although fish silage
has proven to be a good product as an alternative to fish by-
products (Raa and Gildberg 1982). its production in Mexico has
not been established. Notwithstanding, fish silage has been de-
scribed as an ingredient in abalone feed formulation (Viana et al.
1996). The present work demonstrates that fish silage can be ef-
fectively used as an ingredient to formulate lobster baits without
observing any significant difference compared to the fresh fish
regularly use in Mexico as bait.

Fishermen use a piece of fish every day. which can weight
between 500 g to 1 kg (Raul Cells, pers. comm.). This piece is
changed every day in order to be attractant to lobster. The baits can
be supplied from the Cooperative's head office or by the own
fisherman. As stated before it is difficult to estimate the cost for
bait use in the season for all co-operatives, but they need to change
opinion to a better opportunity in order to be able to stand the
market price fluctuations.

The fish silage baits presented here are easy to use and the price
would be far less if fish by-products are used. No differences were
observed in leaching and as attractants between neutralized and
non-neutralized baits (0.84 and 0.88 mg protein/g/h, respectively),
suggesting that neutralization with 5%' sodium carbonate is not
necessary. This means that production of bait will require fewer
procedures while conserving the bait at low pH. Bacteria grow
very easily in fish products due to their highly digestible protein
and soluble carbohydrates. At lower pHs as in the acid silage it is
known that essentially no bacteria will grow (Raa and Gildberg

TABLE 7.
Total bacteria count in lobster baits at different times.

Neutralized Tuna

Time

By-Product Silate

(Days)

(colonies/gl

16
183

<25
<25
<25
<25

262

Chanes-Miranda and Viana

TABLE 8.

Average value of lobsters caught per trap under
experimental conditions.

Fresh Bait
(Mackerel)
Variable (n = 41l

Neutralized Tuna Non-Neutralized
By-Product Tuna By-Product

Silage (n = 41) Silage (n = 15)

Small lobster
(illegal size)

Commercial
lobster
(legal size)

Total

16.32 ±1.58" 12.63 ± l.vg-*"

1.15±0.16''
17.47 ± 1.65"

1.02 + 0.19"
13.66 ± 1.87""

6.91 ±3.32''

1.00 ±0.35"

7.92 ± 3.46*"

Significant differences are indicated with different letters for each variable
(/>< 0.015).

1982). However, when the acid fish silage is combined with other
feed ingredients the pH in the mixture approaches 6 depending of
the type of ingredients (Rivero and Viana 1996). In the present
work the non-neutralized baits mixed together with fishmeal and
starch at low proportions bacteria failed to grow even after 183
days (< 25 colonies/g). which means that the presence of non-
neutraUzed silage could effectively inhibit decomposing bacteria.
This is considered of particular importance since food for human
consumption needs to be free of microbes, it will be necessary to
maintain baits and lobster under hygienic conditions during their
fishery.

When baits are hang they can be reduce to humidity as low as
14% after 40 days. Even if leaching was observed after re-
hydration, the amount that washed out was significant lower (0.55
compared to 0.88 mg protein/g/h). In the present work it was not
tested the effect of dry baits as attractants and therefore it will be
necessary to performed another experiment in order to conclude

their effectiveness. In the meantime, baits can be packed in
vacuum bags to avoid dehydration if longer periods of time are
necessary to store without refrigeration as was done in the bacteria
growth test. Both neutralized and non-neutralized baits performed
similarly in terms of leaching, dry matter loss and for catching
legal size lobster. However, in the field trial, small lobsters (illegal
size) showed to be less attracted by the non-neutralized than that of
the neutralized fish silage and traditional baits (6.91 compared to
12.63 and 16.32 lobster/trap, respectively). The reason is difficult
to explain, but it could also be an advantage to catch fewer illegal
size lobsters.

Although several types of baits have been tried before ( Koyama
et al. 1971; Cange et al. 1986; Huner et al. 1990; Miller 1990;
Brown et al. 1995; Mohan-Rajan and Shahul 1995), none of them
have been successfully used on a commercial scale. This could
have been due to different reasons like the cost of those baits,
hygienic conditions or space in the fishing boats. This bait pre-
sented here could be of importance since uses inexpensive feed
ingredients and stores readily without refrigeration; are microbial
safe and its size make it easy to handle in a small boat. Neverthe-
less, in order to make the present baits commercially available, the
direct involvement of the Cooperatives will be necessary since
they will be the principal or the only buyers of the.se type of baits
since the lobster is on concession to them.

ACKNOWLEDGMENTS

Our thanks to the people of the Co-operatives Emancipacion
and Ensenada for their support in testing the baits in the field.
specially to David Camacho. We also thanks to Dr. Armando
Shimada for his valuable criticism on the statistics. This project
was partially financed by Conacyt Project No. 0237-A9I07. The
author received a fellowship from the DGETl (Direccion General
de Educacion Tecnolosica e Industrial).

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on artificial baits for fishing. .Artificial bait for tuna longline. Bull.

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lobster, Htimant.s gamnuinis: laboratory and field trials. ./. Coits. In!.

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Aqtial. Set. 47:1228-1250.
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Nutr. pp 343-119.
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Artificial Lobster Baits 263

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Aqiuuulnire 140:87-98. dall). Biol. Bull. 164:341-353.

Zar, J.H. 1999. Biostatistical Analysis. 4* edn. Prentice Hall, USA. 663 pp. Zimmer-Faust, R.K., P.B. O'Neill & D.W. Schar. 1996. The relationship

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Journal of Shellfish Research. Vol. 00. No. 0. 265-274. 2000.

DESCRIPTIVE STATISTICS OF FISHING PRACTICES, POSTHARVEST HEALTH STATUS,

AND TRANSPORT CONDITIONS IN THE PRINCE EDWARD ISLAND LOBSTER

{HOMARUS AMERICANUS) INDUSTRY

JEAN LAVALLEE,' * K. LARRY HAMMELL,'
ELIZABETH S. SPANGLER,' RICHARD J. CAWTHORN,^
IAN R. DOHOO'

A VC Lobster Science Centre, and
^Department of Health Management, and
'Department of Pathology and Microbiology

Atlantic Veterinary College

University of Prince Edward Island

Charlottetown. Prince Edward Island

Canada, CIA 4P3

.ABSTRACT This study describes and compares lobster fishing and handling practices on various boats, transportation conditions
between fishing wharfs and processing plants, and health assessments of lobsters followed from the time of harvest to the time of arrival
at the processing plants during the spring and fall fishing seasons of Prince Edward Island. Canada. A total of 2,191 lobsters landed
from 64 boats in 1997 were tagged and included in the study. Over 20 fishing and transport-level factors were monitored, and more
than 10 lobster-level factors were assessed on market-sized lobsters. A significant increase {P < 0.05) of 7.1% in the proportion of
lobsters with open wounds from the time of harvest to the time of entry in the processing plant was found during the spring season;
whereas, the proportion of lobsters with vigor loss significantly increased by 2.5% (P < 0.05) during the same interval. Total hemocyte
counts (THC) and hemolymph total protein (TP) levels were significantly higher in the spring than in the fall {P < 0.001). THC and
TP also increased significantly tP < 0.05) from the time of harvest to the time of arrival at the processing plants, a period in which
the lobsters were held out of the water. The prevalence of Aeroccociis viridans infected lobsters was significantly iP < 0.001 ) higher
in the fall season (10.4'*) than in the spring season (5.5%). Lobster catches experienced significantly warmer, windier, and sunnier
conditions in the fall season (P < 0.05). Mackerel was the bait most commonly used during both seasons, and gaspereaux were only
used during the fall season. In the spring season, lobsters of different sizes were prevented from having mutual contact on more than
63% of the boats, but only on 18% of the boats in the fall season. Most spring fishers (83.1%) added water to the live-tank after all
the traps were hauled, as compared to a majority of fall fishers (72.7%), who had no water in the live-tank at any time (P < 0.001).
Finally, lobsters spent, on average, significantly (P < 0.001 ) more time on board fishing vessels in the fall than in the spring season.

KEY WORDS: Lobster. Homariis americamis. fishing practices, postharvest, health

INTRODUCTION

The lobster (Homarus americamts. H. Milne Ecjwarcjs 1837)
fishery is one of the most economically important fisheries in
Atlantic Canada. Although Canadian landings reached a record
peak in 1991 with more than 48.500 metric tons, they have re-
mained more or less stable since 1992 at about 40.000 metric tons,
with an estimated landed value of almost $400 million (Can) in
1997 (Fisheries and Ocean Canada 1999). In 1997. Prince Edward
Island fishers landed a total of 8.096 metric tons (20.8% of total
Canadian landings) with a value of more than $73,8 million (Fish-
eries and Ocean Canada 1999).

To supply the market with live product year-round, the industry
holds live lobsters in captivity for various periods, ranging from a
few hours to several months. However, preprocessing mortalities
can cause tremendous losses. These losses have been estimated by
the lobster industry to be in the range of 10-15% (Cawthom
1997). The ability of the industry to maximize economic returns is
reduced by lack of knowledge of factors contributing to these
losses. Presently, there are no cohesive strategies that link all par-

*Author to whom correspondence should be addressed. Current address:
Aquatic Science & Health Services, 158 St-Peters Road. Charlottetown.
Prince Edward Island, Canada, CIA 5P8.

ticipants, from fishers to consumers, to detect and quantify pro-
duction inefficiencies.

The lobster fishery is primarily a specialized in-shore small
boat fishery (Pringle and Burke 1993). There is a consensus in the
Canadian lobster industry that substantial variation in the quality
of the product landed by different fishing boats and also among
different fishing wharves is present. Variations in lobster health at
the time of arrival at processing plants and storage facilities, and
when lobsters are removed from short-term or long-term holding,
can be partially attributed to conditions at the time of landing.
Therefore, wharf-level factors and boat-level factors would di-
rectly influence lobster health. To understand fully the total varia-
tion of lobster health that is affected by fishing boats, further
assessment at the boat level is needed. Paterson and Spanoghe
(1997) suggested that sampling lobsters at various points of han-
dling should yield valid information on stressors causing fatigue,
weakness, and death. Following and assessing lobsters through the
different handling points enabled the current study to produce a
precise estimate of variation in individual lobster health. Identifi-
cation of the frequency of losses and correlation to specific fishing
or handling practices could contribute to better management. Sub-
sequent reduction of losses, even by minimal amounts, could pro-
vide significant increases in economic returns to the lobster indus-
try. Fishers, buyers, pound operators, processors, and exporters

265

266

Lavallee et al.

might change their handling methods if provided with results that
demonstrate which handling events affect productivity.

The primary objectives were to assess lobster health immedi-
ately after removal from the fishing traps, to reassess their health
at the different handling points, and to describe fishing practices,
handling events, and lobster transportation conditions with pos-
sible effects on lobster health in Prince Edward Island during the
1997 spring and fall fishing seasons. A subsequent paper (Lavallee
et al. 2000) evaluates relationships between risk factors and lobster
health.

MATERIALS AND METHODS

Several lobster health indices were monitored on fishing boats
during the 1997 spring (May and June) fishing season in federally
designated Lobster Fishing Areas (LFAs) 24 and 26, in Prince
Edward Island. Lobster health was also monitored at wharves and
processing plants where lobsters were handled. Sampling was re-
peated during the early part of the 1997 Prince Edward Island fall
fishing season in LFA 25 (mid-August to mid-October). A total of
12 wharves in the spring season and three wharves in the fall
season were included in the study.

Fishing Boat and Wharf Selection

A list of all lobster fishing wharves located in Prince Edward
Island was provided by the Department of Fisheries and Oceans
Canada (Charlottetown. PEI). A randomized sample of wharves
taken from all lobster fishing wharves with more than 10 boats per
wharf was computer generated (Minitab-. version 1 0.1, Minitab
Incorporated, State College, PA, USA, 1994) and divided into two
components based upon the fishing season: spring or fall. A bro-
chure describing the project was distributed to fishers at each
wharf sampled. Fishers were asked to accommodate an extra per-
son on board to tag and assess lobsters landed on 1 day and also to
monitor fishing practices. Fishers were free to decline participa-
tion. An average of two wharves were sampled each week, de-
pending on weather conditions and fishers" cooperation. Fishers
were selected randomly when more participants than needed
agreed to cooperate. Sample-size determinations were based on the
maximum expected number of boats on which fishing practices
could be evaluated and lobsters tagged and assessed, according to
the length of the fishing season (spring) and the availability of
technical help (fall).

Lobster Selection

Only market-sized lobsters with a minimum carapace length of
81 millimeters (mm) were included in this study. Individual lob-
sters were identified by placing a 25.5 cm onc-piecc prenumbered
plastic tag with a pull-tight seal (Ketchum Manufacturing Inc..
Ottawa, ON) around the knuckle, proximal to the claw. Whenever
sufficient lobsters were available, a .sample size of at least 50
lobsters, based on the average number of individual market-sized
lobsters expected per boat, was identified using this tagging
method.

Physical Examination of Lobsters

Carapace length (measured in mm), sex, shell score (from 0 to
5. depending on (he severity of the lesions), and physical condition
index were recorded for every tagged lobster. Lobster physical
condition index encompassed assessments of damaged or missing
claws, legs and antennae, damaged body, open wounds, and vigor

status (normal, decreased, or dead). These assessments were per-
formed on the boats immediately after lobsters were removed from
the traps. Body weight, recorded in kilograms (Accu- Weigh DSY-
1000, Industrial Scales Ltd., Surrey, BC). shell score, and physical
condition index were also measured on every tagged lobster at
fishing wharves and at arrival at the processing plant, whenever
possible.

Hemolymph Sampling

Hemolymph was sampled from a systematic random sample of
approximately 15 lobsters per boat for determination of
hemolymph total protein (TP). total hemocyte counts (THC). and
evidence of Aerococciis viridans (causative agent of gaffkemia)
infection status. Whenever possible, the same sampling procedures
were repeated when tagged lobsters were landed at the wharf and
after they reached the processing plant.

Using a 3-mL syringe with a 23-gauge needle, 1 .6 mL of
hemolymph was aseptically removed from the ventral sinus after
swabbing the surface with 70% alcohol. THC were performed
according to the method described by Horney et al. (2000): 0.5 mL
of hemolymph was added to a lO-inL plastic tube containing 4.5
mL of artificial sea water (ASW) buffer with 0. 1 % formalin. The
tubes were inverted 20-30 times and placed on ice until cell counts
were performed with a hemocytometer (double dark line Neubauer
improved counting chamber, la Fontaine. Dynatech. Germany) us-
ing light microscopy (Zeiss Standard 16 microscope. Germany)
with a 40 power objective, by counting 20 squares per sample. The
presumptive phenylethylalcohol (PEA) broth test was used for A.
viridans isolation (Stewart et al. 1966) by adding 0.5 niL of
hemolymph to 4.5 mL of PEA broth, vigorously shaking and in-
cubating at 28 °C for 96 h. Duplicate PEA broth cultures were
inoculated for each lobster. Suspicious broth culture tubes were
identified by the typical purple to green to yellow color change of
the broth, and confirmatory testing for the presence of A. viridans
(tetrad-forming Gram-positive cocci) was performed using Gram
stain and microscopic examination. The remaining 0.1 mL of
hemolymph was placed on a temperature compensated refractom-
eter for direct reading of total solid protein. The readings were then
incorporated in the following formula to be converted to total
protein as if measured by the biuret method (Homey et al. 2000):

Lobster TP = 0.898 (refractometer reading) - 7.3
Assessment of Fishing Practices

A three-part data collection fonn was used on board each fish-
ing boat from which lobsters were sampled. The first part. Iden-
tijlcation and Boat Specifications, collected data on the crew size
and number of years of experience of the captain or owner of the
boat.

The second part. Environment, included information on air and
surface water (less than 1 meter deep) temperature (°C). Strength
(from none to storm), duration (in hours), and description of pre-
cipitation (inlerniiltent or continuous) were recorded. In addition,
this part included information on sunshine (in hours) and wa\'e
conditions (from none to >2 meters). Wind speed was also sub-
jectively scored from 0 to 4 (0 = none, 1 = light, 2 = moderate.
3 = strong, and 4 = storm).

The third part of the data collection fonn. Fishini- Practices.
collected data on how lobsters were caught and handled on the
boats. Bait used in the traps was classified as gaspereaux, mack-
erel, herring, flatfish, eel pout, rcdfish, crab, or other. The use of

Lobster Health, Handling, and Fishing Practices

267

a proiective tarp over the lobster crates or totes, possible contact
among lobsters before banding, having the lobsters loose on the
deck at any point, and "packing over" and "dumping" of the lob-
ster, either by the fishers or the buyers, were also evaluated. Pack-
ing lobsters over was defined as completely emptying a crate by
taking each lobster one by one and repacking them into another
container. Dumping lobsters was defined as transferring the entire
crate or tote into another container simultaneously.

Holding units (small containers in which lobsters are first
placed after being removed from the traps and after being graded/
banded), and before and after grading were recorded as wooden
crate, plastic tote, barrel, tray, polyvinyl chloride (PVC) tube,
homemade box. other, or none. The live-tank system (large con-
tainers in which the holding units are placed) used on board the
fishing vessels was recorded as none. "X-Actics"''" box." fiberglass
tank, or other. The presence of a lid while fishing and on the way
back to the wharf was ranked as either none, partially on, or totally
on. In addition, data were collected on the availability of water in
the tank system while fishing and on the way back to shore, with
the following six categories: "none:" "stagnant." where water was
poured on the lobsters but with no flushing: "tlow-through." where
water was constantly pumped in the live-tank: "poured on," when
water was poured on the lobsters and immediately flushed out:
"ice." if the lobster were kept on ice; and "other." Furthermore,
information was recorded on the maximum and minimum time a
lobster could have been on the boat: the maximum time repre-
sented the period from when the first lobster was caught until the
boat arrived at the wharf, while the minimum time was the same
interval for the last lobster caught. The over-all handling of the
lobsters on each boat was graded as either generally "placing" or
"tossing" the lobster from the traps to the temporary holding units.
Finally, the trap setting configuration used by the fishers was
documented with four categories: single (one trap per buoy),
double (two traps per buoy), multiple (more than two traps on a
longline marked by two buoys), or a combination of the previous
three methods. The depth (maximal and minimal) at which the
traps were set was recorded in meters.

Assessment of Transport Conditions

The different transportation vehicles and conditions were also
recorded by the investigators. Data collected included a general
description of the vehicle and whether lobsters were transported in
an open bed truck (pick-up truck), in a permanently closed trans-
portation compartment (without refrigeration), or in a refrigerated
transportation compartment. The outside temperature was recorded
in degrees Celsius, and the weather conditions were subjectively
described. Availability and of ice was noted as yes or no, and the
type of ice during transport was noted as either, freshwater or
saltwater ice. The use of wooden crates or plastic totes was also
recorded. Finally, the time interval between the fishing wharf and
the processing plant and the total time the shipment spent in the
vehicle were recorded in hours.

Statistical Analysis

All data collected were entered in a computer using spreadsheet
software (Quattro® Pro version 7, Corel Corporation Limited, Ot-
tawa, Ontario, Canada. 1996). A random sample of 120 records
was examined for data entry errors by comparing against original
datasheets. The dataset was transferred into a statistical software
package (STATA^^' 5.0, Stata Corporation, College Station,

Texas, USA. 1996) for further analysis. Validation of data was
done by obtaining descriptive statistics and looking for outliers.
Frequency distributions were generated for each categorical vari-
able and collapsed into dichotomous variables if obvious distribu-
tion patterns were seen. Analysis included descriptive statistics,
binomial probability tests for gender ratios. Chi-square tests for
comparisons of proportions. ;-tests for comparisons of means in
continuous variables, multiple comparisons (analysis of variance:
ANOVA) with Bonferroni adjustments for categorical variables.
For all analyses, differences were considered significant when P <
0.05. Results are presented as mean values ± standard deviation
(SD), or proportions with the corresponding binomial exact 95%
confidence interval (CI).

RESULTS

Fishing Wharves, Boats, and Lobsters Assessed

The number of tagged and sampled lobsters was limited by the
daily catch. In total. 2.191 lobsters (1.672 in the spring and 519 in
the fall) from 64 boats (53 in the spring and 1 1 in the fall) were
examined. Approximately 74.5% (1.245 lobsters) of the lobsters
tagged on board fishing boats during the spring season were fol-
lowed to processing plants compared to 6 1 . 1 % ( 3 1 7 lobsters ) in the
fall season. Finally, 36.4% (609 lobsters) of the lobsters examined
on the boats in the spring had hemolymph sampled: whereas, this
proportion was 31.8% (165 lobsters) in the fall. The compliance
was excellent in the spring, with only one fisher out of 52 who
were approached declining to participate: whereas, the fall com-
pliance was fair with five fishers out of 16 declining to participate
in the study.

Physical Examination of Lobsters

The sex ratio of tagged lobsters for the spring and fall seasons
together was not significantly different than 1:1. with 52.0% fe-
males and 48.0% males (n = 2,180 and P = 0.07). However, the
gender ratios differed significantly from a I : I distribution in the
spring (n = 1,665) and the fall (n = 708): 44.5% of the lobsters
were males in the spring compared to 59.4% males in the fall.

No significant seasonal differences in lobster body weight {P
= 0.7081 or carapace length (P = 0.872) were observed. Lobster
body weight for both seasons ranged from 0.264 kg to 2.318 kg
with a mean of 0.599 kg (±0.242 kg), and the mean carapace length
for both seasons pooled together was 89.1 mm (±9.2 mm) and
ranged from 77 mm to 154 mm.

With the exception of vigor, statistically significant differences
were observed between the spring and the fall seasons in every
physical index assessed, and these differences were present at
every source of assessment with a consistent higher proportion of
lobsters with normal indices in the fall (Table 1). Although no
decrease in the proportion of lobsters with normal physical index
was observed in the fall between the time of harvest and the time
of landing or the time of arrival at the processing plant, some
significant differences were recorded in the spring season. The
proportion of lobsters with normal vigor at the processing plant
was significantly lower than at the time of harvest or time of
landing (Table 1 ). A significant increase in the proportion of
wounded lobsters was also observed between the time of harvest
and the arrival at processing plants, as reflected by the decrease in
the proportions of normal legs and antennae at the processing

268

Lavallee et al.

TABLE 1.

Percentage of lobsters with normal physical indices for each Zone,

and at each assessment source during the 1997 spring and fall

lobster fishing seasons in PEI.

Lobsters with Normal Indices ( % )

Physical Indices

Spring Season

Fall Season

Claws

Boat

85.3°'

93.1"'

Wharf

83.5"'

93.8"'

Processing plant

83.7»'

90.2"'

Legs

Boat

93.7"'

98.1"'

Wharf

88.7--

97.9"'

Processing plant

88.6"-

96.9"'

Antennae

Boat

86.8"'

98.7"'

Wharf

85.7'''-

97.9"'

Processing plant

82.8"-

97.8"'

Body

Boat

93.1"'

96.9"'

Wharf

88.5"-

97.2"'

Processing plant

90.6"'-

96.5"'

Wound

Boat

87.9"'

94.8"'

Wharf

84.1"'--

93.1"'

Processing plant

80.8"-

92.1"'

Vigor

Boat

99.7"'

98.1"'

Wharf

99.1"'

98.6"'

Processing plant

97.2" -

96.9"'

Significant differences are represented by different superscripts: letters
within each row and numbers within each column. The normal score for the
index "wounds" indicates the absence of wounds. In the spring, n = 1 ,672
on the boats, 866 at the wharves and 1 ,245 at the processing plants, and in
the fall, n = 516 on the boats, 145 at the wharves and 317 at the processing
plants.

plants. None of the lobsters assessed during this study suffered
from shell disease.

Hematology

Hcmolymph Total Protein (TP)

For every source of assessment (boat, wharf, and processing
plant), the mean TP levels in the spring season were significantly
higher than TP levels in the fall season for both male and female
lobsters, with mean values ranging between 57.6 g/L and 70.5 g/L
in the spring and between 3 1 .9 g/L and 47.5 g/L in the fall (Table
2). Female lobsters had significantly higher TP levels than male
lobsters at ever assessment source in the spring and also at the
processing plant in the fall season, but male lobsters had higher TP
levels at the wharf than females in the fall (Tabic 2). In the spring
season, TP values of female lobsters were significantly higher al
the lime of landing than al ihc lime of harvest (Table 2).

Total Hemocyte Counts (TIK I

•Significantly lower THC values were observed at the boat level
during the spring season, as compared to the fall season; whereas,
higher counts were seen al the lime of landing in the spring, as
compared to the fall season (Tabic 2). Similar to TP, some gender

differences in THC were also observed in both fishing seasons: in
the spring, the only significant difference between sexes was ob-
served at the time of landing, with male lobsters having a mean
THC lower than female lobsters. In the fall season, male lobsters
also had lower THC than female lobsters at the time of arrival at
processing plants, but had significantly higher THC than females
at the time of harvest (Table 2). For both male and female lobsters
during the spring season, significant increases in THC were ob-
served between the time of harvest and the time of landing. In-
creases in females THC in the spring and fall were also significant
between the time of harvest and the arrival at processing plants,
while being significant only in the spring for male lobsters (Ta-
ble 2).

Aerococcus Viridans Prevalence

The spring prevalence (n = 635) of Aerococcus viridans. the
causative agent of gaffkemia was estimated at 5.33'7r (3.88%,
7.61%) and was significantly lower {P < 0.001 ) than the fall preva-
lence (n = 173) of 10.41% (6.28%, 15.95%). No significant dif-
ference in the prevalence of A. viridans according to sex in either
season was observed.

Fishing Practices

Crew Size and Experience

There was no significant difference in the distribution of the
size of the crew between the spring and fall seasons, and overall,
the majority of crews consisted of fewer than three persons (Table
3). A significant difference between the two seasons {P = 0.037)
was observed in the mean number of years of experience of the
captain with fall captains being more experienced (Table 3).

Environmental Factors

The maximum and minimum air temperatures and the water
temperature were all significantly lower (P < 0.001 ) in the spring
than in the fall (Table 3). A significant difference (P = 0.024) was
also found in the amount of sunshine during fishing, between the
spring and fall seasons, but not in rainfall (Table 3). The amount
of sunshine was greater than 6 hours on a majority of boats in the
fall; whereas, the majority of spring boats experienced 6 hours or
less. No significant difference was observed in the wave conditions
or the wind velocity between season, and overall, the waves were
estimated to be of an approximate height of one meter or less;
whereas, on a majority of boats, the wind was scored as being none
to light (Table 3).

Setting Cunllguralion of Traps and Hail

Fishers used four differcnl trap setting methods: single, double,
muhiplc, or a combination of the previous three methods. A sig-
nificant difference (P < O.OOll in the methods used was found
between seasons (Table 3). For example, multiple traps per line
were used on 95.6% of the boats in the spring, but never used in
the fall fishing season. Significant differences were present be-
tween .seasons {P = 0.005 for the minimum depth and P < 0.001
for the maxinnmi depth), and both the maximum and minimum
depths at which traps were set during the fall were greater than
during the spring (Table 3l. A significant difference (P < 0.001)
was observed in the distribution of baits used between the two
fishing seasons; although mackerel was the bait most commonly
useil in both seasons, gaspereaux was only used in the spring, and

Lobster Health, Handling, and Fishing Practices

269

TABLE 2.

Range, mean, and standard deviation of total hemolymph protein ITP) and total hemocvte counts (THC), according to the source of
assessment and the Tishing zone during the 1997 spring and fall lobster Pishing seasons in PEL

Variable

Spring

Fall

Source

Gender

Mean (SD)

Gender

Mean (SD)

Boat

Male

57.6(14.3r'-''t

Male

39.8(12.3r---t

Female

65.4(21.9f-'-t

Female

40.5 (22.7f'--t

Wharf

Male

60.4(12.5r'-t

Male

38.9 (7.9)"--t

Female

70.5 (20.0)'''-:t:

Female

31.9(I1.5r'--1:

Processing plant

Male

59.5(12.4r'-t

Male

40.7(1 0.8 )-'---t

Female

65.6 {20.Sr-'-t

Female

47.5 (22.9)"---1:

Boat

Male

20.4iS.5r-'-f

Male

25.3 (7.9r--t

Female

20.8(10.7)^'t

Female

20.6(7.3)''-'-t

Wharf

Male

30.2 (9.2 ^'t

Male

27.8 (8.2f -'-t

Female

33.4(11.3)*''1:

Female

26.9(3.7r'''-t

Processing plant

Male

30.3 (lO.S)"'!

Male

27.5(8.6f't

Female

32.0(15.8)''-'-t

Female

31.3(13.8)''-'t

Total protein (g/L)

Hemocvte counts (xlO* cells/ml)

Significant differences are represented by different superscripts: letters for the differences among sources of assessment for each both variables, in each
gender and each season: numbers for the differences between season for both variables, in every source and each gender; and symbols (t and |) for
differences between gender for both variables, in every source and each season.

herring was used more often in the fall than in the spring lobster
fishing season (Table 3).

Contact Before Banding and 0> er-all Handling of Lobsters

The proportion of boats on which physical contact among lob-
sters was prevented until they were measured and had their claws
banded was significantly higher (P < 0.001) in the spring than in
the fall (Table 3). Market-sized lobsters, after being removed from
the traps, were generally tossed rather than placed into the tem-
porary holding units on only 25'7f of the boats in total, and no
difference was observed between seasons (Table 3).

Holding Unit and Live-Tank System

In both fishing seasons, the traditional plastic tote with a stor-
age capacity of approximately 35 kg, represented the holding unit
mostly used onboard fishing boats, whether it was before or after
the lobsters were measured, graded, and banded (Table 3). Al-
though no significant difference was noted between seasons in the
distribution of the holding units used after grading, a difference {P
< 0.001) was observed before grading; during the spring, the
wooden crate was not used, as compared to more than 45*^ for the
fall fishers. No difference between seasons was observed in the
type of live-tank used on board the fishing boats, and overall, the
fiberglass tank was mostly used (Table 3).

Water .■\vailability and Lid Cover with the Live-Tank

In the spring, a majority of the fishers waited until all traps had
been retrieved before adding stagnant water into the live-tank, and
this practice was significantly different (P < 0.001 ) than during the
fall season during which, most fishers never added water into the
live-tank (Table 3). However, most fall fishers had the lid cover
completely on the live-tank at any time; whereas, only 38% of the
spring fishers had the cover completely on during fishing (Table
3). The difference between season in the lid availability distribu-
tion was only significant during fishing (P < 0.00 1 ), and not once
all the traps had been retrieved.

Time on Board Fishing Boats

Only the maximum amount of time lobsters spent on board
fishing boats was significantly different between the spring and the
fall seasons {P < 0.001), with longer maximum time on board
fishing vessels in the fall season (Table 3).

Transport Conditions

Transportation Vehicles and Ice with Transportation

A significant difference {P < 0.001 ) was present in the different
vehicles used between spring and fall seasons to transport lobsters
from wharves to processing plants (Table 4). During the spring, the
vehicles mostly used consisted of trucks with refrigerated trans-
portation compartments; whereas, in the fall, only closed trucks
without refrigeration were used. Whenever ice was used around
lobster crates or totes, it was freshwater ice, and during both sea-
sons, a majority of shipments were sent to processing plants with-
out ice.

Transportation Interval — Traveling and Shipping Intervals

The traveling and the shipping intervals showed significant
differences (P < 0.001 for both intervals) between seasons, being
shorter in the fall season than in the spring season (Table 4).

Air Temperature and Wind Velocity During Road Transport

The average temperature during transportation of lobsters from
the wharf to the processing plant was significantly (P < 0.001)
lower in the spring than in the fall (Table 4). It was also signifi-
cantly (P = 0.037) windier in the fall fishing season than in the
spring (Table 4).

DISCUSSION

The over-all compliance from the fishing and processing sec-
tors of the lobster industry during this study was good, suggesting
increasing concerns from the industry in lobster health-related is-
sues. Although only market-sized lobsters were to be included in

270

Lavallee et al.

TABLE 3.

Fishing factors assessed during the 1997 spring and fall lobster

fishing season in PEI, with corresponding distribution (proportion)

for each category or corresponding mean (SD).

TABLE 3.
continued

Fishing Season

Fishing Factor

Spring

Fall

Overall

Crew size

Less than 3 persons 26 (57.8%) 5 (45.5%) 31 (55.4%)

3 persons or more 19 (42.2%) 6 (55.4) 25 (44.6%)

Captain's experience,

in years 18.2(11.0) 27.1(13.0) 19.1(10.9)

Maximum air temperature,

in °C 15.4(4.0) 21.8(4.8) 16.5(5.1)

Minimum air temperature,

in °C 8.2(3.8) 13.7(1.7) 9.3(4.2)

Surface water temperature,

in X 7.9(3.2) 17.2(1.2) 9.1(4.8)

Rain

None 33 (73.4%) 7 (63.6%) 40 (61.4%)

Light to moderate 12 (26.6%) 4 (36.4%) 16 (38.6%)

Sun

None 11 (24.4%-) 2 (18.2%) 13 (23.2%)

6 hours or less 29 (64.5%) 3 (27.3%) 32 (41.1%)

More than 6 hours 5 (11.1%) 6 (54.5%) 11 (19.7%)

Wave

1 meter or less 36 (80.0%) 6 (54.5%) 42 (75.0%)

More than 1 meter 9 (20.0%) 5 (45.5%) 14 (25.0%)

Wind

None to light 33 (73.4%) 7 (63.6%) 40 (61.4%)

Moderate to strong 12 (26.6%) 4 (36.4%) 16 (38.6%)

Trap setting method

Single or double 1 (2.2%^) 7 (63.6%) 8 (14.3%)

Multiple 43 (95.6%) 0 (0.0%) 43 (76.8%)

Combination 1 (2.2%) 4 (36.4%) 5 (8.9%)

Max. trap setting depth,

in m 17.7(6.0) 28.0(3.1 1 19.3(6.8)

Min. trap setting depth,

in m 6.0(4.3) 12.0(7.6) 6.9(5.3)

Bait

Gaspereaux 11 (24.1%) 0 (0.0%) 11 (14.9%)

Mackerel 19 (41.4%>) 13 (46.4%) 32 (43.2%)

Herring 4 (8.7%) 7 (25.0%) II (14.9%)

Flatfish 5 (10.3%) I (3.6%) 6 (8.1%)

Other 7 (16.1%) 7 (25.0%) 14 (18.9%)

Contact before banding

Yes 14 (34.1%) 9 (81.8%) 23 (44.2%)

No 27 (65.9%) 2 (18.2%) 29 (55.8%)

Lobster handling

Placed 30 (73.2%.) 9 (81.8%) 39 (75.0%)

Tossed II (26.8%) 2 (18.2%) 13 (25.0%)

Holding unit before grading

Tole 37 (69.8%) 6 (54.5%) 43 (67.2%)

Crate 0 (0.0%) 5 (45.5%) 5 (7.8'/r)

Other 7 (13.2%) 0 (0.0%) 7 (10.9%)

None 9 (17.0%) 0 ((),()'?) 9 (14.1%)

Holding unit after grading

Tote 51 (96.2%) 9 (81.8%) 60 (93.8%)

Crate 2 (3.8%) 2 (18.2%) 4 (6.2%)

Live-tanl<

Fibergla.ss 32 (71.1%) 4 (36.4%) 36 (64.3%)

X-Actics'" 10 (22.2%) 5 (45.4%) 15 (26.8%)

Other 3 (6.7%) 2 (18.2%) 5 (8.9%)

Fishing Season

Fishing Factor

Spring

Fall

Overall

continued on next page

Water availability during fishing

Stagnant 19(35.9%) 1(9.1%) 20(31.3%)

Other 6(11.3%) 2(18.2%) 8(12.5%)

None 28(52.8%) 8(72.7%) 36(56.2%)

Water availability after fishing

Stagnant 44(83.0%) 1(9.1%) 45(70.3%)

Other 2(3.8%) 1(9.1%) 3(4.7%;)

None 7(13.2%) 9(81.8%) 16(25.0%)

Lid on live-tank during fishing

Completely on 17(37.8%>) 10(90.9%) 27(48.2%)

Panially on 18(40.0%) 0(0.0%) 18(32.1%)

None 10(22.2%) 1(9.1%) 11(19.7%)

Lid on live-tank after fishing

Completely on 28(62.2%) 10(90.9%) 38(67.9%)

Partially on 13 (28.9%^) 0(0.0%) 13(23.2%)

None 4(8.9%) 1 (9.1%r) 5(8,9%)

Maximum time on board'

Between 4 and 6 hours 28(62.2%) 3 (27.3%o) 31(55.4%)

6 hours or more 17(37.8%) 8(72.7%) 25(44.6%)

Maximum time on board"

2 hours or less 44(97.8%) 10(90.9%)) 54(96.4%.)

More than 2 hours 1(2.2%) 1(9.1%) 2(3.6%)

' The maximum time represented the period from when the first lobster
was caught until the boat arrived at the wharf.

- The minimum time represented the period from when the last lobster was
caught until the boat arrived at the wharf.

this study, one canner-sized lobster (carapace length < 8 1 mm) was
tagged and probably represents one canner-sized lobster that was
misplaced by the fisher with the market-sized lobsters. Only the
handling and fishing practices that showed substantial results are
discussed. These include the lobster-level factors of gender, physi-
ological indicators of health and vigor, environmental conditions,
and boat-level factors related to traps and bait, holding and live-
tank systems, and. finally, the over-all lobster handling. All re-
maining factors, including all transportation condition factors, did
not show in)portant variations and are not discussed further. The
uneven distribution of wharves between the spring and the fall
seasons was attributable to the limited time the investigators had to
carry the study into the fall season. Because of the variation in
sample sizes in the different fishing seasons, caution must be taken
when comparing results from these lobster fishing seasons.

Physical Examinalioti of Lobsters

Lobster Physical Condition and Vigor

The initial assumption was that lobster health status would
being to decrease only after lobsters entered the traps. Most of the
vigor loss occurred between wharves and processing plants, not
between boats and wharves. Perhaps these vigor losses were in-
duced by injuries inflicted on the wharves or even on the boats, but
their effect was not detected until later. The fall fishing season is
occasionally considered a lesser quality fishery because a majority
of lobsters are in a postmolting softer-shelled condition. Therefore,
the significant higher proportion of lobsters with normal physical

Lobster Health, Handling, and Fishing Practices

271

TABLE 4.

Transportation factors assessed during the 1997 spring and fall

lobster fishing seasons in PEI, with corresponding distribution

(proportion) for each category, or corresponding mean (SD).

Fishing Season

Transport Factor

Spring

Fall

Overall

Vehicle

Direct' 7(17.5%) 0(0.0%) 7(14.3%)

Refrigerated truck 26(65.0%) 0(0.0%) 26(53.1%)

Nonrefrigerated truck 4(10.0%) 9(100.0%) 13(26.5%)

Unknown 3(7.5%) 0(0.0%) 3(6.1%)

Road shipment on ice

Yes 8(25.0%) 3(33.3%) 11(26.8%)

No 24(75.0%) 6(66.7%) 30(73.2%)

Travelling interval

Direct'" 7(17.5%) 0(0.0%) 7(14.3%)

1 hour or less 15(37.5%) 9(100.0%) 24(49.0%)

Between 1 and 4 hours 15(37.5%) 0(0.0%) 15(30.6%)

4 hours or more 3(7.5%) 0(0.0%) 3(6.1%)

Shipping interval

Direct' 7(17.5%) 0(0.0%) 7(14.3%)

1 hour or less 6(15.0%) 6(66.7%) 12(24.5%)

Between 1 and 4 hours 13(32.5%) 3(33.3%) 16(32.6%)

4 hours or more 14 (35.0%) 0 (0.0%) 14 (28.6%)

Outside air temperature.

in "C 15.3(6.6) 20.6(1.1) 16.1(6.4)

Wind during transport

Light 32(88.9%) 4(57.1%) 36(83.7%)

Moderate 4(11.1%) 3(42.9%) 7(16.3%)

' Direct meant that no vehicle was used; the lobsters were landed directly
at the processing plants.

indices in the fall compared to the spring season was unexpected,
and the explanation is unclear.

Gender

Ennis (1978) found that the male-female ratio of large lobsters
when sampled by scuba divers was 1:1, but when estimated
through trap capture, the ratio was 3:1 in favor of male lobsters.
The over-all sex ratio of all tagged lobsters included in this study
was 52% females versus 48% males, but significantly more female
lobsters were observed in the spring season than in the fall season.
Assuming a normal distribution of the sex ratio among all hatched
lobster eggs, and equal survival rates among female and male
lobsters to market size, a 507f male. 50% female population should
be available for harvest. Federal regulations require that all oviger-
ous females must be returned to the water, and the removal of their
eggs is prohibited (Miller 1995). By protecting ovigerous females,
fewer females should be landed, because on average, female lob-
sters will carry eggs externally for almost a year (Waddy et al.
1995), thus increasing the likelihood of catching more males at any
point in lime. The reason for the higher proportion of females
landed in the spring season is still unclear, but could reflect a
competitive behavior among males, as suggested by Campbell
(1986), or perhaps a difference in the feeding behavior of male
versus female lobsters.

In the early part of the Prince Edward Island fall season, a
significant proportion of lobsters are in postecdysis. Because fe-
male lobsters mate shortly after the ecdysis (Talbot and Helluy

1995); a higher proportion of males caught in this period could
reflect shelter-restricted behavior of females who recently molted
and mated. Waddy and Aiken (1990) reported a higher relative
activity of mature male lobsters than females. These behavioral
differences could explain the gender ratio of landed lobsters fa-
voring males over females observed during the fall season.

Hematology

Some hemolymph parameters have been used to define lobster
health (Homey et al. 2000. Jussila et al. 1997). The range for THC
and TP reported here were very large, mostly attributable to the
sampling done during two different fishing seasons. In this study,
95% of the lobster population had TP levels measured on the boats
were between 18.6 g/L and 99.8 g/L. The levels of TP reported by
Homey et al. (2000) were within the range of these data, with
mean values of 19 to 65 g/L, and the values reported by Chen and
Chia (1997) in the mud crab (Scylla serrata) were also similar, at
81.0-88.4 g/L. Because many external factors can affect the TP
and THC levels postlanding. the values obtained when lobsters
were taken onto the boats were probably the most representative
assessments of the natural situation. Homey et al. (2000) reported
mean values for THC in their laboratory study of 6.6 to 3 1 . 1 x 10'"
hemocytes/mL. while that reported by Cornick and Stewart ( 1978)
was 18.1 X 10'" hemocytes/mL. These values are also within the
mean THC ± 2 SD (95% of the population) reported in this study
(5.0 to 50.5 X 10" hemocytes/mL). Jussila et al. (1997) reported a
range for THC in western rock lobster (Pamdirus cygnus) of 2.5 to
15.9 X 10'' hemocytes/mL. with the highest mean THC in lobsters
freshly arrived at the factory tanks, and suggested stress from
handling or exposure to air as causative factors for the high mean
of THC.

Lobster TP levels are influenced by the time of year, the molt-
ing cycle, the water temperature and probably by many other fac-
tors including diet, size, and gender (Chen and Chia 1997, Ennis
1973, Homey et al. 2000; Paterson and Spanoghe 1997). Imme-
diately following the completion of the ecdysis, lobsters will ingest
and absorb substantial volumes of seawater to expand their volume
to often 50% greater than they were before the ecdysis (Aiken and
Waddy 1992). This would dilute the hemolymph and result in
lower TP levels and lower THC. The fall fishing season of Prince
Edward Island is timed to occur after most newly molted lobsters
have achieved stage C of the molting cycle, and. therefore, lower
TP in the fall were expected. Chen and Chia (1997) reported the
lowest protein levels for the mud crab (Scylla serrata) during stage
B, and the highest levels during stages D-, and D,. High level of
feeding activities could be necessary to enable lobsters to recover
from the immediate low postmolt condition, as suggested by vary-
ing serum protein in field-captured lobsters (Ennis 1973).

Female lobsters had significantly higher TP levels than males at
every assessed handling point of the industry during the spring
season: boat, wharf, and arrival to the processing plant. Homey et
al. (2000) only reported a minimal influence of sex on the different
components measured in lobster hemolymph held under laboratory
conditions. Chen and Chia (1997) reported no significant differ-
ence in TP levels between male and female mud crabs {.Scylla
serrata). During the fall, female lobsters had significantly higher
TP levels than males only when asses,sed at entry to processing
plants.

Another important finding was the apparent dehydration ob-
served in lobsters kept out of the water, demonstrated by increas-

272

Lavallee et al.

ing THC, and by TP levels to a lesser degree, from boats to
wharves, and additionally from boats to arrival at processing
plants. Perhaps lobsters kept out of the water for extended periods
are losing considerable amounts of body fluids, resulting in hemo-
concentration. and higher THC and TP values. Recently, Jussila et
al. (1999) reported that only emersion periods greater than 2 hours
induced significant changes in western rock lobster (PanuUrus cyg-
mis) THC. This correlates with the present findings, because the
average emersion period calculated from the time of landing to the
time of arrival at processing plants was approximately 3 hours and
15 minutes in the spring. In the fall season, this period averaged
only 1 hour and 10 minutes, but because almost 829r of the fishers
did not add any water at all in the live-tank after fishing, the total
mean period for lobsters were kept out of the water is estimated to
be at least 2 hours and 20 minutes. Dehydration has been demon-
strated in prawns (Penaeiis japonicus) to be up to O.li'Jc loss of
body weight per hour of exposure to air, at 75-859^ relative hu-
midity (Samet et al. 1996). Newsom et al. (1994) suggested that
spraying red swamp crawfish (Procambarus clarkii) with seawater
could probably protect them against dehydration through evapo-
ration, and may also help replace some of the body fluids lost.
Jussila et al. (1999) also suggested that regular handling of lobster
(Pamdinis cygnus) as it happens during postharvest manipulation
can elevate THC, and when sampling is performed during han-
dling, especially if delays in sampling are occurring, significant
elevation in THC may result after only a few minutes. It is likely
that the combination of stress inflicted through increasing handling
procedures from harvest time through to arrival at processing
plants, and the extensive emersion periods explains the elevated
THC observed at the time of arrival at the plant compared to THC
estimated directly on fishing boats.

During the spring season, a significant gender difference in the
THC values was seen only at the wharves, with females having
higher counts. For the fall season, significant differences in THC
between male and female lobsters were observed on the boats and
at the time of entry into processing plants; females having higher
counts than males at the plants, but lower at the boats. Overall, no
consistent patterns in the THC according to sex were recorded,
similar to the findings of Jussila et al. (1997) on rock lobsters
(Pamdinis cygnus). Cornick and Stewart ( 1978) did not assess sex
differences in THC, although they looked at differential hemocyte
counts and reported no significant difference according to gender.
Female lobsters do not molt as often as males of comparable sizes
because of the reproductive cycle, some variation between gender
in both TP and THC values could be present before, during, and
shortly after ecdysis, which could explain some of the results of
this study.

It may be more appropriate to report TP or THC range instead
of mean tor indicating or predicting lobster health at the population
level. The wide range of TP and THC values observed may be
helpful, if certain factors affecting these ranges can be identified,
and, therefore, used as indicators of health. Further studies of
factors influencing the range instead of the mean values of either
TP or THC are required if these factors can first be identified.

The significantly higher prevalence oi Aerococcus viridans in
lobsters from the fall fishing season compared to the spring season
is likely a reflection of the warmer water temperatures in the late
summer and fall. The only factor that seems to affect the preva-
lence of clinical disease in A. viridans infected lobsters is the water
temperature. A decreased mean time to death with increasing water
temperatures would result in increased clinical disease incidence
and mortality (Bayer and Daniel 1987. Stewart 197.5). Therefore.

it may be possible that at higher water temperatures, more infective
particles are released in the environment via decomposition of
dead infected lobsters, thus increasing the likelihood of capturing
live infected lobsters. Gaffkemia is a disease endemic to lobster
populations of North America, and has also been reported in Eu-
ropean waters (Alderman 1996). Huang and Bayer ( 1989) reported
A. viridans prevalence of 6.7% in freshly caught lobsters off the
coast of Maine. The prevalence of /A. viridans in Atlantic Canada
was estimated at almost 5% by Stewart et al. (1996), while site
specific levels ranged from 0 to 22% according to Vachon et al.
(1981). Keith et al. (1992) reported that 0 to 40% of lobsters
caught in Canadian locations were infected with A. viridans. Lob-
sters cannot control or efficiently respond to infection with A.
viridans (Marks et al. 1992. Stewart and Zwicker 1974). In A.
viridans infected lobsters, there is no agglutination and the bacte-
ricidal ability of the hemolymph deteriorates in the presence of
virulent strains oi A. viridans (Johnson et al. 1981. Stewart 1975).
Lobsters infected with A. viridans will eventually die of gaffkemia,
which explains some of the confusion in the literature when re-
porting prevalence oi A. viridans versus prevalence, or incidence,
of gaftl<emia in wild lobsters. Nevertheless, these reported infec-
tion levels agree with the prevalence estimated in this study that
was 5.5% in the spring and 10.4% in the fall.

Environmental Factors

Lobsters are sensitive to freshwater exposure (Ennis 1995, Jury
et al. 1994, McMahon 1995), and therefore, heavy exposure to rain
will likely be detrimental to lobsters, especially for prolonged
exposure times. Furthermore, exposure to sunshine could result in
more rapid drying of the external surface of the lobsters, resulting
in a loss of body fluids. Newsom et al. (1994) suggested that water
is lost more rapidly than other components of the hemolymph, and
that the hemolymph concentration should increase.

Fishing Practices

Stress from harvesting and handling of western rock lobsters
has been blamed for productivity losses in the lobster (PanuUrus
cygnus) industry of Western Australia (Paterson and Spanoghe
1997). Similarities with the Canadian lobster fishery are most
likely, and the various fishing practices should also have profound
impacts on further industry losses. Therefore, a higher proportion
of lobster injuries on boats where fishers were less careful with
their catch was expected.

Setting of Traps and Bait

Cooler water temperatures at greater depths might have differ-
ent impacts on lobster health than warmer waters from shallower
bays. Greater changes in vsater temperature and hydrostatic pres-
sure are experienced by lobsters from deeper traps when brought to
the surface. Perhaps this change in pressure removes some of the
oxygen from the hemocyanin. and could result in anoxia.

The type of bait, its composition, and its quality may physi-
ologically affect lobsters postcapture. During the fall season, most
fishers used two or three different types of bait, explaining why the
total number of bait-types used exceeded the total number of boats
(28 bait-tvpes used by 1 1 boats). Bait quantity and quality has been
reported to influence trap catch yields in crawfish (Romaire 1995).
However, Addison and Bell ( 1997) reported that reduced bait at-
tractiveness had less influence on catch rates than behavioral in-
teractions. L'nforlunately. correlations between types of bait used
and numbers of lobsters caught were not investigated in this study.

Lobster Health, Handling, and Fishing Practices

273

Different trap attraction rates were evaluated when mussels, sea
urchins, cattle hocks, animal guts, and diesel oil were used as bait
in spiny lobster {Panuliriis sp.) traps, with mussels and diesel oil
being, respectively, the most effective baits (Mohan Rajan et al.
1993). Mussels as bait could represent an interesting alternative in
Prince Edward Island, with the important number of mussel farms
and processing plants; mussels being culled at harvest could be
used in the lobster fishery. Crawfish fishers typically use "fish
bait" in cold waters (< 20 °C), but when fishing in warmer waters
(< 20 °C). they either use "formulated bait" or a combination of
fish and formulated baits (Romaire 1995), and. therefore, a for-
mulated bait could also represent another alternative for the fall
lobster fishery.

Holding Unit and Live-Tank System

The tolerance of aquatic crustaceans to exposure to air is
greatly increased by cooling the animals, and by having a high
relative humidity (Samet and Nakamura 1997). Having ice in the
live tank can be an advantageous alternative when no water is
used. However, if the water stays stagnant for long periods, oxygen
is depleted, and lobsters become hypoxic. Lobsters usually survive
for longer periods if they are kept out of water, in a cool environ-
ment where their gills stay wet, compared to maintenance in stag-
nant water with no aeration. Keeping aquatic crustaceans dry is
harmful, especially if they are exposed to wind or direct sunshine,
and their gills could eventually collapse if the relative humidity
drops beyond a critical value for an extended period (Samet and
Nakamura 1997). The use of a lid offers some protection from
direct sunlight or rain, and can help maintain the relative humidity
high and constant in the live tank. The lid may also help in reduc-
ing the temperature variation inside the live-tank. Sudden and
drastic changes in water temperature are stressful to lobsters, and
can strongly infiuence their behavior (Crossin et al. 1998), and
most likely their health status as well. Unfortunately, the water
quality of the live-tanks was not assessed during this study and its
direct effect could not be documented.

CONCLUSIONS

By tagging and following individual lobsters during the Prince
Edward Island spring and fall season, lobster health was assessed
at the different handling points of the fishing industry. THC and
TP showed increasing levels from the time of harvest of the time
of landing and subsequently, to the time of arrival at processing
plants, suggesting a possible influence of handling related stress,
dehydration, or possible a combination of the two. Fishing prac-

tices handling events, and lobster transportation conditions were
thoroughly described. Some environmental conditions and fishing
practices showed substantial variation between the spring and fall
fishing seasons. Fishing conditions were warmer, sunnier, the
waves were greater, and traps were set in deeper water in the fall.
While fishers from both season used mainly mackerel as bait,
gasperaux was only used in the spring season. In the spring season,
lobsters of different sizes were prevented from having mutual con-
tact on most boats, but only on a small proportion of boats in the
fall season. Most spring fishers waited after all the traps were
hauled before adding water to the live-tank compared to a majority
of fall fishers who had no water in the live-tank at any time.
Finally, lobsters spent on average significantly more time on board
fishing vessels in the fall than in the spring season.

Attempting to follow lobsters from fishing boats through
wharves and processing plants is a logistical challenge for evalu-
ation of lobster health or productivity. In many field studies, these
logistical problems have limited the ability to obtain valid results.
This study enabled the investigators to identify the frequency with
which a series of different fishing practices, and transportation
conditions was u.sed. Paterson (1999) suggested that analytical
models could be used to predict certain outcomes, such as lobster
weakness state or death from varying lobster stress indicators. By
using epidemiological methods where lobster-level factors (stress
indicators) and group-level factors (wharf or boat-level), a model
predicting associations between factors and lobster survival would
be advantageous. In a subsequent paper (Lavallee et al. 2000), the
results from this study were used to build an epidemiological
model assessing relationships between fishing and handling risk
factors for decreased lobster vigor upon arrival at processing
plants.

ACKNOWLEDGEMENTS

Special thanks to Drs. J. Davidson. G. Johnson, D. Speare, and
J. VanLeeuwen for their advice and editorial comments. The au-
thors also thank A. MacKenzie, R. MacMillan, K. Whitman, and
T. Rogers for their support and help with many parts of the project,
and Drs. M. Boucher, P. Campbell. A. Eraser, A. Johnson. A.
Jackson, H. MacMillan, and A. Schneider for their assistance in
the data collection process. This work was funded in part by the
Industrial Research Assistance Program of the National Research
Council of Canada, by the Canadian Atlantic Lobster Promotion
Association, and by the Aquaculture & Fisheries Research Initia-
tive of Prince Edward Island.

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Campbell. A. 1986. Implications of size and sex regulations for the lobster
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Journal of Shelljhh Rcseiinli. Vol. 19, No. 1, 275-281. 200(1.

ANALYTICAL ASSESSMENT OF HANDLING, FISHING PRACTICES, AND TRANSPORTATION
RISK FACTORS ON LOBSTER (HOMARVS AMERICANUS) HEALTH IN PRINCE EDWARD

ISLAND, CANADA

JEAN LAVALLEE,'* ELIZABETH S. SPANGLER,'
K. LARRY HAMMELLL' IAN R. DOHOO," AND
RICHARD J. CAWTHORN^

A VC Lobster Science Centre, and
^Department of Health Management
'Department of Pathology and Microbiology
Atlantic Veterinaiy College
University of Prince Edward Island
Charlottetown. Prince Edward Island
Canada. CIA 4P3

ABSTRACT The objective of this study was to identify lisk factors for productivity losses in the Canadian lobster industry. Lobster
handling and fishing practices onboard 64 fishing boats and transportation conditions on 49 vehicles subsequently transporting these
lobsters from fishing wharves to processing plants were assessed during the spring and fall fishing seasons of 1997 on Prince Edward
Island. These practices and conditions were then related to the health of the lobsters, estimated by the vigor status on arrival at the
processing plants. A generalized estimating equation logistic regression model was used to assess the impact of handling, fishing, and
transportation practices on lobster vigor. Significant risk factors for loss of vigor included the following boat-level factors: the use of
mackerel for bait (odds ratio. OR. of 7.1; /• = 0.003). tossing (as opposed to placing) lobsters from traps to temporary holding units
on board the fishing boats (OR = 3.6. P = 0.048), and exposure to rain while on board fishing boats (OR = 3.6, P = 0.01 1). while
greater maximal depths at which the traps were set had a protective effect on lobster vigor (OR = 0.85/m. P = 0.010).

KEY WORDS: Lobster. Homanis americamis. risk factors, postharvest. health

INTRODUCTION

Among Canadian fisheries, the lobster {Homarus americamis.
H. Milne Edwards 1837) fishery is one of the most important, both
in volume and in landed value. It consists primarily of a special-
ized inshore small boats fishery (Pringle and Burke 1993). Pater-
son and Spanoghe ( 1997) suggested that sampling lobsters at vari-
ous points of handling should yield information on stressors influ-
encing lobster health. The traditional lobster industry in Atlantic
Canada is usually represented by the fishing sector that sells its
daily catch to buyers located directly at the fishing ports, and these
buyers can either sell their live product to processing plants or to
seafood companies specializing in live holding. Lobsters can then
be shipped live or processed before being exported. Variations in
lobster health, before and after short or long-term holding, are
hypothesized to be associated with conditions experienced while
on fishing vessels and perhaps also during wharf-le\el handling
events.

Several potential risk factors for productivity losses in the Ca-
nadian lobster industry were identified by monitoring handling and
fishing practices on board fishing boats and during subsequent
transport from fishing wharves to processing plants during the
1997 spring and fall fishing seasons in Prince Edward Island (La-
vallee et al. 2000). These factors included some crew and boat
specifications, such as crew size and years of experience of the
captain. They also included the environmental variables rain, sun-
light, wind. wave, water, and air temperatures. Fishing practice
risk factors investigated included trap setting configuration and

♦Current address: Aquatic Science & Health Services. 158 St-Pelers Road.
Charlottetown. Prince Edward Island. Canada, CIA 5P8.

depth at which traps were set. lobster handling methods, type of
bait, contact among lobsters before grading and banding, type of
temporary storage unit and holding tank, water and lid availability
for holding tanks, methods of transferring lobsters, and maximum
and minimum periods spent on boats. Transportation conditions
included the following risk factors: type of vehicle, presence of ice
during transportation, type of shipping unit, outside air tempera-
ture, and other weather conditions, time interval between wharves
and processing plants, and maximum duration shipments stayed in
transport vehicles.

The objective of this study was to develop an epidemiological
model to assess the impact of different fishing and handling prac-
tices previously identified as potential determinants of the health
and quality of lobsters upon arrival at processing plants.

MATERIALS AND METHODS

Data Collection and Variable Selection

Data on lobster physical and physiological status, and on fish-
ing practices, handling practices, and transportation conditions that
may have a significant impact on lobster health before holding,
after holding, or prior to processing and marketing were collected
between May 1997 to August 1997 in Prince Edward Island,
Canada. Date were obtained by monitoring fishing practices on
boats and subsequent transportation conditions as lobsters were
moved to processing plants. Fishing and handling practices were
monitored daily in 64 groups of lobster. Each group consisted of
all market-sized lobsters (carapace length 281 mm) caught on one
boat during one day. These groups were subsequently transferred
to processing plants by 49 different vehicles. Lobster health was
assessed directly on board the fishing vessels, and later when

275

276

Lavallee et al.

groups were landed, and eventually at the processing plants (see
Lavallee et al. 2000 for further description of the data collection
process).

Variables recorded were a mixture of continuous, ordinal,
nominal, and dichotomous variables. Continuous variables were
converted to dichotomous variables with two levels when the in-
dependent variable showed a tendency to group around discrete
values. The outcome variable for the model was a dichotomous
variable describing the vigor of each lobster upon arrival at pro-
cessing plants. Every lobster was individually observed, and a
lobster showing any delay or absence of response (i.e., tail flip-
ping, claw(s) rising, antenna(e) movement) to physical stimuli was
given a score of "I" (decreased vigor); whereas, a lobster showing
immediate response to physical stimuli was given a score of "O"
(normal vigor).

Statistical Analysis

The dataset was transferred into a statistical software package
(ST ATA 1^' version 3.0, Stata Corporation, College Station, Texas,
USA, 1996). After transformation of all categorical variables into
dichotomous variables, 47 explanatory variables were retained.
Unconditional associations between the outcome (lobster vigor
score at arrival at the processing plant) and the predictors were
evaluated by Chi-square tests for dichotomous variables (with cor-
responding relative risks) and by f-tests for the continuous vari-
ables. For all analyses, observed associations were considered sig-
nificant when P < 0.05.

Variables with significant unconditional associations with the
dependent variable (lobster vigor) were selected for inclusion in a
multiple variable model building process. The model used was a
logistic regression model using a generalized estimating equation
(GEE) procedure (Liang and Zieger 1986). The model assumed a
binomial error distribution, calculated robust standard errors, and
used a logit link function with an exchangeable correlation struc-
ture for the correlations among lobsters within a boat.

A forward stepwise procedure under the control of the inves-
tigator (i.e., not computer generated) was used to identify variables
having important associations with lobster vigor at the processing
plants. Initially, a model containing only the main effect for each
variable showing significant association was fit. Then, all possible
two-way interactions among these individually significant vari-
ables were explored. Finally, three-way interactions were consid-
ered for inclusion in the model. Models were compared using the
deviance statistic, and the model with the lowest deviance was
selected. The fit of the model was assessed using a Hosmer-
Lemeshow goodness-of-fit test. For comparison purposes, the final
model developed using GEE was subsequently fit as a multilevel
model (MLwiN Version 1, University of London) with lobster as
the level 1 identifier and boat as the level 2 identirier. Second-
order PQL (penalized quasilikelihood) estimates were obtained
using the RIGLS (restricted iterative gcncrali/cd least-squares)
estimation procedure.

RESULTS

Descriptive Slalislics

From 64 groups of lobsters assessed during this study. 17
groups were rejected because of missing information at the pro-
cessing plants. The outcome, lobster vigor upon arrival at the
processing planl. was assessed on a total of 2.191 lobsters from 47

different groups or fishing boats. Because of the limited number of
boats sampled in the fall and the obvious differences in environ-
mental characteristics, only data from 38 boats in the spring season
were used in developing the model. In the spring sampling, vigor
was assessed on 1,181 lobsters from 38 boats. A total of 35 lob-
sters had decreased vigor upon arrival at processing plants in the
spring season, with 12 groups having at least 1 lobster with de-
crea.sed vigor.

Measure of Association

Three continuous variables (date, maximal, and minimal depths
at which traps were set) and 19 of the 39 dichotomous variables
were found to be significantly associated with lobster vigor
(Tables I and 2). and retained for further analysis. The vigor of
lobsters assessed directly on boats was significantly associated
with vigor upon arrival at the processing plants. However, this
variable was not kept as predictor for further analysis because of
the small number of cases (n = 3) of decreased vigor observed on
boats and the plant. Similarly, flatfish as bait was also dropped:
although 76 lobsters were fished with flatfish as bait, it represented
only I fisher.

Handling and Fishing Practices

The minimal and maximal depths at which traps were set
showed significant unconditional associations with lobster vigor
status assessed upon arrival at processing plants (Table 1 ). Re-
duced vigor lobsters tended to come from more shallow water
(minimal and maximal depths of 3.5 m and 14.8 m, respectively)
compared to normal vigor lobsters (minimal and maximal depths
of 5.4 m and 17.6 m, respectively).

Boats with smaller crews and older captains tended to have a
higher risk of producing low vigor lobsters (Table 2). Lobsters
landed from boats with crews of two or fewer members were 2.4
times more at risk of having a decreased vigor at the processing
plant than lobsters landed by larger crews, while the same risk was
3.0 times greater when landed by a captain with more than 20 years
of experience compared to less experienced captains.

A number of weather-related variables had significant uncon-
ditional associations with lobster vigor (see Table 2). Warm
weather, rain, sunlight, and rough weather (waves) all increased
the risk of low vigor. After dichotomization, both the inaximum
OlO °C or <10 °C) and minimum (>9 °C or <9 °C) air tempera-
tures had significant unconditional associations with lobster vigor
at processing plants. When the maximum temperature was above
10 °C, lobsters were 3.6 times more likely to experience loss of
vigor at the plants than when the maximum daily temperature was
10 °C or below. A similar situation was noted with the minimal
daily air temperature, with relative risk of observing decreased
lobster vigor at the plant of 3.1 when the minimal air temperature
was above 9 °C. When caught on rainy days, lobsters were 6.3
limes more likely lo have decrea.sed vigor upon arrival al the plant
compared to nonrainy days. Also significant was the exposure to
sunlight, with relative risk for lobster decrease in vigor of 5.3 if
landed on sunny days as compared to cloudy days. When the
waves were classified as moderate to strong, lobsters were 3.3
times more likely to suffer from loss of vigor at the plants com-
pared lo when waves were calm to minimal.

Boats tishing with mackerel or flatfish halts were at higher risk
oi delivering lobsters with lower vigor: whereas, boats fishing with
gaspercaux halt landed livelier lobsters (Table 2). When fished

Risk Factors Affecting Lobster Health

277

TABLE 1.

Unconditional associations between fishing practices and lobster {Homanis americanus) characteristics that were measured on a continuous

scale, and a measure of lobster vigor at the processing plant.

Mean

Variable

Description

Normal

Decreased

'-value

vigor'

vigor'

0.026

162.9

166.3

0.181

7.9

8.6

0.111

289

300

0.001

17.6

14.9

0.003

5.4

3.5

0.381

0.58

0.63

0.414

88.2

89.4

0. 1 90

77.4

84.8

0.422

198.3

221.3

Date

Water temp.
No. traps
Ma.\. depth
Min. depth
Weight
Length
Protein
Hemocyte

Date of the boat sampling (# of days from January 1 )

Surface water temperature (°C)

Number of traps hauled

Maximum depth at which the traps were set (m)

Minimum depth at which the traps were set (m)

Individual weight as measured at the wharf (kg)

Individual carapace length as measured on the boat (mm)

Total hemolymph protein, on the boat (g/L)

Total hemocyte counts, on the boat (x 10'' hemocytes/ml)

Normal and decreased vigor as assessed at the processing plant with the dichotomous variable "'vigor.'
The /"-values were obtained by r-test.

with mackerel, lobsters were almost 4 times more likely to express
loss of vigor compared to fishing with alternate baits, while gas-
pereaux had a protective effect whereby lobsters fished with gas-
pereaux were only one-third as likely to have reduced vigor. Lob-
sters tlshed with Hatfish as bait seemed to be 5.3 times more likely
to suffer loss of vigor upon arrival at the processing plant. How-
ever, flatfish bait was used by only one fishing boat included in the
study and, therefore, its unconditional effect may be overesti-
mated. For a complete description of the frequency distribution of
the baits used see Lavallee et al. (2000).

Rough handling, physical contact among lobsters before mea-
suring the carapace, the use of plastic totes as temporary storage
units, and packing lobsters over at the wharves, were all practices
that induced higher risk for loss of lobster vigor (Table 2). Lobsters
coming from boats where physical contact before carapace mea-
surements was possible had an 1 8 times greater chance of suffering
loss of vigor at the processing plants than if they were landed from
boats in which physical contact between lobsters was purposefully
prevented. Lobsters landed from boats in which lobsters were gen-
erally tossed from the traps to the temporary holding units were
almost 3 times more likely to suffer vigor loss at plants as com-
pared to lobsters that were placed into temporary holding units. If
these temporary holding units were the traditional plastic totes,
then lobsters were 4 times more likely to express loss of vigor at
processing plants than if other types of temporary storage units
were used (Table 2). Furthermore, the practice of packing over the
lobsters once at the wharf showed that lobsters that went through
this process were more than 3 times more likely to have decreased
vigor at the processing plants than lobsters that did not go through
this process. All other handling and fishing practices did not show
any significant unconditional association with the lobster vigor
status at processing plants (Table 2).

Transportation Conditions

Three transportation variables individually showed statistically
significant association with lob.ster vigor loss when assessed at
arrival at processing plants: the use of closed compartment ve-
hicles, warmer outside air temperatures, and windy conditions dur-
ing transport between wharves and processing plants (Table 2).
When the transport vehicle was a nonrefrigerated closed compart-

ment truck, lobsters were almost four times more likely to have
decreased vigor than if other types of vehicles (open-bed truck or
pick-up truck, and vehicle equipped with a closed refrigerated
transportation) were used. In the presence of moderate to strong
winds during transportation, the proportion of lobsters that suf-
fered from loss of vigor upon arrival at the processing plants was
7.3% as compared to 1.5% in the presence of calm or light winds;
lobsters transported during windier days were more than five times
more likely to have vigor loss at the plants. Finally, if the outside
air temperature was above 18 °C, lobsters became seven times
more likely to suffer vigor loss at the processing plants than if the
air temperature was 18 °C or less. No other transportation condi-
tions showed significant association with lobster vigor at the pro-
cessing plants.

Regression Models

The Generalized Estimating Equation logistic regression model
(GEE) identified four significant variables predicting lobster vigor
at the processing plant: maximum depth at which traps were set,
occurrence of rain during fishing, tossing of the lobsters from traps
to temporary storage units, and the use of mackerel as bait (Table
3). While controlling for other factors, lobsters landed from boats
in which mackerel bait was used instead of alternate baits were 7.1
times more likely to have decreased vigor at the processing plants;
lobsters were 6.3 times more likely to have decreased vigor if
landed on rainy days as compared to nonrainy days; and lobsters
from boats on which they were generally tossed into the temporary
holding units were 3.6 times more likely to experience vigor loss
at the plant. Finally, lobsters caught in deeper waters were less
likely to have loss of vigor at the processing plants than lobsters
caught from shallower waters, with the risk of experiencing loss of
vigor decreasing by 1.2 for every meter increase in depth, while
controlling for other variables in the model.

The deviance for the multivariable model was 240.81, the Pear-
.son dispersion coefficient for the model was 1.03. with a total of
1,148 observations, and the Chi-square value for the over-all sig-
nificance of the model was 36.56 (P < 0.001 ). The standard errors
were adjusted for clustering on the boat, using the GEE model. The
Hosmer-Lemeshow goodness-of-fit test yielded a Chi-square
value of 8.1 1 with a P-value of 0.423 (critical value = 15.507, P

278

Lavallee et al.

TABLE 2.

Unconditional associations between lobster (Homarus americanus) handling, fishing practices, transportation conditions and lobster
characteristics that were measured on a dichotomous scale, and a measure of lobster vigor at the processing plant.

Variable description

Frequency distribution if vigor' is
decreased at the plant (%)

P-value

RR

Crew: size of the crew on the boat, including the captain

Experience: years of fishing experience of the captain

Maximum air temp.: maximum daily air temperature

Minimwn air temp.: minimum daily air temperature

Wind: wind strength while fishing

Rain: raining during fishing

Sun: sunshine during fishing

Water during: water available in the live tank, while fishing

Water after: water available in the live tank, after fishing

Casperaux: fresh gaspereaux used as bait

Mackerel: fresh mackerel used as bait

Herring: fresh herring used as bait

Flatfi.':li: fresh flatfish used as bait

Fiberglass: fiberglass box as live tank

X-Actic: X-Actic box as live tank

Tate before: plastic tote used for holding unit before grading

Wood box: wooden box used for holding unit before grading

Tote after: plastic tote used for holding unit after grading

Contact: physical contact among lobsters before being banded

Handling: overall lobster handling procedure on the bout

Wave: moderate to high waves vs. small or calm sea

Tra/i: comhinatiim of iiuilliple. single, or double trap setting

Lid before: presence of a lid on the live tank while fishing

Tiiiw max: maximum time one lobster spent on the boat

Time mill.: minimum time one lobster spent on the bo:il

Packing: packing over of the lobsters at the wharl

Truck: transportation between wharf & plant in closed compartnienl

Direct: lobsters landed directly at the plant, no transport vehicle

Wind tnuk: wind strength during road transportation

2 or +

28/776(3.61%)

0.027

2.4

3 or +

7/473(1.48%)

2 1 yrs or +

27/652(4.14%)

0.003

3.0

20 yrs or -

8/597 ( 1 .34%)

1 1 °C or +

20/340 (5.88%)

< 0.001

3.6

10°Cor-

15/909(1.65%)

10°Cor +

21/412(5.10%)

0.001

3.1

9 °C or -

14/837(1.67%)

Moderate-strong

2/201 (1.00%)

0.090

0.3

calm-light

3.3/1,048(3.15%)

Yes

21/2.39(8.79%)

0.000

6.3

No

14/1,010(1.39%)

Yes

14/972(1.44%)

< 0.001

0,2

No

21/277(7.58%)

No

26/795 (3.27%)

0.185

1.7

Yes

9/454(1.98%)

Yes

29/1,004(2.89%)

0.709

1.2

No

6/245 (2.45%)

Yes

4/400 ( 1 .00%)

0.008

0.3

No

31/849(3.65%)

Yes

29/700(4.14%)

0.001

3.8

No

6/549(1.09%)

Yes

2/127(1.57%)

0.377

0.5

No

33/1,122(2.94%)

Yes

6/47(12.77%)

< 0.001

5.3

No

29/1.202(2.41%)

Yes

5/249(2.01%)

0.396

0.7

No

30/1.000(3.00%)

Yes

30/907(3.31%)

0.078

2.3

No

5/342(1.46%)

Yes

31/817(3.79%)

0.012

4.0

No

3/317(0.95%)

Yes

0/95 (0.00%)

0.073

na

No

34/1,039(3.27%)

Yes

35/1,176(2.98%)

0.135

na

No

0/73 (0.00%)

Yes

.34/895(3.80%)

0.002

na

No

0/2.W(0.00%)

Tossed

31/859(3.61%)

0.033

2.9

Placed

4/322 ( 1 .24%)

Moderate-strong

14/212(6.60%)

< 0.001

3.3

calm-small

21/1,037(2.03%)

Yes

35/1,176(2.98%)

0.1.35

na

No

0/73 (0.00%)

Yes

.^1.083(3.14%)

0.065

5.2

No

1/166(0.60%)

4-6 hours

2.V730(3.15%)

0.377

1.4

6-8 hours

12/519(2.31%)

>2 hours

35/1,193(2.93%)

0.194

na

2-1 hours

0/56 (0.00%)

Yes

3/283(1.06%)

0.043

0.3

No

32/966(3.31%)

Yes
No

4/108 (3.70%)
10/986(1.01%)

0.018

3.7

Yes

1/2.W(().42%)

0.180

0.3

No

I.VS55(1.52%)

Moderate-strong

21/286(7.34%)

< 0.001

5.1

calm-light

14/963(1.45%)

continued on next page

Risk Factors Affecting Lobster Health

279

TABLE 2.
continued

Variable description

Frequency distribution if vigor' is
decreased at the plant (%)

P-value

RR

Air temp nuck: air temperature during road transportation

Sex: gender of the lobsters

Gajfkfmia: gaffkemia test result, on the boat (for Acrococciis viiiduns)

Liveliness: lobsters liveliness, as assessed on the boat

Woiiiul: wound or active lesion, as assessed on the boat

CUiw: quality of the claws, as assessed on the boat

Leg: quality of the legs, as assessed on the boat

Antennae: quality of the antennae, as assessed on the boat

Body: quality of the overall body, as assessed on the boat

19 "C or +

29/506 (5.73%)

< 0.001

7.1

18°Cor-

6/743(0.81%)

Female

23/672(3.42%)

0.162

1.6

Male

12/570(2.11%)

Positive

2/23 (8.70%)

0.070

3.6

Negative

12/494(2.43%)

Decreased

1/3 (33.33%)

0.001

12.2

Normal

34/1,245(2.73%)

Present

5/145 (3.45%)

0.617

1.3

Absent

30/1,103(2.72%)

Normal

29/1,051 (2.76%)

0.832

0.9

Abnormal

6/198 (3.03%)

Normal

33/1,162(2.84%)

0.768

1.2

Abnormal

2/87 (2.30%)

Normal

29/1,061 (2.73%)

0.726

0.8

Abnormal

6/188(3.19%)

Normal

3.3/1,152(2.86%)

0.645

1.4

Abnormal

2/97 (2.06%)

Outcome.
The P-values were obtained by Chi-square tests.
RR is the relative risk associated with each variable.

< 0.05, df =8). Therefore, it was concluded that the model was
a reasonable fit for the data.

All parameter estimates from the tnultilevel model (results not
shown) were virtually identical to those obtained from the GEE
procedure. In addition, the level two (boat) variance was zero once
the fixed effects in the model had been accounted for. This indi-
cates that once the variables maximum depth at which traps were
set, occurrence of rain during fishing, tossing of the lobsters from
traps to temporary storage units, and the use of mackerel as bait
were controlled, the probability of reduced vigor was essentially
independent of the boats.

DISCUSSION

It is important to understand the difference between causation
and association. A risk factor is associated with the outcome when
the distribution of the outcome is significantly different between
the "exposed" and "nonexposed" (Martin et al. 1987); whereas,
causation must additionally include a set of guidelines to assess the
likelihood of the association to be causal. Among these guidelines
(but not limited to) are the following: the exposure to the risk
factor must precede the outcome; the exposure to the risk factors
should also be more common in the individuals expressing the
outcome than in those individuals without the outcome; the re-
moval of the risk factor should decrease the incidence of the out-
come; and. the modification of the host's response should also
decrease the incidence of the outcome (see Evans 1978 for a com-
plete description of these guidelines).

Some correlation among the variables in the GEE model was
present. This explains the multicollinearity problems encountered
during the model building process, especially among dummy vari-
ables. Multicollinearity concerns relationships among predictor
variables, but does not directly involve the outcome (Kleinbaum et
al. 1988), and GEE models do not take multicollinearity problems
into account. When a predictor is included in the model, adding

another predictor that is correlated to the previous one contributes
relatively little to the explanatory model and would seem to be
nonsignificant. Care was taken during the model-building process
to avoid this.

GEE models are more efficient if variables are independent
(Liang and Zeger 1986). Although lobster-level predictors were
clustered within fishing boats, GEE models account for factors up
to two levels of clustering (Pendergast et al. 1996). However, this
study did not result in any lobster-level predictors remaining in the
final model.

More than 457c of all potential determinants measured showed
significant crude association with the outcome, loss of vigor. Hav-
ing multiple observations for every cluster; that is, multiple lob-
sters per fishing boat, can artificially increase the significance of
many determinants (Kleinbaum et al, 1988), and a conservative
approach must be taken when drawing conclusions, especially with
crude associations. Although the distribution of the event of con-

TABLE 3.

Results of the generalized estimating equation regression models

with 95% confidence interval to predict lobster (Homarus

americanus) vigor at the processing plant.

Vigor

Odds Ratio

z

P>lzl

95% Confidence Interval

Max depth

0.85

-3.008

0.003

-0.085

-0.018

Rain

3.63

2.538

0.011

0.294

2.286

Handling

0.28

-1.977

0.048

-2.559

-0.01 1

Mackerel

7.07

3.018

0.003

0.686

3.226

Max depth; maximum depth at which the traps were set (m).

Rain: if it was raining during fishing.

Handling: if the lobsters were placed (versus tossed) from the traps to the

temporary storage unit.
Mackerel: if fresh mackerel was used as bait.

280

Lavallee et al.

cem was binomial and took on only one of two values (normal or
decreased vigor), the frequency of the event meant that the number
of boats with decreased lobster vigor was sufficiently rare to have
little statistical power if the data had been collapsed to the boat
level. Because the data were clustered at the boat-level, an ordi-
nary logistic regression would not account for this clustering
(Kleinbaum et al. 1988). and would likely overestimate the sig-
nificance of predictor variables. The investigators wanted to keep
individual lobsters as the unit of evaluation, so both lobster-level
and boat-level factors could be considered. Consequently, a GEE
logistic modeling approach was chosen.

The final GEE model included four significant predictors. The
significant predictors for loss of vigor at airival at processing
plants did not include any lobster-level factors, but only consisted
of boat-level factors. Lobsters landed from boats using mackerel
bait were more than seven times more likely to suffer from loss of
vigor when arriving at the processing plant as compared to lobsters
landed from boats using alternative baits, when controlling for the
other variables. The biological or physiological explanation for
this phenomenon is unclear. Scombroid fish, such as mackerel,
contain high levels of the amino acid histidine (Nenietz and Shotts
1993). Decomposition of such fish, which is more likely to occur
when carcasses are not refrigerated for extended periods, produces
significant amounts of histamine by bacterial decarboxylation of
the histidine (Barancin et al. 1998). Bacterial histamine contami-
nation has been associated with adverse health effects when scom-
broids are consumed directly by humans (Barancin et al. 1998). If
histamine is present in lobster baits, it could possibly adversely
affect the health of harvested lobsters. Mackerel were not tested for
histamine levels in this study, and. therefore, this is speculation
without any real evidence. Further investigation of the effect of
mackerel with different states of decomposition and histamine lev-
els on lobster health would be necessary before this speculated link
could be seriously considered. More recently, Castonguay et al.
(1997) demonstrated the presence of small amounts of paralytic
shellfish poisoning (PSP) toxins in the Atlantic mackerel. Perhaps
the slight accumulation in mackerel of toxins related to paralytic
shellfish poisoning may intluence lobster health. It is also possible
that mackerel can intluence lobster health for other reasons.

Using less care in the over-all handling process of lobsters on
board the boats is likely to result in loss of vigor. Tossing lobsters
made them 2.9 times more likely to have loss of vigor at the
processing plant than lobsters placed into the temporary storage
unit, when the effect of other variables was controlled.

Groups of lobsters landed on rainy days were 6.3 times more
likely to lose vigor compared to landings on days without rain.
Lobsters are .sensitive to fresh water exposure (Jury et al. 1994.
Ennis 1993, McMahon 1995), and heavy exposure to rain will
likely be detrimental for lobsters, especially for prolonged expo-
sure times.

The odds ratio for the maximinn depth at which traps were set
was 0.8.'i for each meter. For each 3 m o[' additional depth at which
traps were set, the lobsters became approximately 1.6 times less
likely to suffer from loss of vigor upon their arrival at processing
plants. Colder water temperatures found at greater depths are per-
haps closer to those preferred by lobster (Crossin et al. 1998). and
thus may have beneficial impacts on lobster vigor. Also. Lawlon
and Lavalli (199.5) reported that lobsters may occasionally expe-
rience hypoxia in warm waters, especially in intertidal environ-
ments. Because of their aggressive and territorial behaviour (Aiken
anil Waddy 1995), perhaps some lobsters were forced to move

toward less optimal habitats resulting in a precapture bias toward
less healthy lobsters when caught in more shallow waters.

Lobster health predictors at the group-level (i.e., boat-level)
would probably be more accessible to the industry, because they
would not require individual assessments of lobsters. However,
predictors at the individual (i.e., lobster-level) should yield more
accurate estimates of population health, assuming a valid sample
that is representative of the wild populations. None of the lobster-
level parameters assessed in this study proved to be good predic-
tors of lobster health at the processing plant, as assessed by the
vigor state. Other lobster level predictors, such as physiological
parameters, should be assessed to help define lobster health. The
quantification of the Crustacean Hyperglycaemic Hormone (CHH)
being correlated with stress levels in some decapods including the
American lobster, may have potential (Chang et al. 1998, Paterson
and Spanoghe 1997). Inorganic ions, such as magnesium, calcium,
or potassium, metabolites and waste products such as glucose con-
centration and ammonia levels in the hemolymph should also be
considered for further research, as proposed by Paterson and Span-
oghe ( 1997). Although total hemolymph protein (TP) is used as a
lobster health predictor by the industry, these data did not indicate
that individual lobster vigor upon arrival at the processing plants
was predicted by TP measurements. No association was found
between low TP levels and decreased vigor at processing plants.

Perhaps the lack of correlation between the physiological pa-
rameters used in this study and the vigor state of lobsters at the
processing plants was because of the lack of statistical power, with
only 35 lobsters with decreased vigor in the dataset. Although the
internal validity of these results is acceptable, it may not be valid
for lobster health and fishing practices outside of Prince Edward
Island. The lack of correlation with the outcome may also be partly
explained by the utilization of a subjective outcome (lobster vigor)
not sensitive enough to detect lobster stress, or loss of liveliness.
Recently. Paterson ( 1999) suggested that analytical models may be
useful in predicting certain outcomes, such as lobster weakness
state or even lobster death. Epidemiological models using survival
analysis methods to correlate lobster-level, boat-level, or transport-
level parameters to lobster survival poststorage may be very in-
formative, but are not appropriate for Prince Edward Island. Live
lobster holding is rarely for more than 10-14 days in Prince Ed-
ward Island, and, therefore, the frequency of reduced lobster sur-
vival would probably not be sufficiently high to justify using sur-
vival models.

CONCLUSION

The main objective o\' this study was to establish an epidemio-
logical model assessing the impact of different transportation, fish-
ing, and handling practices on the health and quality of lobsters
upon arrival at processing plants. Four lobster health predictors
were identified, although no transportation condition factors were
significantly associated. The use of different baits inlluenced lob-
ster vigor with mackerel being detrimental. Further investigations
of the types and quality of bail used and its influence on lobster
health should be conducted. Lobster fishers should be encouraged
not to toss lobsters, because gentle handling seems to enhance
lobster vigor. Protection from rainy weather conditions should
reduce industry losses, because lobsters directly exposed to rain
experienced vigor loss. Setting lobster traps in deeper waters also
seemed to be beneficial to lobster health. However, changing the
water ileplli oi' traps is not readily altered by fishers because of

Risk Factors Affecting Lobster Health

281

iiKiiiN Other uncontrollable influences on areas available for fish-
ing. When building such epidemiological models, it is always pos-
sible that there may be unmeasured or unmeasurable factors that
are more related to the outcome (in this case, loss of vigor upon
arrival at the processing plants), or that were indirectly measured
with the factors included in the model-building process.

Lobsters caught in Prince Edward Island generally experience
very low mortality rates preprocessing. Therefore, an examination
of factors associated with survival was not feasible in Prince Ed-
ward Island. This low level of mortality is likely a result of several
factors; most canner-sized lobsters (approximately 63..'i-8l mm)
are held in pounds for extremely short periods, and the majority of
lobsters caught in Prince Edward Island waters are canner-sized

lobsters. To examine handling factors on the boat and their asso-
ciation with survival, a similar evaluation should occur in other
areas of Atlantic Canada where market-sized lobsters and weak
lobsters may exist in greater proportions and where they are held
in captivity for longer periods.

ACKNOWLEDGMENTS

Special thanks to Drs. J. Davidson. G. Johnson. D. Speare. and
J. VanLeeuwen for their advice and editorial comments. This work
was funded in part by the Industrial Research Assistance Program
of the National Research Council of Canada, by the Canadian
Atlantic Lobster Promotion Association, and by the Aquaculture &
Fisheries Research Initiative of Prince Edward Island.

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Barancin. C. E., J. C. Smoot, R. H. Findlay & L. A. Actis. 1998. Plasmid-

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Joiirmil of Shellfixh Research. Vol. 19. No. 1. 283-291. 000.

FORAGING BEHAVIOR OF CARCINUS MAENAS (L.): COMPARISONS OF SIZE-SELECTIVE
PREDATION ON FOUR SPECIES OF BIVALVE PREY

M. MASCARO* AND R. SEED

School of Ocean Sciences
University of Wales. Bangor
Memii Bridge
Anglesey LL59 5EY, United Kingdom

ABSTRACT Experiments were designed to investigate size-selective predation by medium (40-55 mm carapace width) and large
(55-70 mml Carcinm maenas when feeding on four bivalves of contrasting shell morphologies, mussel. Mytiliis ediilis. tlat oyster
Ostrcci eiiiilii. Pacific oyster Crassostrea gigas. and edible cockle Ceraswdenna edute. Medium-sized crabs preferred mussels 5-15
mm .shell length (ma.\imum shell dimension) and cockles 5-10 mm long, whereas large crabs preferred mussels 15-25 mm and cockles
10-20 mm long. Generally, no preference was shown for any particular size of either species of oyster. Comparisons amongst the
preferred size ranges of prey showed that crab preference for a particular size range of prey was more strongly related to the minimum
than to the maximum shell dimension, and that the minimum shell dimension was always equivalent to, or smaller than, the maximum
cross section of the crabs' chelae. The size ranges of M. ediilis and C. ediile selected by C maenas either clearly corresponded to, or
were slightly smaller than, the size ranges of prey with the highest profitability ( = dry weight consumed per unit of handling time).
Profitability values of M. edidis and C. edide. however, showed considerable scatter; whereas those of O. edidis and C. gigas were even
more variable. This variation seems to be the result of behavioral strategies by which crabs attack all encountered prey but reject those
that remain unbroken after a certain number of opening attempts. Our results emphasize the mechanistic nature of size-selective feeding
in C. maenas, and suggest that the differences in the observed patterns of size-selection were mainly determined by the contrasting
morphological features of the bivalve shells, and the way these features influence the vulnerability of prey to crab predation.

KEY WORDS: Foraging beha\'ior. CarciiiKs maenas. size-selection, bivalve

INTRODUCTION

Behavioral and mechanical aspects of predation by crabs that
forage extensively on bivalve populations have been recurrent top-
ics in research (e.g.. Blundon and Kennedy 1982. Hughes and Seed
1995, Seed and Hughes 1995). Selective foraging is a major aspect
within this topic, given the direct influence that the removal of
certain prey types has on the abundance and distribution of the
populations involved in the predatory interaction and of other spe-
cies in the system that are related to them (Ebling ei cil. 1964).
Many authors studying crab selective foraging behavior have re-
lated their findings to the Optimum Foraging Theorem, whereby a
predator chooses its diet to maximize net energy intake per unit of
handling time (Chamov 1976, Hughes 1980). Given a choice of
different sizes and species of prey, a predator should select that
with the highest dietary value (Pyke et al. 1977). However, mini-
misation of handling time { Hughes and Seed 1981) and the risk of
claw damage associated with attacks on larger, more resistant prey
items (Juanes 1992) have also been suggested as causal factors of
prey selection in several crab-mollusk predator-prey relationships.

Because handling times, and. hence, prey values, have a com-
plex variation related to the morphological characteristics of the
crab chelae and the prey shell, foraging tactics can vary when crabs
feed on different species of prey (Creswell and McLay 1990).
Moreover, foraging strategies can also be related to differences in
the patterns of prey dispersion that crabs encounter in their natural
habitats (Hughes and Elner 1979). Thus, comparisons of foraging
behavior when crabs are presented with different hard shelled prey
can provide enlightening information regarding the basis of prey
selection. Whereas numerous studies have addressed the dietary

* Current address: Laboratorio de Biologia Marina Experimental. Depto.
de Biologia. Fac. de Ciencias. UNAM. Apdo. Post. 69. Cd. del Carmen.
Campeche. Mexico. E-mail: mmm@hp.fciencias.unam.mx

and energetic consequences of selective feeding (reviewed by
Hughes 1990), few have identified the cues that make such forag-
ing behavior possible (e.g.. Kaiser el al. 1993).

The shore-crab, Carcinus maenas (L.), is abundantly distrib-
uted in the North Atlantic and is particularly common around the
British Isles (Ingle 1980). Predation by shore-crabs can influence
the abundance and distribution of commercially important bivalves
when these are an integral part of the crab's natural diet (e.g.. Dare
el al. 1983; Sanchez-Salazar et al. 1987a). We investigated the
foraging behavior of adult C. maenas when feeding on four species
of bivalve moUusks with contrasting shell morphologies: the mus-
sel, Mytiliis edidis L.; the flat oyster, Ostrea edidis L.; the Pacific
oyster, Crassostrea gigas (Thunberg); and the cockle, Cerasto-
derma edide (L). Comparisons amongst the foraging strategies
adopted when feeding on these species are used to identify the
stimuli involved in prey selection and to determine the basis of
their size-selective feeding behavior.

MATERIALS AND METHODS

Samples of intertidal Mytiliis edidis and Cerastoderma edide.
covering as wide a size range as possible, were collected from
naturally occurring populations at various sites around the Isle of
Anglesey, North Wales. Samples of Ostrea edidis and Crassostrea
gigas were obtained from CEFAS commercial oyster beds located
in the Menai Strait in North Wales. Shells were cleaned of any
attached fouling organisms and shell length (SL: maximum linear
dimension of the shell), shell height (SH; maximum linear dimen-
sion of the axis at right angles to SL). and shell width (SW;
minimum linear dimension of the shell) of each individual were
measured to the nearest 0. 1 mm using vernier calipers. Soft tissues
were removed following brief iminersion in boiling water and
dried to constant weight at 60 °C. Dry tissue weight (W) was then
determined to the nearest 0.01 mg on a top loading balance. Re-

283

284

Mascaro and Seed

lationships between shell length (.v) and width, height, and dry
weight (V) were best described by the allometric equation y = a •
a'' where a & b are constants. Linear relationships amongst these
variables were obtained by least-square regressions on logarithmi-
cally transformed data. Regression lines were compared by analy-
sis of variance using the General Linear Model with shell length as
the covariate. Pairwise comparisons between the regression slopes
and intercepts were then performed using Tukey's method.

Carcimis maenas 40-70 mm carapace width (CW) were col-
lected by hand from the low shore in the Menai Strait, and main-
tained individually in plastic aquaria (30 x 20 cm) filled to a depth
of 10 cm with running sea water. Water temperature in the aquaria
varied between 12-17° C, and photoperiod was kept constant at
approximately 14 h light: 10 h dark using 40 W fluorescent lights.
Only undamaged male crabs in the later intermoult stage were used
in the experiments in order to avoid any potential bias caused by
morphological and behavioral differences associated with sex and
moult stage. Following their capture, crabs were starved for 48 h
before experiments in order to standardize hunger levels. Size-
selection experiments were carried out by presenting medium-
sized (40-55 mm CW) and large (55-70 mm CW) crabs with prey
species ranging from 5^0 mm SL. Only one species of prey was
offered to the crabs during any single feeding experiment. Each
crab was simultaneously offered five prey items in each 5-mm size
class. Prey items were scattered randomly over the floor of the
aquaria and monitored twice a day. Any item consumed within
each 1 2 h period was recorded and replaced by another of similar
size in order to maintain constant prey availability. Experiments
were run continuously until a consistent feeding pattern emerged
(=10 d).

The number of prey consumed within the different size classes
was analyzed using a chi-square test to determine whether these
deviated from random choice (Peterson and Renaud 1989). Be-
cause the number of size classes of prey offered to crabs was never
less than five, in the event of chi-square tests being significant, the
preferred size classes would be those consumed in >20'7f of the

total number of prey consumed. Comparisons of the size ranges of
prey preferred by both size categories of crabs were made on the
basis of: (1) shell length; (2) width; and (3) relative prey size,
which was obtained by dividing the median value of shell width
within each of the size classes of each prey species offered by the
height (maximum cross section) of the major chela. The height of
the major chela in C. inuenas was estimated using the allometric
equation MH = 0.13 CW' -' (r = 0.98; « = 61) where MH
(mm) is master chelal height and CW (mm) is carapace width
(Mascaro, 1998). To compare the biomass corresponding to the
total number of items of each prey species that were consumed
daily by each crab, the dry flesh weight (mg) of the ingested prey
within each size class was estimated from the median lengths of
each size class using the appropriate allometric equations.

Handling time experiments were carried out by offering each
medium-sized and large crab a prey item of known shell length,
and recording: (1) breaking time (Tb), the time from the first
physical contact with the prey item, through the period of manipu-
lation to the point where the shell was finally opened and the flesh
exposed; (2) eating time (Te), the period from when the prey was
opened to the point where the meal was completed and the empty
shell abandoned; and (3) handling time (Th; i.e., the sum of Tb and
Te). If the crab was successful in the attack, another prey item was
presented, and this procedure repeated until data for a wide size
range of prey were obtained. If crabs were reluctant to eat, they
were starved for 1-3 days until hunger levels recovered. Least-
squares regressions on previously log-transformed handling times
were fitted to the exponential model y = a • e'*' where a and b are
constants, and handling time curves were predicted for each crab
using parameters a and b. Prey profitability was estimated as dry
flesh weight per unit of observed handling time (mg ■ sec"').
Profitability curves were, therefore, obtained by dividing the esti-
mated dry flesh weight of a prey item of known shell length by the
handling time predicted by the exponential model. Analysis of
variance (ANOVA) and Scheffe's method for pairwise compari-
sons of breaking times and profitability values between prey spe-

TABLE 1.

Equation coefficients of the allometric relationships between shell length (SL mm) and shell width (SW mm), shell height (SH mm), and dry
flesh weight (VV mg) in Mytilus edulis (A/), Oslrea edulis (O), Crassostrea gigas (C), and Cerasloderma edule (£).

Relationship

Equation Coefficients

Turkey's Comparisons

M

O

Los SW on loL' SL

Log SH on log SL

Log W on log SL

M. eihiHs
O. edulis
C. gigas
C. edule

M. edulis
O. edulis
C. gigas
C. edule

M. edulis
O. edulis
C. gigas
C. edule

0.42

1.03

0.99

35

M

0.23

0.70

0.82

35

O

0.39

0.96

0.97

42

C

0.35

1.15

0.99

35

E

0.13

0.88

0.99

35

M

0.26

1.12

0.98

35

0

0.02

0.91

0.94

42

C

0.04

LOO

0.99

35

E

4.94

2.69

0.99

35

M

5.99

2.89

0.96

35

O

6.50

3.30

0.95

oT

C

4.86

2.82

0.99

25

F.

ns
ns
ns

Cocincienls a (inlercepll and h (slope) in ihe linear model (log y = log a + b log .v) were obtained by Icasl square regressions; r is the coefficient of
deterniinallon. Results ot Tukey's pairwise comparison tests pert'ormod on Ihe regression coefficients (slopes: normal type; intercepts; bold type) of each
allometric relationship are also prcsenlcd; */■' < 0.05. ns = not significantly dilferenl.

Size-Selective Predation of Carc/nus Maenas

285

20
1 5
1 0 -
05

-" 1

- Ol C

= -2-

a

ac -3

0,3

06

— r-

09

1—
1 3

T"

n

log Shell length

Figure 1. Relationships between (A) shell width (mm), (B) shell height
(mm), (C) dry flesh weight (mg) and shell length (mm) for a wide size
range of Mylilus ediilis (solid squares), Ostrea ediilis (open circles),
Crassostrea gigas (open triangles), and Cerastoderma edule (solid tri-
angles). Solid lines represent predicted values based on the parameters
of the allometric equations presented in Table 1.

cies were performed on the basis of the size ranges of prey pre-
ferred by crabs during the size-selection experiments. In those
cases where crabs exhibited no apparent size preference, the size
range used was comparable to that for the pi'eferred size range of
mussels. Breaking times and profitability values were log-
transformed before analysis of variance was applied to the data.

RESULTS

Analysis of variance of the allometric relationships between
shell length and shell width, height and dry flesh weight resealed
significant differences amongst the four bivalve species (Table I ).
Analysis of variance showed significant differences amongst the
slopes (f= 34.12: P< 0.001) and intercepts (F = 2.87: P< 0.05)
for the regressions between shell width and length of the four
species examined (Table I ). With increase in shell length, C edide
increased in shell width more rapidly than M. ediilis and C. gigas.
which in turn increased in shell width more rapidly than O. ediilis.
Analysis of variance on the regression lines of shell height and
length showed significant differences amongst the slopes (F =
11.5; P < 0.001) and intercepts (f = 6.88: P < 0.001). With
increase in shell length. O. ediilis increased in shell height more
rapidly than C. edule. which, in turn, increased more rapidly than
M. ediilis and C. gigas. Thus, among small individuals shell width
and height of the four bivalves were broadly similar. However.
among larger prey. C. edule had a significantly wider shell than
both M. ediilis and C. gigas of comparable shell length: whereas.

O. edulis had the narrowest shell of the four species (Fig. lA).
Large O. edulis and C. edule. however, had significantly higher
shells than both M. edulis and C. gigas of comparable length (Fig.
IB).

Results of the analysis of variance on the regression lines of
weight and shell length showed significant differences among the
slopes (F = 6.82: P < 0.001 ) and intercepts (F = 46.92: P <
0.001). Pairwise comparisons between regression parameters re-
vealed that the regression lines for M. edulis and C. edule are not
significantly different from each other (Table 1 ). and that both
have the same slope but a higher elevation than the regression line
for O. edulis. These results suggest that both M. edulis and C.
edule had significantly more biomass than any O. edulis of com-
parable shell length (Fig. IC). The regression line for C. gigas
intersects that for O. edulis. suggesting that among smaller prey.
Pacific oysters had less biomass than mussels, cockles, and flat
oysters. However, with increase in size, the biomass of C. gigas
increased more rapidly than in the other species (slope: 3.30 ±
0.34) so that among prey > 20 mm long. C. gigas had more flesh
than O. edulis of comparable shell length.

Both the degree of crab selectivity (i.e.. pattern of size selec-
tion) and the maximum length of prey consumed by C. maenas
varied from one prey species to another (Fig. 2). Although the
percentage of mussels consumed by all crabs decreased slowly
among mussels of increasing shell length, the percentage of cock-
les consumed declined steeply among the larger size classes of
prey. The maximum shell length of M. edulis opened by medium-

B

80
60

M. edulis

^

£ 0.0
(J

I 10

I '

E

9

Z 1

ik

kLkM

d^

80
60

40
a 20

I

O. edulis

^Mtjtl

^

nd

i

fe

fe^

rn^Hi

C. gigas

nd

L

KK^g?;^

C edule

5-10 15-20 25-30 35-40 5-10 15-20 25-30 35-40

Shell length (mm)

Figure 2. (A) Number (± se) and (B) percentage of Mytilus edulis.
Ostrea edulis, Crassostrea gigas, and Cerastoderma edule that were
consumed daily by each Carcinus maenas 40-55 mm CVV (black col-
umns) and 55-70 mm CW (hatched columns) during a period of 8-10
days. Note that C. gigas 5-10 mm in shell length were not available
during these experiments (nd = no data).

286

Mascaro and Seed

TABLE 2.

Results of chi-square (x') tests on the total number of prey consumed by medium (40-55 mm CW) and large (55-70 mm CW) Carcinus

maenas during size-selection experiments.

Species

Medium

Large

Preferred Size Ranges

Preferred Size Ranges

SL

SW

RPS

SL

SW

RPS

M. edulis

699.5*

5-15

2.0-6.3

0.15-0.46

235.9*

0. edulis

7.2 ns

4.0 ns

C. gigas

22.8*

10-15

3.7-5.4

0.27-0.40

3.1 ns

C. edule

282.6*

5-10

2.9-6.4

0.21-0.47

165.1*

15-25

10-20

6.3-10.6

6.4-14.1

0.33-0.56

0.34-0.75

RPS was calculated as the median value of shell width in each size class of prey divided by the height of the ma.ster chela in each size category of crab.
The preferred (i.e., consumed in >20'7f ) size ranges of each prey species are expressed in terms of shell length (SL mm), shell width (SW mm) and relative
prey size (RPS) when chi-square tests proved statistical significance; *significant at P < 0.001. ns = no significant departure from a random choice.

sized and large crabs was 20-25 and 35-40 mm. respectively:
whereas, the maximum shell length of C. edule opened by both
size categories of crabs was 20-25 mm. By contrast, crabs in-
cluded O. edulis and C. gigas of up to 35-40 mm in their diet and
were less size selective than when feeding on mussels and cockles,
resulting in relatively more uniform distributions in both oyster
species.

Chi-square analysis on the number of prey consumed by each
size category of crab revealed that medium-sized crabs (40-55 mm
CW) significantly preferred mussels of the two smallest size
classes (5-15 inni SL). and cockles of the smallest size class
offered (5-10 mm SL: Table 2). Larger crabs (55-70 mm CW)
showed a significant preference for slightly larger mussels ( 15-25
mm SL) and cockles (10-20 mm SL). Neither medium nor large
crabs included cockles >25 mm long in their diets. When feeding
on oysters, crabs generally showed no preference for any particular
size class of either species, the only exception being medium-sized
C. maenas that consumed C. gigas 10-15 mm long in significantly
higher numbers, but included Pacific oysters of all size classes
offered. Differences between the preferred size ranges of prey
were larger when expressed in terms of shell length than in terms
of shell width. For example, medium C. maenas preferred mussels
5-15 mm and cockles 5-10 mm long, but these measured 2.0-6.3
mm and 2.9-6.4 mm in shell width, respectively. In addition, crabs
always preferred prey with a relative prey size <1. suggesting that
preferred prey was always smaller than the height of the largest
chela.

The total number of each prey species consumed daily by each
size category of crab and the corresponding biomass (mg) varied

TABLE 3.

Ingested dry biomass (mg • crab"' • day"') corresponding to the

total number (No.) of Mytilus edulis, Oslrea edulis, Crassoslrea gigas,

and Cerastoderma edule of all size classes consumed by medium

(40-55 mm CW) and large (55-70 mm CW) Carcinus maenas.

from one prey species to another (Table 3). Carcinus maenas
consumed more mussel flesh than any of the other bivalves of-
fered, and differences in consumed biomass were greatest between
M. edulis and O. edulis. There was a general trend toward higher
biomass consumption among crabs 55-70 mm CW, but these
larger crabs generally consumed fewer prey items than crabs 40-
55 mm CW, presumably because large crabs fed on larger prey and
these would have proportionately greater biomass.

Handling times and profitability curves for each size category
of crab feeding on each prey species and the estimated parameters
of the exponential relationships varied among the four bivalve prey
(Table 4, Fig. 3. The strong reluctance of C. maenas to feed on
large C. edule did not allow for handling curves to be predicted for
cockles >20 mm long. However, handling times for small cockles
were overall much lower than for any of the other prey species.
From the handling time data it is clear that large crabs required less
time to handle prey of any particular shell length than did medium-
sized crabs, and, consequently, had access to larger prey items of
any of these prey species. Results showed a considerable scatter
within the handling time data for each prey species, and this be-
came more extreme when profitability values were plotted, par-
ticularly among crabs feeding on O, edulis and C. gigas. None-
theless, profitability plots suggest that larger crabs were consis-
tently capable of obtaining higher profitability than medium-sized
crabs, regardless of the size and species of prey offered. There al.so
seemed to be an opliiiium size of prey, below and above which

TABLE 4.

Equation parameters for the exponential relationships between

handling time ( Th seel and shell length (SL mml lor medium (40-55

mm CW) and large (55-70 mm CW ) Carcinus maenas feeding on a

size range of Mytilus edulis, Oslrea edulis, Crassoslrea gigas, and

Cerastoderma edule.

Medium

Large

Species

M. edulis
O. edulis
C. gigas
C. edule

a

2.93
1.94
2.99

3.27

b

0.24
0.21
O.IS
0.19

r'

0.87
0.74
O.SI
0.69

n

50
26
26
23

a

2.41
3.28
3.18
2.21

b

0.21
0.14
0.14
0.24

Medium

1

urge

r- n

Species

M. edulis
O. edulis

Biomass

211.2
11.8
34.5
60.4

No.

19.4
1.8
3.0
7.1

Biomass

417.6

9.2

37.2

252.5

No.

12.0
1.1
2.1

10.1

0.80 33
0.56 1 5
0.90 2 1
0.68 24

C. gigas
C. edule

a and b were estimated as:
determination.

InTh =

In a +

bSL;

r is the coefficient of

Size-Selective Predation of Carcinus Maenas

287

lOn

2 8

M
W

^ 6

V

E

03

0-

M. edulis

O. edulis

10

6

2-

0

lOi

C. gigas

C. edule

lOn

5-

. ;

o

08

o

0.6

oo

. /

04-

/

//o

02

Jf^^"

0.0-

B

0.20n

S 0.15

E

0 10

0.00

0,20

0.15

0.10

0.05

0.00

0.20n

0.15-

0 10

005

0.00

5 10 15 20 25 30 35

5 10 15 20 25 30 35 5 10 15 20 25 30 35

Shell length (mm)

0.20

0.15

0.10

0.05

0.00

5 10 15 20 25 30 35

Figure 3. (A) Handling time and (Bl profitabilitv for a size range ot Mylilus edulis, Oslrea edulis. Crassostrea gigas. and Cerastoderma edule
consumed by Carcinus maenas 40-55 mm CW (solid symbols) and 55-70 mm CW (open symbols). Solid lines represent predicted values based
on the parameters of the exponential equations presented in Table 4, and on the dry weight-shell length relationships presented in Table 1. Note
scale change for C. edule handling time.

profitability decreased, and this optimal size seemed to increase as
a function of crab size. In both oyster species, however, the size
range at which profitability was maximized was wide, and peaks
were relatively broad. Both medium and large C. maenas obtained
slightly higher profitability when feeding on cockles than on mus-
sels of similar shell length, and crabs would, therefore, need to
feed on slightly larger mussels than cockles to obtain similar prof-
itability.

The ranking order of prey profitability paralleled the order in
which prey were consumed by both size categories of crabs during
feeding experiments (Fig. 4). Analysis of variance showed that
profitability of the preferred size range of M. edulis was signifi-
cantly greater than that of O. edulis and C. gigas (Table 5). The
preferred size ranges of mussels and cockles, however, provided
similar profitability for crabs in both size categories. No significant
differences were detected between profitability of the preferred
size classes of O. edulis and C. gigas for either medium or large
crabs. Analysis of variance of breaking time of the preferred size
range of each prey species revealed that medium-sized crabs took
significantly less time to break open cockles than mussels of the
preferred size range (Table 5). Among large crabs, however, sig-
nificant differences were only found between prey with extreme

values (C gigas > C. edule). No significant differences were ever
found between the breaking times of mussels and oysters or be-
tween the two oyster species. Because crabs generally took similar
times to open all four bivalves, these results suggest that differ-
ences in profitability between the selected size ranges of prey were
mainly because of differences in their biomass.

DISCUSSION

Optimal Foraging Theory assumes that predators are able to
rank prey in the order of their dietary value, and predicts that prey
should be selected accordingly (Charnov 1976, Fyke el al. 1977,
Hughes 1980). When, in the present study, Carcinus maenas were
fed Mytilus edulis and Cerasloderma edule. there was an optimum
size range of prey above and below which profitability ( = dry
flesh weight ingested per unit of handling time) decreased (Fig. 3).
Moreover, the size ranges of M. edulis and C. edule actually se-
lected by these crabs either closely corresponded to. or were
slightly smaller than, the size ranges of prey with the highest
profitability (Table 2). Results similar to these have previously
been demonstrated for C. maenas when feeding on mussels (Finer
and Huahes 1978) and cockles (Sanchez-Salazar et al. 1987b).

288

Mascaro and Seed
B

9-,

40-55 mm CW

1— M

13

6-

^— H

7»

u

3-

T /~

i 0

•
B

Si

0-

-^^ 0

01

a

"S ^ 55-70 mm CW

E

I 4

0-

— -^— c
o

0,04

0.03-

002

0.01

M 0.00
S

2 0 100
e

o

*" 0,075-

0050-

0.025

0.0 0.2 0.4 0.6 0.8 10

0000

E
M

s

u

aa

i°

/u-

90-

-■

60-

^L

M

30-

» E

n-

i^V-

240-

c

■ 0

160-

80-

1 -r

- £

n-

OO 0.2 0.4 0.6 0 8 1 0

0.0 0.2 0.4 0.6 08 1.0

Relative prey size (RPS)

Figure 4. (A) Mean number (± se), (B) profltabilitv l± se) and (C) breaking time (± se) of Mytilus edulis (A/), Ostrea edulis (O), Crassostrea gigas
(C), and Ceraslodenna ediile (E) of the size range that were preferred by Carcinus maenas 40-55 mm CW (upper panels) and 55-70 mm CW
(lower panels). For comparative purposes prey size is expressed as relative prey size (RPS) = shell width/chela! height.

TABLE 5.

Results of analysis of variance and selected pairwise comparisons using Scheffe's method on profitability and brealting time data of Mytilus
edulis (M), Osirca ciliili.s (O), Crassostrea gigas (C) and Cerastoderma edule (El consumed by medium (40-55 mm CW ) and large (55-70 mm

CW) Carcinus maenas.

Size category

F

Pairwise Comp.

Diff. Mean

SE

LCI

HCI

P

Result

Profitability

40-55 mm

30.98

M-O

0.31

0.089

0.05

0.56

*

M>0

***

M-C

0.58

0.070

0.37

0.78

*

M>C

O-C

0.27

0.102

-0.03

0.56

ns

O = C

M-E

-0.12

0.065

-0.31

0.07

ns

M = E

55-70 mm

60.00

M-O

0.60

0.072

0.39

0.80

:1c

M>0

***

M-C

0.66

0.078

0.44

0.89

*

M>C

O-C

0,07

0.0S3

-0.17

0.305

ns

0 = C

M-E

-0.17

0.062

-0.35

0.004

ns

M = E

Breaking lime

40-55 mm

16.92

M-O

-0.17

0.1.38

-0.57

0.23

ns

M = O

***

M-C

-0.23

0.109

-0.54

0.09

ns

M = C

O-C

-0.05

0.158

-0.51

0.41

ns

O = C

M-E

0.58

0,101

0.29

0.87

*

M>E

55-70 mm

4.30

M-O

-0.19

0.136

-0.58

0.20

ns

M = O

**

M-C

-0.24

0.148

-0.66

0.19

ns

M = C

O-C

-0.05

0. 1 57

-0.50

0.40

ns

0 = C

M-E

0.18

0.118

-0.16

0.52

ns

M = E

DilT. Mean = difference between means; SE = standard error of the mean; LCI = low limit of confidence interval; HCI

interval: */■■ < 0.05. **P < 0.01. ***p < 0.001; ns = no significant differences between pairs of variables.

high limit of confidence

Size-Selective Predation of Carcinus Maenas

289

Elner and Hughes (1978) suggested that the feeding strategy ex-
hibited by C. jmienas maximized net energy intake: whereas.
Sanchez-Salazar el al. (1987b) suggested that preference for
smaller size classes of cockles probably reflected shorter absolute
breaking times.

Profitability values in the present study, however, showed con-
siderable variation, particularly from the preferred to the largest
size ranges of prey consumed (Fig. 3). Examination of the data
showed that small differences in shell length could result in large
variations in handling time, suggesting that differences in the
thickness and fracture resistance between individual prey items of
similar linear length probably accounted for much of the observed
variation in handling time and profitability. Moreover, the patterns
of variation seem to be the result of behavioral strategies that crabs
present when feeding on prey that vary little in size, yet vary
markedly in vulnerability. When crabs opened smaller prey within
the size range offered, breaking time was approximately constant,
indicating that crushing techniques used by crabs were equally
effective on all small size classes of these bivalves. This resulted
in short handling time but low profitability values, although eating
time, and hence handling time, constantly increased with prey size
as a result of small increments in flesh weight. When crabs at-
tempted to open a prey item of an intermediate size, the shell
would sometimes yield in a short period of time, making that
particular prey item highly profitable because of its relatively
larger biomass. However, when the shell did not yield in the first
few crushing attempts, crabs took a considerably longer time to
access the flesh, resulting in reduced profitability. Microfractures
in the shell substructure and possible leakage of body fluids as the
shell started to break probably constituted a reinforcing cue for
crabs, causing them to persist with that prey item, thereby increas-
ing substantially handling time. Thus, prey of an intennediate but
similar shell length had extremely variable profitability, although
among these prey, profitability achieved its maximum value.

Because flesh weight increases approximately as the cube of
shell length (Table 1 ): whereas, handling time increases exponen-
tially with respect to shell length (Table 4), the time taken by crabs
to handle prey successfully ultimately increased more rapidly than
the biomass obtained. The lowest profitability values, therefore,
generally corresponded to prey items of a larger shell length,
which, although offering the greatest biomass. could only be
opened by crabs using more complex and time-consuming han-
dling techniques.

Variations in breaking time of a narrow size range of prey
could be attributable to differences in hunger levels (Hughes and
Elner 1979). or to learning mechanisms developed by crabs in
order to manipulate prey of a particular shape (Cunningham and
Hughes 1984). Age-specific differences in shell strength and mor-
phology of individual prey items can also influence size-related
preferences (Boulding 1984). Our results indicate that the lack of
precision in predicting prey value could be attributable to one or
more of these factors rather than to experimental designs that fail
to measure handling time accurately. Furthermore, our results sug-
gest that size selection may be the result of a mechanical process
in which all encountered prey items are attacked but rejected if
they remain unbroken after a certain number of opening attempts.
If this latter view is correct, the decreasing order of crab preference
for different size classes of prey should reflect the decreasing order
of their vulnerability to crab attack. A pattern of size selection that
decreases monotonically with increase in prey size cortesponds to
the behavioral strategy described above, and has been reported for

several brachyuran crabs feeding on a variety of hard-shelled mol-
luscan prey (e.g.. Pearson et al. 1981. Davidson 1986, Juanes and
Hartwick 1990). Moreover, authors have suggested that selection
of small size classes of hard-shelled prey can minimize handling
time (Hughes and Seed 1981. Seed 1990) as well as the risk of
claw damage (Juanes and Hartwick 1990), thereby increasing sur-
vival of foraging crabs that may themselves be vulnerable to pre-
dation.

In the present study, however, large C. maenas selected inter-
mediate size classes of M. edidis (Fig. 2). Earlier studies have
reported that larger prey items may be less preferred, because of
their robustness to crab attack: whereas, the smaller size classes of
prey are less preferred simply because they are encountered less
frequently, misidentified among shell debris, or frequently
dropped (e.g.. Elner and Hughes 1978, Rheinallt and Hughes
1985). This latter would be most likely for crabs with large, less
dextrous chelae that do not allow for the efficient manipulation of
small prey items. Although our study did not include experiments
that presented crabs with altered proportions of different size
classes of prey, observations during handling time experiments
showed that large crabs often had difficulty encountering, identi-
fying, and grasping small prey items.

Variability in handling time and profitability was even more
pronounced among C. maenas feeding on oysters, and the size
ranges of Ostrea edulis and Crassostrea gigas at which profitabil-
ity attained its highest values were not cleariy delimited (Fig. 3).
The precise orientation of the shell within the chelae when force is
first applied further influenced oyster handling time, presumably
as a result of their irtegular shape and resistance at different points
of the shell. Furthennore, large variations in oyster profitability
probably explain the lack of size-related preferences by crabs
when feeding on these particular bivalves. If crabs attempted to
open oysters as they encountered them, rejecting those that did not
yield to the first few crushing attempts, then the oysters consumed
would be those that took the least amount of time to open. Such a
feeding strategy would eventually result in the more uniform pat-
terns of size selection we observed for both O. edulis and C. gigas.
Our results, therefore, emphasize the mechanistic nature of size-
selective feeding in C. maenas. Because the diets of crabs feeding
on all four prey species corresponded approximately to those pre-
dicted by optimal foraging theory, a behavioral strategy by which
crabs attack all encountered prey but reject those that remain un-
broken after a certain number of crushing attempts probably maxi-
mizes feeding efficiency.

Despite the variability in handling time, profitability curves
varied among the species of bivalve prey used: the patterns of size
selection similarly varied from one prey species to another. These
results strongly suggest that differences in size-selective predation
among these bivalve prey are related to the contrasting morpho-
logical features of their shells and the way these features influence
the vulnerability of such prey to predation by shore crabs. Not only
do these four bivalves have contrasting shell shapes, but. as they
increase in size, their flesh content increases at different relative
rates (Table 1 ). Differences in the total biomass consumed by crabs
when offered each of these four prey species individually partly
reflects the variations in their flesh content (Table 3). However,
differences in the total number of prey items consumed suggest
that consumption rates among prey species also reflect crab feed-
ing preferences.

Sanchez-Salazar et al. (1987b) attributed differences in the size
selection of M. edulis and C. edule by C maenas to variations in

290

Mascaro and Seed

shell morphology and strength per unit length. These authors
showed that the shell dimensions of cockles that could be opened
by crabs of a given chelal strength were less, but the energy ob-
tained was greater than when feeding on mussels. Accordingly,
they suggested that crabs could obtain better yields by consuming
cockles than mussels of a similar linear size. In the present study,
profitability values for C. maenas feeding on cockles were over-all
higher than when feeding on mussels (Fig. 3). However, the size
classes of both prey species that were selected by crabs yielded
similar biomass per unit time (Table 5, Fig. 4). Crabs of both size
categories preferred cockles of a shell length that was slightly
smaller than for the preferred mussels, but the preferred size ranges
of both species were of similar shell width (Table 2). In addition.
the percentage of prey consumed by all crabs decreased more
steeply for cockles than for mussels among the larger size classes
of prey (Fig. 2). suggesting that the ability of crabs to crush prey
decreased more abruptly for cockles than for mussels as these
increased in size. As the more globular-shaped cockles increase in
length, shell width increases more rapidly than in the more elon-
gate mussels: consequently, cockles have a significantly wider
shell than mussels of similar shell length (Fig. I). Because dome-
shaped shells are intrinsically stronger than flatter shells (Wain-
wright 1969), the presence of a higher dome in the more convex
cockle shell probably increased force applications required by
crabs to open this infaunal bivalve. Such shell features as large
size, increased thickness, greater inflation, and the absence of gape
reduced the vulnerability of clams to predation by Cancer prodiic-
tiis (Boulding 1984), and have been shown to influence size-
related preferences of crabs (Blundon and Kennedy 1982. Seed
199.3. Walne and Dean 1972) and other decapods (Griffiths and
Seiderer 1980) feeding on bivalve prey.

The importance of shell shape and volume also became evident
in our experiments with O. ediiUs. As prey increased in length. O.
ediiUs shells became significantly higher, but much narrower than
M. edidis. C. edide, and C. gificis (Fig. 1 ). The total shell height of
O. edidis constituted a shape-related restriction that made crabs
unable to accommodate the shell within the widest aperture of the
chelae, while its smaller width prevented crabs from firmly grasp-
ing the shell in a horizontal position and applying an effective
crushing force. The shape-related restriction imposed by the shell
dimensions of O. edulis resulted in similarly long handling times
over all the size ranges of oysters offered, thus explaining the lack
of size selection by C. maenas (Fig. 2).

The patterns of oyster size-selection in our experiments accord
with those previously reported for C. maenas when feeding on C.
gigos and Tiostrea I = Ostrea) liilaria (Richardson cral. 1993); the
latter, as with O. edulis. has a very flat shell. Similarly. C. maenas
has been reported to open C. giitas of up to 50-60 mm SL (Dare
el at. 1983). Although Richardson el al. (1993) suggested that the
reluctance of C. maenas to feed on T. Itiiaria was perhaps related
to characteristics in the shape of its shell. Dare el al. (1983) ex-
plained their own results in terms of the presence of thin "window"

areas in the valves of C. gigas, which made these oysters particu-
larly vulnerable. Our results, therefore, emphasize the importance
of shell shape in determining the vulnerability of different prey
species and suggest that crab preference for a particular size range
of prey is more strongly related to the minimum, than to the
maximum, linear dimension of the shell. As bivalves grow, their
increased minimum shell dimension makes them too large to be
accommodated completely within the chela, decreasing the amount
of force that can be applied (Boulding 1984). Moreover, results
expressed in terms of relative prey size showed that the size of
prey preferred by C. maenas were all within the range of 0.15-
0.75. The initial position of the prey within the chela during the
first application of force was always with the widest, most volu-
minous part of the shell in the region where the dactylus and
propus close to form a distinct gape. If crabs selected prey that
yielded in the shortest time relative to their crushing efforts, then
it is likely that they would consistently select those prey with a
shell width that is equivalent to, or smaller than, the maximum
height of their major chela. This is not surprising, because size-
selective feeding is strongly related to chelal height (Seed and
Hughes 1997) and relative prey size reflects the importance of the
shape, volume, and position of prey when handled by crabs in
diverse attack strategies. Thus, when crab foraging behavior is
being examined, prey size based solely on shell length is not an
appropriate indicator of the shell characteristics associated with
crab preference; the geometry and crushing resistance of prey
shells should also be taken into account.

Interspecific differences in shell shape and thickness between
Lillorina riidis and L. nigrolineata determined prey vulnerability
to predation by C. maenas (Finer and Raffaelli 1980). and are.
therefore, expected to influence crab preference among other prey
species. Whether C maenas exhibits a species-selective feeding
behavior regarding these bivalve prey and whether this behavior
involves an active choice will be addressed in a subsequent pub-
lication. However, in this study, crabs consumed the four prey
species at different rates (Fig. 2), and the order in which prey
species ranked according to consumption rates clearly paralleled
the rank order of prey profitability (Fig. 4). Because differences in
profitability between the selected size ranges of prey were attrib-
utable mainly to differences in their biomass, these results provide
an indication that prey value could influence prey species-selection
by C. maenas.

ACKNOWLEDGMENTS

We are grateful to Mr. Peter Millican. The Center for Environ-
ment. Fisheries, and Aquaculture Science, Conwy, North Wales
for supplying the oysters used in these experiments. We thank Dr.
C. Whittaker and Dr. J. Martins for their advice concerning sta-
tistical analysis. M. M. acknowledges the financial assistance pro-
vided by the Universidad Nacional Autonoma de Mexico, the Brit-
ish Council, and the Overseas Research Students Awards Scheme.

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Jimnial of Shellfish Research. Vol. 19. No. 1. 293-300. 2000.

FORAGING BEHAVIOR OF CARCINVS MAENAS (L.): SPECIES-SELECTIVE PREDATION

AMONG FOUR BIVALVE PREY

M. MASCARO* AND R. SEED

School of Ocean Sciences
University of Wales, Bangor
Menai Bridge
Anglesey LL59 5EY. UK

ABSTRACT Species-selective predation by medium (40-50 mm carapace width) and large (55-70 mm) Carcinus maenas was
investigated by presenting individual crabs with paired combinations of mussels Mytihis edulis. flat oysters Ostrea edulis. Pacific
oysters Crassoslrea gigas. and cockles Ceniswdenna ediile in various proportions. When offered mussels and either oyster species
simultaneously, both size categories of crabs consistently selected mussels, and food choice was independent of prey relative abun-
dance. By contrast. C. maenas selected mussels and cockles as expected by the frequency in which each size category of crab
encountered the preferred size ranges of prey. Crab preference clearly paralleled the rank order of prey profitability, which, in turn,
was mainly determined by prey biomass. suggesting that active selection takes place at some point of the predation cycle. Decisions
by crabs on whether to attack oysters and mussels initially were not influenced by the flavor or odor of their tlesh. Both mussel and
oyster filtrates had a similar reinforcing effect on crab perseverance to open prey. However, species-related preferences exhibited by
crabs feeding at or near the optimal size suggest that foraging decisions are partly based on evaluations of over-all prey shape and
volume, and that the minimum dimension of the shell constitutes an important feature that crabs recognize and associate with prey
value.

KEY WORDS: Foraging behavior. Carcinus maenas, species-selection, bivalve

INTRODUCTION

Patterns of prey selection are the result of a sequence of spe-
cific behavioral components that a predator performs during a
predation event, including the location, attack, capture or attack
success, and ingestion, of prey (Hughes 1980). Feeding prefer-
ences can be partially attributable to passive or mechanistic con-
sequences of physical properties in a predator-prey system that
determine encounter rate and prey vulnerability (Rodrigues el al.
1987: Sponaugle and Lawton 1990). However, decisions by preda-
tors on whether to attack an encountered prey item or to continue
searching, or to reject or accept a prey item that is being manipu-
lated, are still the result of an active choice (Barbeau and Scheib-
ling 1994; Sih and Moore, 1990). To comply with Optimal For-
aging Theory, these decisions must be based in part on the ability
to recognize prey characteristics that correlate with profitability,
defined as the potential energy yield per unit of handling time.
Mechanisms of prey recognition are relatively well understood
among visual hunters such as fish (e.g.. Ibrahim and Huntingford
1989). Crabs, on the other hand, have been the subject of com-
paratively few works relating these mechanisms to optimal forag-
ing behavior (e.g.. Kaiser el al. 1993).

Although size-selective predation in the common shore-crab.
Carcinus maenas (L), has been extensively documented (e.g.. Fi-
ner and Hughes 1978. Finer and Raffaelli 1980. Jubb et al. 1983),
little information is available for selective predation amongst dif-
ferent species of prey. This paper examines the prey species-
related preferences exhibited by C maenas when feeding on a
variety of combinations and proportions of the mussel. Mytihis
edidis L.. the fiat oyster. Ostrea edulis L.. the Pacific oyster, Cras-
soslrea gigas (Thunberg). and the cockle, Cerastoderma edute (L).
A series of experiments testing the importance of shell shape and

*Current address: Laboratorio de Biologfa Marina Experimental. Depto. de
Biologi'a. Fac. de Ciencias. UNAM. Apdo. Post. 69. Cd. del Carmen.
Campeche, Mexico. E-mail: mmmCffhp.fciencias.unam.mx

flesh odor/flavor in prey species selection was designed to inves-
tigate which of these characteristics determined crab decisions
throughout a foraging bout.

MATERIALS AND METHODS

Species-selection experiments were performed by presenting
medium (40-55 mm carapace width: CW) and large (55-70 mm
CW) C. maenas with paired combinations of a wide size range of
prey species (Table 1 ). Five prey items in each 5 mm-size class
were scattered randomly over the floor of the aquaria and moni-
tored twice a day. Any item consumed within each 12-h feeding
period was recorded and replaced by another of similar size to
maintain constant prey availability. Fxperiments were run continu-
ously until a consistent feeding pattern emerged ( = 10 d). Crabs and
prey were collected and maintained as described in Mascaro and
Seed (2000).

To establish whether crab preferences resulted from a passive
response to the rate in which the prey species were presented,
medium and large crabs were individually offered equal and un-
equal numbers of the preferred size ranges of prey in the combi-
nations M. edulis-O. edulis and M. edulis-C. edule. The size
classes of preferred prey were established on the basis of those
most frequently consumed in single prey species experiments
(Mascaro and Seed, 2000; Table 1 ) The proportions of presented
prey were altered prey so that the prey species that had been
preferentially selected in the previous experiments was now at the
lower relative abundance of 1:2 and 1:4 with respect to the less
preferred species. Each time a prey item was consumed by a crab
it was immediately replaced by another of similar size, and the
precise order in which prey items were taken was recorded. Once
a prey item was encountered and recognized as potential food, a
crab could either reject (i.e., touch, manipulate and finally aban-
don) or accept (i.e., successfully open and consume) the prey.
Because satiation, among other factors, can strongly influence the
behavioral sequence adopted by an individual crab, each trial was
run for 1 h, the average period of time taken for crabs to become

293

294

Mascaro and Seed

TABLE 1.

Size ranges (mm in maximum sliell dimension) of Mytiliis ediilis (A/),
Ostrea edulis iO), Crassostrea gigas (C), and Cerastoderina ediile (£),

tliat were offered to two size categories (mm of carapace width,

CW) of Carciniis maenas in experiments with a wide size range of

prey, and with equal and altered proportions of prey species.

Experiment

CW

M-O

M-C

o-c

M-E

Wide size
range of prey

40-55
55-70

40-55
55-70

5-30
5-30

M

10^0
10-40

o

10-40
10^0

c

10-40
10-10

E

Equal and altered
proportions

10-15
15-20

10-15
15-20

10-15
15-20

5-10
10-15

Size classes were established on the basis of those most frequently selected
by crabs in experiments where prey were presented individually (Mascaro
& Seed 1999).

satiated during preliminary observations. Experiments were re-
peated on a daily basis until a consistent pattern emerged (=5 d).
The total number of times that crabs encountered each prey species
were then tested for goodness-of-fit to the expected values (as-
suming a probability of encounter: 1;1, 1:2, and 1:4). Differences
in the total number of prey of each species that were successfully
opened by each individual crab were tested using the same proce-
dure.

Two experiments were designed to examine whether prefer-
ence for a certain prey species was influenced by the odor/flavor of
the flesh. First, five individually maintained C. maenas (50-60 mm
CW) were presented with 10 agar cylinders (9x10 mm), 5 mussel-
flavored and 5 oyster-flavored. Gels were made using a filtrate of
10 g of either mussel or oyster flesh homogenized in 100 mL of
seawaler; this was then mixed with 20 g of agar and poured to a
depth of 10 mm into petri dishes. When set, the cylinders were cut
using a 9-nim diameter cork borer and stored at -10 °C. The
number of cylinders that were attacked was monitored every
10 min during a period of 1 h. Differences among the number of
each type of cylinders destroyed within 10, 30 and 60 min were
tested for goodness-of-fit to equal expected numbers.

The second experiment presented individually maintained C.
maenas of 50-60 mm CW with models made of mussel and oyster
shells 15-20 mm in shell length (maximum linear dimension of the
shell) filled with either mussel or oyster gel. Intact bivalves were
briefly immersed in boiling water to remove all the flesh and
thoroughly clean the shells. Empty shells were then dried, and a
hole was drilled through the valves so that these could be closed
together with fuse wire. Rectangular pieces of gel cut using scalpel
were carefully held between the valves, which were then pressed
and tied together. Five individually maintained crabs were offered
5 mussel shells + mussel gel and 5 mussel shells + oyster gel; a
further Ave crabs were offered 5 oyster shells + mussel gel and 5
oyster shells + oyster gel. Each time a crab opened a model. Ihe
model was replaced by another of the same type to maintain con-
stant availability. The time thai elapsed from the moment a crab
grasped a model, until that model, whether it was intact or no[. was
finally abandoned was recorded with a stopwatch, and defined as
persistence time. The number of models thai were accepted and
rejected within I h was recorded. The total number of times thai
each crab encountered each model type (i.e., models with similar
shells but diffcrcnl gel) was (hen tested for goodness-of-fil to the

expected values (assuming an equal probability of encounter). Dif-
ferences in the total number of models that were accepted by each
individual crab were tested using the same procedure. Compari-
sons of persistence time between models of similar shell types but
different gel contents were examined using 2-sample Mests on
previously log-transformed data.

To assess the importance of shell shape on crab feeding pref-
erences, experiments were carried out by presenting medium and
large C. maenas with inedible models of comparable size and
weight to those of the preferred live prey (Table 1 ). Models of zero
profitability were constructed by filling empty shells of the pre-
ferred size range of each prey species with epoxy resin. Models
were left 24 h in sea water to eliminate the smell of resin. The
combinations of models offered were the same as for the live
prey experiments. Trials consisted of presenting four C. maenas
(50-60 mm CW), each maintained separately, with a sequence of
six models of the same prey .species. As soon as a crab finished
manipulating one model in the sequence, it was presented with the
next. On day 1, two crabs were presented with six models of a
certain species, and two crabs were presented with six models of
another. On day 2. the treatment was reversed so that each crab
was presented with a paired combination of model prey (M-O,
M-C, O-C, and M-E) Persistence time with each model was re-
corded with a stopwatch and data were logarithmically trans-
formed before analysis of variance using a balanced design with
"model in sequence" and "model type" as fixed factors, and "crab"
as a random factor.

The importance of shell shape in detemiining crab persistence
was further examined by presenting C. maenas of 50-60 mm CW
with epoxy resin models of three contrasting geometric shapes: a
"cockle" (sphere: 904 mm'), a "mussel" (wedged rectangle: 420
mm^), and an "oyster" (flat disc: 530 mm'; Fig. 1). The resin
models were similar in length (maximum linear dimension, all:
12-15 mm) but differed in Iheir height (maximum linear dimen-
sion of the axis at right angles to their length: sphere: 12 mm;

Length: 15 mm

\Height

6 mm
Width: 6 mm

B

Length and
Height: 15 mm

Lengtli, Height
and Width: 12 mm

Figure 1. Epoxy resin models of contra.sting geometric shapes that were
uttered to Ciinimis iiuicinis (50-60 mm CW) to examine the importance of
shell shape in crab forging behavior: A) wedged rectangle (420 mm'), B)
Hal disc (530 mm"); C) sphere (904 mm').

Species-Selective Predation of Carcinus Maenas

295

Medium

• Mylilus edulis vs

6

Large

Oslrea edulis

' Mylilus edulis vs

6

— • — Crassoslrea gigas

E

Ostrea edulis vs

6
5

1

Crassoslrea gigas

^^'''tesCXS—r L.

- Mylilus edulis vs

Cerastoderma edule

Shell length (mm)

Figure 2. Number of prey of various size classes that were consumed by
medium (40-55 mm CW) and large (55-70 mm CW) Carcinus maenas
during experiments where crabs were offered prey species in paired com-
binations: (A) M. ediilis-0. edulis, (B) M. edulis-C. gigas. (C) O. edulis-
C. gigas. (D) M. edulis-C. edule. Values are mean consumption rates •
crab"' day"'. Note change in scale in (C).

rectangle: 6 mm; disc: 15 mm) and width (minimum linear dimen-
sion: sphere: 12 mm; rectangle: 6 mm; disc: 3 mm). On each day,
six individually maintained crabs received a sequence of five mod-
els of one of the three inodel types and their persistence times with
these models were recorded. Over a 3- d period, each crab had
experienced each of the different model types. After each trial,
crabs were fed on mussel flesh for 1 h before being starved until
the following day. The order in which each crab experienced the
different model types was random. Data were logarithmically
transformed before differences in persistence time were examined
by analysis of variance (ANOVA) using a balanced design with
"model in sequence" and "model type" as fixed factors and "crab"
as a random factor.

RESULTS

When M. edulis was offered in combination with either O.
edulis or C. gigas, both medium (40-55 mm CW) and large (55-70
mm CW) C. maenas showed a strong preference for mussels (Fig.
2a.b). When offered a choice between O. edulis and C. gigas.
neither size group of crabs showed any preference for either oyster
species (Fig. 2c). When given a choice between M. edulis and C.

edule. medium-sized C. maenas clearly preferred mussels;
whereas, large crabs consumed similar numbers of both prey of
10-15 nun long, but only consumed mussels from the larger size
classes (Fig. 2d).

When crabs were presented with both equal and unequal num-
bers of the preferred size ranges of M. edulis-O. edulis. the per-
centage of mussels opened by medium and large C. maenas (77%-
100%) was always significantly higher than that of oysters (Table
2). By contrast, the proportion of O. edulis accepted by crabs was
never greater than 23%. even when the alternative species was
scarce. Of all the mussels encountered, very few were rejected;
whereas, any encountered O. edulis were only occasionally con-
sumed, and were frequently rejected before the crabs had at-
tempted to open them. Although the percentage of rejected oysters
was always high, the already low percentage of rejected mussels in
the 1:2 ratio experiments decreased even further in the 1:4 ratio
experiments, where mussels were at their lowest relative abun-
dance (Table 2). Results of goodness-of fit tests showed that the
number of observed encounters was not significantly different
from those expected in all mussel-oyster combination trials (x"
from 0.22-2.84, all at F > 0,05), suggesting that consumption rates
were not infiuenced by prey encounter rates.

In experiments with equal and unequal numbers of the pre-
ferred size ranges of M. edidis-C. edule. however, the percentage
of accepted and rejected prey varied according to the rates in
which prey species were encountered (Table 2). Of the total num-
ber of prey accepted by large crabs, the percentage of M. edulis
decreased when mussels were less abundant. Similarly, the per-
centage of accepted C. edule incieased as their relative abundance

TABLE 2.

Percentage and numbers (in parenthesis! of Mylilus edulis, Ostrea

edulis. and Cerastoderma edule that were accepted or rejected by

Carcinus maenas 40-55 and 55-70 mm CW in experiments where

crabs were presented with the preferred size classes of mussels and

oysters and mussels and cockles in proportions of 1:1, 1:2, and 1:4.

Outcome

M. edulis O. edulis

M. edulis C. edule

Crab Size

1:1

1:1

40-55 mm

ace

88(7)*

13(1)

70(8)*

30(3.4)

rej

36(3.6)

64(6.4)

0(0)

100(0.4)

55-70 mm

ace

88(7.4)*

12(1)

50(2.6)"

- 50 (2.6)

rej

6(0.4)

94(6)

44(0.8)

56 ( 1 )

ace

1:2

1:2

40-55 mm

91 (1 1.4)*

19(2.6)

54(8.4)*

46(7.2)

rej

4(0.8)

96(17.8)

10(0.2)

90(1.8)

55-70 mm

ace

100(2)'^"

0(0)

38(4.2)"

" 62 (6.8)

rej

33(3.8)

67(7.8)

50(0.8)

50 (0.8)

ace

1:4

1:4

40-55 mm

77 (4.6)*

23(1.4)

49(8.2)"

51 (8.6)

rej

4 (0.6)

96(15.4)

8(0.4)

92 (4.6)

55-70 mm

ace

100(1.4)''"

0(0)

9 (0.6)"

' 91 (6.2)

rej

11 (0.6)

89(4.8)

56(1)

44(0.8)

Values are mean consumption rates per crab over 1 -h periods during 4-5
consecutive days; * denotes prey species accepted in significantly higher
numbers than expected (P < O.OI ): NTA = cases in which results did not
allow for chi-square tests to be applied; ns = no significant differences;
ace = accepted; rej = rejected.

296

Mascaro and Seed

.a

I

Mm

Mo

Om

Oo

Model type

Figure 3. Mean persistence time (sec ± se) taken by Carciinis maenas
(50-60 mm CW) to open shells of either Myliliis ediilis (M) or Ostrea
edulis (O) filled with gels made from either mussel (m) or oyster (o)
filtrate.

increased. Large crabs encountered both prey species as expected
by the proportions in which they were presented (x" from 0.03-
1.63, all axP> 0.05). Although medium-sized crabs also accepted
mussels and cockles in the same proportions as they encountered
them, the encounter rates of mussels and cockles did not corre-
spond to the relative abundance in which prey were offered (1:1
trial: x" = V.48, P < 0.05; 1:2 trial: x" = 9.6, P < 0.01 and 1:4
trial: x" = 25.77, P < 0.001, respectively). Medium-sized crabs
encountered mussels and cockles in statistically indistinguishable
numbers in the 1:2 ratio trial (x" = 0.05, P > 0.05), and encoun-
tered mussels at approximately half the rate that they encountered
cockles in the 1:4 ratio trial (x" = 1-85, P > 0.05).

When C. maenas (50-60 mm CW) were offered a choice be-
tween mussel and oyster agar cylinders, these were readily ac-
cepted whenever they were encountered. No significant differ-
ences in the number of mussel and oyster agar cylinders accepted
after 10 tnin, 30 min. and 1 h were detected (x' from 0.08-0.89, all
at P > 0.05). When crabs were presented with mussel shells filled
with either mussel or oyster gel, they encountered and accepted
both model types in similar proportions (encountered: x" = 0.31;
P > 0.05; accepted: x" = 0.20; P > 0.05). When oyster shells filled
with either mussel or oyster gel were offered, the encounter rates
for both model types were again similar (x^ = 2.25; P > 0.05). and
crabs again accepted similar numbers of oyster shells filled with
either type of gel (X" = 0.15; P > 0.05). Because crabs showed no
apparent preference for one type of gel over the other, results from
experiments with different types of gel but similar types of shell
were combined. Comparisons of results among experiments with
mussel shells and those with oyster shells showed that, although
crabs encountered significantly more oyster shells than mussel
shells (x~ = 16.46; P < 0.001 ), they accepted mussels and oysters
in similar proportions (x" = 1.13; P > 0.05). Crabs, however.
rejected a signilicanlly higher proportion of oyster shells than mus-
sel shells (x" = 26.28; P < 0.001), so that in experiments with
oyster shells, crabs actually rcjeclcd most of the models encoun-
tered.

Crabs persisted for similar periods of time with luussel shells
filled with either mussel or oyster gel (133.9 + 21.5 and 176.5 ±
10.8 sec. respectively; / = -1.84; P > 0.05; Fig. 3). Persistence
limes were also similar amongst oyster shells filled with either
mussel or oyster gel (45.9 ± 6.5 and 58.4 ± 10.2 sec, respectively;
( = -1.01: /' > 0.05). Because persistence times v\ith different gel

types were in each case similar, results for similar types of shell
were combined. Persistence times for mussel shells were signifi-
cantly longer than for oyster shells regardless of the type of gel
with which they had been filled it = 7.79, P < 0.001 ). In summary,
our results indicate that prey choice is based on shell shape and
strength rather than flesh odour.

When offered five epoxy resin models in sequence, C, maenas
(50-60 mm CW) persisted for significantly shorter periods of time
through the sequence of the first to the last model in all species
presentations (M-O: F = 6.78; P < 0.001; M-C: F = 47.16; P <
0.001; 0-C: F = 13.23; P < 0.001; M-E: f = 6.31; P < 0.001;
Table 3; Fig. 4). Crabs persisted significantly longer with the first
model of M. edulis presented than with either the O. edulis or C.
gigas models in the M-0 and M-C combinations (F = 131.76;
P < 0.001; F = 104.60; P < 0.001, respectively), and persisted
significantly longer with the first C. gii;as than with the first O.
edulis in the O-C combination (F = 1 8.53; P < 0.001 ). However,

TABLE 3.

Results of ANOVA for the sequential presentation of epoxy resin

models of prey species in four combinations to Carcinus maenas of

50-60 mm CW.

Mytiliis ediilis-Ostrea edulis

Source

df

SS

MS

F

P

Sequence

5

1 .943X7

0.3XX77

b.li

<0.00l

Model type

1

7.55.544

7.55.544

131.76

<0.00l

Sequence ■ model

type

5

0.3S005

0.07601

1.33

0.278

Crab

3

0.32239

0.10746

1.87

0.153

Error

33

1.89235

0.05734

Total

47

12.09411

Mytilus edulis-Crassoslrea gigas

Source

df

SS

MS

F

P

Sequence

6.79175

1 .35

47.16

<0.00l

Model type

3.01310

3.01310

104.6

<(}.00l

Sequence ■ mode

type

0.(IS96S

(1.01794

0.62

0.6X3

Crab

U.3196I

0.10654

3.70

0.021

Error

33

0.95057

0.028X1

Total

47

11.16471

Ostrea edulis-Crassoslrea gigas

Source

df

SS

MS

F

P

Sequence

3.23497

0.64699

13.23

<0.00l

Model tvpc

().9()(i()4

0.90604

1 8.53

<0.00l

.ScL|Lience ■ mode

type

0. I9S73

0,03975

0.81

0.549

Crab

2.S4772

0.94924

1941

<0.()01

Error

33

I.6I3SS

().()4X9I

Total

47

S. SO 1,^6

Mytili

.V edul

s-Cerasloderma edule

Source

df

SS

MS

F

P

Sequence

5

3.1709

0.6342

6.3 1

<0.()01

Model tvpe

1

0.0004

0.0004

0.00

0.95 1

Sequence • Mode

1 tvpe

.S

0.2725

0.0545

0.54

0.743

Crab

3

0.9271

0.3090

3.07

0.041

Error

33

3.3 IS4

0.1006

Total

47

7.6X93

ANOVA: Persistence time = sequence • model type (fixed factors); crab
(randtHii l;ictor).

Species-Selective Predation of Carcinus Maenas

297

e

4>

B

600

400

200

0

600
400-1
200
O-l

"•ni « ^ .» ^

600

400

200

0

600

400
200
0-.

Mytilus edulis

(— •— )

vs

Ostrea edulis

(-0-)

Mytilus edulis

(— )

vs

Crassostrea gigas

(— *— )

Ostrea edulis

(-o-)

vs
Crassostrea gigas

Mytilus edulis

(— )

vs

Cerastoderma edule

Number of model in sequence

Figure 4. Mean persistence time (sec ± se) for inedible models of Mytilus
edulis. Ostrea edulis. Crassostrea gigas, and Cerastoderma edule when
presented sequentially to Carcinus maenas of 50-60 mm CW.

crabs did not differ in their persistence times with the first models
of either type in the M-E combination (F < 0.01; P > 0.05). The
lack of any significant contribution of the interaction between the
model in sequence and model type to the over-all variance of the
data for all prey combinations (F from 0.54-1.87: P > 0.05) indi-
cates that the decrease in persistence times throughout the se-
quence of presentation was similar regardless of the type of prey
handled by the crabs. Significant differences in the persistence
time between individual crabs in the M-C {F = 3.70; P < 0.05). the
0-C (F = 19.41; P < 0.001), and the M-E {F = 3.07; P < 0.05)
combinations indicates that some of the variability in persistence
time is attributable to unexplained differences among individual
crabs. However, no significant differences were detected among
the crabs in the M-O combination (F = 1.87; P > 0.05; Table 3).
When C. maenas (50-60 mm CW) were presented with epoxy
resin models of three contrasting geometric shape, persistence time
decreased significantly through the sequence of models regardless
of their shape (Fig. 5; F = 20.46; P < 0.001). Although the
decrease in persistence time was similar for all three geometric
shapes (F = 0.75; P > 0.05). crabs persisted for a significantly
longer period of lime with the first sphere and wedged rectangle
than with first flat disc (F = 7.31; F < 0.001). No significant
differences in persistence time among individual crabs were de-
tected (F = 2.00; P > 0.05).

Taken overall, the results of the experiments with epoxy resin
models indicate that crabs initially preferred those with a larger
minimum dimension (i.e., models of mussels and cockles, and
models in the shape of a sphere and a wedged rectangle). Persis-
tence time, however, decreased with increasing number of models
in the sequence in a similar rate regardless of model type, sug-
gesting that shell shape does not influence the rate at which pref-
erence declines when crabs handle food items of zero profitability.

DISCUSSION

Many studies have related crab preference to the selection of
prey with a higher value or profitability, expressed as the net
energy intake per unit of handling time (Elner and Hughes 1978,
Elner and Raffaelli 1980). However, authors have noted that se-
lection of optimum prey could result from a greater chance of
encountering items with a larger surface area and/or reduced han-
dling times because of a lower predatory resistance (Barbeau and
Scheibling 1994). in which case, prey selection should be consid-
ered to be a consequence of passive choice. Passive or mechanistic
prey selection usually results from physical properties and behav-
ior of both predator and prey (Hughes 1980) that determine the
probability of encountering and opening alternative prey types.
Body size, speed of movement, and density of predator and prey
can influence the probability of encountering, recognizing, and
predicting valuable prey (Lawton 1989); whereas, contrasting mor-
phologies in bivalve shells and the mechanical properties involved
in crab attack can affect prey vulnerability (Boulding 1984. Brown
and Haight 1992), and hence the probability of opening encoun-
tered prey (Sih and Moore. 1990).

When crabs were offered a wide size range of oysters and
mussels simultaneously, both medium (40-55 mm CW) and large
(55-70 mm CW) crabs consistently selected mussels (Fig. 2). Fur-
thermore, results of experiments with M. edulis and O. edulis in
equal and altered proportions suggest that preference for mussels,
and the apparent lack of preference for oysters, are independent of
the relative abundance in which either prey species are presented
(Table 2). Previous authors have shown that C. maenas consumed
M. edulis at more than twice the rate at which they consumed C.
gigas (Dare et at. 1983); whereas. Cattcer novaezelandiae also
preferred mussels when offered a choice of mussels and gastro-
pods (Creswell and McLay. 1990).

Barbeau and Scheibling (1994) indicated that active selection
can be considered to be an important component of predation when
a predator selects a prey type more often than expected when given
a choice of prey types than when not given a choice (see also
Liszka and Underwood 1990). In our study, comparisons of prey
con.sumption rates in single and multiple choice experiments could
not be made, thus, active and passive components could not be
analyzed in this way. However, the preference for M. edulis ex-
hibited by C. maenas was consistent throughout experiments
where prey types were encountered in varying and contrasting
proportions (Fig. 2; Table 2). In earlier single species experiments
involving M. edulis, O. edulis. C. gigas, and C. edule designed to
establish whether prey selection in C. maenas was size related, the
order in which prey species were ranked according to consumption
rates clearly paralleled the rank order of species profitability (Mas-
caro and Seed 2000). We reported that differences in profitability
between prey species were mainly attributable to differences in
their biomass, rather than to differences in breaking time. These
results suggest that prey value can influence prey species-selection

298

Mascaro and Seed

and thai crab preference for mussels in the present study involves
an active component of selection at some point of the predation
cycle.

When C. maenas were offered M. edulis and C. edule in various
proportions, species selection vv'as strongly influenced by the fre-
quency in which each size category of crabs encountered prey
(Table 2). Although active selection could not be invalidated
{sensii Barbeau and Scheibling 1994), the close agreement in the
proportions of accepted and encountered prey items suggests that
the active component of selection in this particular prey combina-
tion is not important in determining crab preference. The observed
differences in the foraging behavior of C. maenas when feeding on
a combination of mussels and oysters and a combination of mus-
sels and cockles further supports the view that the relative impor-
tance of active and passive selection in explaining prey choice may
differ with each predator-prey system (Abele el al. 1981).

For active prey selection, mechanisms must exist by which
crabs are able to recognize prey characteristics that correlate with
their potential value. Among prey near the optimal size, such char-
acteristics as shell shape or flesh odor/flavor may have an impor-
tant effect on foraging decisions. Amino acids can be readily dif-
ferentiated by C. maenas during searching (Shelton and Mackie
1971) and feeding phases (Case and Gwilliam 1961). Our results
show that crabs were not attracted any more often to gel cylinders
made from mussel-flesh filtrate than to those containing oyster-
flesh filtrate. Crabs attacked both types of cylinders whenever
these were encountered and this behavior persisted throughout the
duration of the feeding trials, suggesting that preference for either
type of flavor was not modified once crabs had experienced the
gels.

A study of the factors affecting diet selection in C. maenas
demonstrated that shore-crabs are sensitive to different concentra-
tions of mussel flesh filtrate, and suggested that, despite dilution
effects, crabs may be able to distinguish mussels of varying quality
by responding to odor (Kaiser et al. 1993). In addition, these
authors indicated that olfactory stimuli had a reinforcing effect on
crab preference, because crabs readily picked up and manipulated
both models with and without mussel filtrate, but more quickly
rejected those that did not incorporate the chemical stimulus. Our
experiments did not include any examination of different concen-
trations of flesh filtrate, but mussel and oyster tlesh filtrates had a
similar reinforcing effect on the perseverance with prey, and crabs
initially attacked either type of model irrespective of their flavor
(Fig. 3). These results provide further evidence of the reinforcing
effect of olfactory and taste stimuli, and comparisons with previ-
ous reports suggest that perhaps the concentration, rather than the
specific chemical composition, of llesh might be involved in prey
attractiveness to crabs (Shelton and Mackie 1971 ). Our results do.
however, suggest that certain characteristics of the shells of M.
edulis and O. edulis might provide mechanical stimuli that influ-
ence decisions by crabs initially to attack and to persist with prey
that is already being manipulated (Fig. 3).

When attacking hard-bodied, resistant prey, predatory crabs
show relatively stereotyped patterns of behavior (Lau 1987, Seed
1993), but details of this behavior can be influenced by specific
morphological characteristics among prey of different size (Finer
and Hughes 1^78). Over a relatively narrow range of prey size;
however, shape can have a marked influence on the handling pro-
cess (Boulding 1984. Griffiths and Seiderer 1980). and informa-
tion gained by crabs while rotating prey with the chelae and mouth
parts can be crucial in deciding whether or not the attack is con-
tinued or aborted (Akumfi and Hughes 1987). In experiments

where C. maenas were offered a sequence of five models of each
prey species, crabs persisted longer with the first model of a mus-
sel to be offered than with the first model of an oyster, and they
also persisted longer with the first model of C. gigas than with O.
edulis (Table 3; Fig. 4). These results suggest that the initial re-
luctance of crabs to attack flat oysters and their propensity to
attack mussels, are not associated with differences in the ultimate
energetic reward, but could be based on an evaluation of the over-
all shape and/or volume of the shells of these bivalves during a
first recognition phase.

Further investigation of the importance of shell shape in species
selection showed that C. maenas persisted much longer with the
first models that resembled both the shape of a cockle (sphere) and
a mussel (wedged rectangle) than with the first model that re-
sembled a flat oyster (disc; Fig. 5). The resin models used had a
similar length, but differed in their height and width, the flat disc
having the smallest width of the three models (Fig. 1). If crabs
evaluate prey on the basis of shell width rather than length, they
would be expected to persist longer on those shells having the
greatest width. A strong association between shell width and vol-
ume is suggested by the significantly greater increase in shell
width with increasing shell length in cockles and mussels, com-
pared to flat oysters, and by flat oysters having significantly less
flesh weight than mussels and cockles of comparable shell length
(Mascaro and Seed 2000). Furthermore. C. maenas obtained the
greatest profitability when feeding on cockles and mussels (Mas-
caro and Seed 2000). and C. edide and M. edulis were included in
crab diets more frequently than O. edulis throughout the species-
selection experiments (Table 2. Fig. 2). These results suggest that
shell width; that is, the minimum linear shell dimension, probably
constitutes an important morphological characteristic that crabs are
able to evaluate and associate with potential prey value.

Previous studies have reported that the minimum shell dimen-
sion is an important characteristic determining prey size and spe-
cies selection in crabs (Boulding 1984) and in other decapods
(Griffiths and Seiderer 1980). In experiments where C. maenas
was presented with Perspex models of different shape and size.
Kaiser el al. ( 1993) found that changes in model length had little
influence on handling time, because the mechanical efficiency of
the chelae was detennined by the cross-sectional profile of the
prey. These authors further suggested that those models that more
closely resembled the shape of a mussel (wedged rectangle) al-
lowed the chelae to operate at maximum mechanical ad\antage
and improved handling efficiency. Mascaro and Seed (2000) re-

-• — Wedged rectangle
•A- - - Flat disc
■♦■ Sphere

12 3 4 5 6

Number of model in sequence

Figure 5. Mean pcrsisicncc lime (sec ± se) for inedible models of three
conlrastinj; geiimetric shapes (wedged rectangle, flat disc and sphere! thai
were presented sequenlially to C{ircinii.\ maenas of .iO-fiO mm CWi.

Species-Selective Predation of Carc/nus Maenas

299

ported that the relative prey size ( = shell width/maximum cross-
sectional dimension of the major chela) preferred by C. maenas
consistently had values <1, suggesting that the position in which
the width of the shell fitted within the chela was significant in
determining attack success of the preferred size classes of prey. In
the present study, flat oysters were more difficult to accommodate
within the chela than were mussels. Although flat oyster shells are
very narrow, their large shell length and height reduce their han-
dling efficiency. In contrast, mussel shells with only one of its
linear dimensions being large, can be accommodated within the
chela either w ith its height or its width in the gape between propus
and dactylus.

If foraging decisions by crabs were based on shell width, then
some patterns of species selection by C. maenas observed in this
study could be explained. The observed similarity in consuinption
rates of M. edulis and C. edule might be attributable to the simi-
larity in shell width of the (different) size classes of these prey
offered (Table 1 ). When crabs were offered similar size classes of
M. edulis and O. edulis, they preferentially consumed mussels
probably because of the differences in shell width between the
(similar) size classes of prey presented (Table 1 ).

Although some portunids can use vision to locate their prey
(Hughes and Seed 1981, Seed and Hughes 1997), crabs are essen-
tially nonvisual predators. This makes olfactory stimuli the pre-
dominant factor directing crabs toward potential prey (Barber
1961). Although different concentrations of chemical compounds
elicit different components of the searching response (i.e., loco-
motory or probing and grasping reflexes; Zimmer-Faust and Case
1982), our results suggest that during this initial phase, differences
in flesh odor/flavor between different prey species did not seem to
influence prey selection. Once crabs have physically encountered

a prey item, shape and size of the shell assume a greater signifi-
cance than flesh odor, interacting with chemical and mechanical
stimuli from alternative prey touched by the pereiopods (Jubb et al.
1983). Our results emphasize the importance of certain shell char-
acteristics and suggest that those dimensions more strongly asso-
ciated with prey volume (e.g.. shell width), and, hence, the amount
of potential flesh, might be predominant in deciding whether a crab
attacks. Once a food item has been recognized and an attack ini-
tiated, further decisions can be influenced by several factors, in-
cluding microfractures within the shell structure (Boulding and
LaBarbera 1986) and leakage of body fluids that stimulate crabs to
persist with the attack. Stimuli from alternative prey may also deter
crabs in their opening attempts. Moreover, it is at this stage, when
a prey item that does not yield easily to the crab's crushing efforts
can be abandoned. Thus, prey vulnerability, together with the te-
nacity and experience of crabs might be particularly relevant at this
point of the encounter. Although our results do not allow a hier-
archical order of crab responsiveness to various prey stimuli to be
defined, they do emphasize that odor and shape assume different
importance at several decisive moments of the foraging bout, and
that by using a variety of stimuli to assess prey quality, crabs
probably enhance their predatory efficiency.

ACKNOWLEDGMENTS

We are grateful to Mr. Peter Millican, The Centre for Environ-
ment, Fisheries, and Aquaculture Science, Conwy, North Wales
for supplying the oysters used in these experiments. We thank Dr.
C. Whittaker and Dr. J. Martins for their advice concerning sta-
tistical analysis. M.M. acknowledges the financial assistance pro-
vided by the Universidad Nacional Autonoma de Mexico, the Brit-
ish Council, and the Overseas Research Students Awards Scheme.

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Joiinuil ofSlwllfhh Research. Vol. 19. No. 1, 301-31 1. 2000.

THE EFFECTS OF HYPERCAPNIC HYPOXIA ON THE SURVIVAL OF SHRIMP
CHALLENGED WITH VIBRIO PARAHAEMOLYTICUS

CHRISTINA M. MIKULSKI,' LOUIS E. BURNETT,' *
KAREN G. BURNETT-

University of Charleston. South Carolimi
Grice Marine Laboratory
205 Fort Johu.son
Charleston, South Carolina 29412
'Department of Microbiology and Immunology
Medical University of South Carolina
221 Fort Johnson
Charleston. South Carolina 29412

ABSTRACT Estuarine organisms routinely encounter fluctuations in dissolved oxygen, carbon dioxide, and pH, which can vary both
seasonally and diumally. Such environmental stresses as hypoxia can affect the immune response of invertebrates and vertebrates and
have been linked to increased disease incidence. This research investigated the effects of hypoxia, hypercapnia, and low pH on disease
susceptibility in both penaeid and palaemonid shrimp. Juvenile penaeid shrimp Litopenaeus vuimamei and adult grass shrimp Palae-
moneu's piigio were challenged by intramuscular injection with a previously determined LDju dose of a known pathogenic strain of
Vibrio paraliaemolyriciis. Mortalities were monitored for shrimp held under normoxia (Po, = 150-15.5 torr, Pco, = 0.23 torr, pH =
7.6-8.0 for L vannamei, Po, = 150-155 torr, Pco, = 0.23 torr, pH = 8.0-8.2 for P. pitgio) and two levels of hypoxia. The penaeid
shrimp were challenged under normocapnic hypoxia (Po, = 45 torr, Pco, = 0,23 torr, pH = 7.8-8.1) and hypercapnic hypoxia (Poj
= 30 torr. Pco, = 15.2 torr, pH = 6,8-7.0). Grass shrimp were challenged under two levels of hypercapnic hypoxia (Po, = 45 torr
and 30 torr, Pco, = 15.2 torr, pH = 6.7-7.0). Both the juvenile L vannamei and the adult P. pugio held under hypercapnic hypoxia
at 30 torr oxygen displayed significantly lower 48-hour survival (15.7 and 3.1%, respectively) than animals held in normoxic water
(28.7 and 29.4%. respectively). There was no significant decrease in survival in L. vannamei under normocapnic hypoxia at 45 ton-
oxygen or in P. piigio under hypercapnic hypoxia at 45 torr oxygen. Total hemocyle count (THC/niL) significantly decreased in adult
L vannamei held under hypercapnic hypoxia when compared to normoxic controls. Oxygen level had a significant effect on total
hemocyte density; whereas, time and the interaction of time and oxygen did not. The reduction in THC/mL may contribute to an
increased rate of mortality in shrimp held under hypoxic conditions and challenged with V. paraliaemolxticus. These results show that
hypercapnic hypoxia decreases survival following bacterial challenge in both L. vannamei and P. pugio and decreases total hemocyte
count in L vannamei. These data provide direct evidence that naturally occurring variations in oxygen, CO, and pH can place estuarine
organisms at increased risk from opportunistic pathogens

KEY WORDS: LD,,,, hypercapnia, hypoxia, palaemonid, penaeid, shrimp, Vibro

INTRODUCTION

Penaeid and Palaemonid shrimp in estuarine waters frequently
encounter levels of dissolved oxygen, carbon dioxide, and pH that
vary dramatically on a diurnal and seasonal basis. Shallow coastal
regions in the southeast and in the Gulf of Mexico often experience
dissolved oxygen concentrations less than 3.0 nig/L (Breitburg
1990, Rabalais et al. 1994, Burnett 1997, Summers et al. 1997). In
South Carolina tidal marshes, tidal creek oxygen pressures can
fluctuate between 9 and 170 torr (6% and 110% air saturation)
within a 24-hour period (Cochran and Burnett 1996). Oxygen lev-
els as low as 1 .2% air saturation (approximately 2 torr) have been
measured in the nearby Savannah River estuary (Winn and Knott
1992). Moreover, hypoxia is almost always accompanied by an
increase in carbon dioxide pressure (Pco,), or hypercapnia, pro-
duced by respiration. Elevated levels of water CO, then drive a
decrease in water pH. Cochran and Burnett (1996) reported that
Pco, varies from 0.3 to 12 torr. and pH ranges from 6.5 to 7.6 in
South Carolina tidal marshes. Thus, hypoxia and low pH often
co-occur in the natural environment (Burnett 1997).

*Corresponding address: Louis E. Burnett. Grice Marine Laboratory. 205
Fort Johnson. Charleston, SC 29412. E-mail: bumettl@cofc.edu

Shrimp raised in aquaculture ponds also experience severe
changes in O,. CO,, and pH because of high density and nutrient
input from feed (Browdy et al. in press. Madenjian 1990). Dis-
solved oxygen levels are routinely measured in well-managed farm
ponds, with the general understanding that low O, levels may be
lethal to shrimp. Supplemental aeration is used to reduce fluctua-
tions in dissolved oxygen; however, periods of hypoxia and hy-
percapnia still occur in routine management (Chang and Ouyang
1988, Garcia and Brune 1991).

Although extreme hypoxia or anoxia can cause mass mortali-
ties in estuarine organisms (Garlo et al. 1979, Winn and Knott
1992, Diaz and Rosenberg 1995, Lenihan and Peterson 1998),
sublethal hypoxia may have an adverse impact on normal physi-
ological functions in shrimp, such as osmoregulatory capacity
(Charmantieret al. 1994) and molting (Clark 1986). Hypoxia also
can suppress immune function in shrimp. Direkbusarakom and
Danayadol (1998) found that hypoxia (1.8-2 ppm) decreased
phagocytosis and bacterial clearance efficiency in the black tiger
shrimp, P. monodon. In addition, Le Moullac et al. (1999) reported
a decrease in hemocyte numbers and respiratory burst activity of
Litopenaeus .sryUrostris exposed to severe hypoxia (1 mg Oo/L).
These observations suggest that chronic sublethal hypoxia might
suppress the ability of shrimp to resist infections with opportunis-

301

302

MiKULSKI ET AL.

tic pathogens such as environmental bacteria, viruses, and fungi.
Indeed. Le Moullae (1999) demonstrated that the levels of hypoxia
that decreased hemocyte numbers and suppressed respiratory burst
activity in L. stylirostris also increased pathogenicity of Vibrio
alginolyticus in that shrimp species.

Unfortunately, most studies of hypoxia ignore changes in CO,
and pH associated with hypercapnic hypoxia (Burnett 1997).
Where the effects have been assessed, low pH and high CO,
enhanced mortality rates of extreme hypoxia (Martinez et al. 1998)
and altered metabolic activity (McCulloch 1990). Low pH inde-
pendently and additively with hypoxia suppressed the respiratory
burst of oyster hemocytes (Boyd and Burnett 1999). The latter
study strongly suggested that hypercapnic hypoxia suppresses the
resistance of wild and aquacultured estuarine organisms against
such naturally occurring opportunistic pathogens as bacteria, vi-
ruses, and fungi.

In the last 10 years, several highly lethal bacterial pathogens
have had a serious impact on both wild and aquacultured popula-
tions of penaeid shrimp (Karunasagar et al. 1994, Mohney et al.
1994, Hiney 1995, Liu et al. 1996. Alapide-Tendencia and Dureza
1997, Lavilla-Pitogo et al. 1998). The most frequently reported
bacterial infection in penaeid shrimp is vibriosis, caused by bac-
teria from the family Vibrionaceae (Adams 1991, Sahul Hameed
1995). Bacteria in the family Vibrionaceae comprise 10-50'7f of
the marine heterotrophic bacteria found in coastal waters (Thune et
al. 1993). Among several Vibrio species associated with this dis-
ease, Vibrio parahaemolyticus is frequently associated with dis-
ease outbreaks in aquaculture (Mohney et al. 1994. Sahul Hameed
1995) and is sometimes found at high densities in coastal waters
(Buck 1990, DePaola et al. 1990).

In this study, we evaluated the impact of hypoxia and hyper-
capnic hypoxia on resistance to the opportunistic bacterial patho-
gen V. parciluieinolyticiis in two commercially and recreationally
important species of shrimp. The Pacific white shrimp, Litope-
naeus vannamei, is the species of choice for penaeid shrimp aqua-
culture in South Carolina. L. vannamei occurs naturally from the
Gulf of California to northern Peru (Perez Farfante and Kensley
1997), but is imported for use in aquaculture because of its faster
growth over native species (Sandifer et al. 199j!), The grass
shrimp, Palaemonetes pugio. serves an important role in the estu-
ary as a delritivore by consuming and breaking down Spartina and
aiding in trophic level energy transfer (Welsh 1975). They also
serve as prey for many important commercial and recreational
fishes and crustaceans, which use the marsh as nursery grounds
(Welsh 1975). In the first phase of these experiments, an intra-
muscular bacterial challenge model with survival endpoini was
developed and applied in both shrimp species to determine LD,„
values for V. parahaemolyticus. Then, to evaluate the contribution
of hypoxia and hypercapnic hypoxia to disea.se resistance, survival
was monitored in shrimp challenged with LD,„ doses of V. para-
haemolxucus and exposed to varying levels of water O,. CO,, and
pH. Finally, to determine whether hypercapnic hypoxia might alter
cellular components of the shrimp immune system over the time
course of these bacterial challenges, total hemocyte densities of the
hemolymph (THC/mL) were compared in animals exposed to nor-
moxia and hypercapnic hypoxia.

MATERIALS AND METHODS

Experimental Animals

Litopenaeiis vannamei (Boone) from specific pathogen-free
stocks were provided by the Waddell Mariculture Center in Bluff-

ton, South Carolina, by Island Fresh Seafood in Yonges Island,
South Carolina, and by Dixieland Maricultural Farms in Holly-
wood, South Carolina. Shrimp were maintained in well-aerated
recirculating seawater at 28-32 ppt salinity, 23-25 °C, and pH
8.0-8.2. Water quality variables (pH. salinity, and temperature)
were measured every other day. Ammonia was monitored twice a
month and remained lower than 0.25 mg/L. Animals were fed once
daily with shrimp feed (Zeigler Brothers, Inc). All necessary pre-
cautions were followed for possessing a nonindigenous shrimp
species as outlined in the nonindigenous shrimp possession permit
#NI98-0565 granted by the South Carolina Department of Natural
Resources.

Grass shrimp Palaemonetes pugio (Holthuis) were collected
with a dip net in a nearby tidal creek. These shrimp were held in
a 50-gallon aquarium at 25-27 ppt salinity and 23-25 °C for at
least 2 days before use in an experiment. Animals were fed Marine
Tetra Flakes daily.

Bacteria

A known pathogenic strain of Vibrio parahaemolyticus (90-
69B3) was streaked on a Tryptic Soy Agar (TSA) plate with 2.5%
NaCI added and allowed to grow overnight at room temperature.
Aliquots (0.5 mL) of the bacteria were stored in freezing media
(Tryptic Soy Broth (TSB) + 2.5% NaCl and 20% glycerol) at
-70 °C. These aliquots were used as the working stock.

For each assay, V. parahaemolyticus was streaked onto TSA +
2.5% NaCl plates from the frozen aliquots and allowed to grow at
room temperature for 24 hours before use. A different aliquot was
used for each assay to avoid excessive passages of the bacteria on
plates. Bacteria were transferred from the plates to 2.5% NaCl
buffered with 20 mmol/L HEPES using wooden applicator sticks.
Bacterial densities were quantified by optical density (OD) at 540
nm and then serially diluted in the saline to obtain the test dosages.
OD values were confirmed by counting colony-forming units on
double layer plates ( 10 niL of marine agar containing the bacterial
dilution overiaid onto 10 mL of TCBS agar). OD values of 0.1 and
1.0 were determined to be equal to 1.0 x 10** colony-forming units
per mL (CFU/mL) and 1.0 x 10" CFU/mL, respectively. Koch's
postulates were satisfied to confirm the pathogenicity and relation-
ship between V. parahaemolyticus and \'ibriosis (Prescott et al.
1996).

The identity of the bacteria used in challenge tests and after
each isolation of Koch's postulates was confirmed using Gram
strains, motility tests, characterization of growth on TCBS plates,
cytochrome oxidase tests, and AP1-20NE test strips for Gram-
negative, nonfermentative bacteria (API resultant bacteria
#7276644). Aseptic techniques were used when working with the
bacteria. Waste material was either autoclaved or disinfected with
1% chlorine bleach.

LPfi, Tests for Litopenaeus vannamei

Vibrio parahaemolyticus was streaked onto TSA -I- 2.5%^ NaCl
plates from the frozen aliquots as described above. Juvenile ani-
mals (5.8 to 8.9 cm and weighing from 1.0 to 4.2 g) were injected
intramuscularly near the fourth \entral abdominal segment using a
Hamilton syringe with 50 (xL of bacterial suspension (ranging
from 5x10' to 5 x lO' CFU/shrimp) or with 2.5% NaCl buffered
with 20 mmol/L HEPES without bacteria as a control. Animals
were then placed in 3.5 L, wide-mouth, screw-lid, glass jars with
700 mL of filtered (0.45 \xm) artificial seawater (ASW) adjusted to
30 ppt. Lids of the lest containers were fitted with tubes for in-

Survival of Shrimp Challenged with Vibrio

303

coming air and an air release tube (61 cm) with two cotton plugs
to contain Vibrio aerosol. Seven animals were placed in each jar
with three replicates for each dose. LD,,, tests were performed
under normoxic conditions (155 torr o.xygen). with low CO, (less
than 1 torr) and high pH (pH 7.7-7.9). and mortality was recorded
at 2, 4, 8. 1 2. 24, and 48 hours after injection of Vibrio. Water was
changed in all jars at 1 2 and 24 hours after feeding and then when
necessary in individual jars (i.e.. when the water became cloudy
because of shrimp mortality). Animals were fed commercial
shrimp food (as above) every 12 hours. The L. vannanwi LD,„ test
was repeated three times. LD50 and confidence intervals for both
species were calculated using the EPA Trimmed Spearmann-
Karber program (Hamilton et al. 1977).

LDf^i, Tests for Palaemonetes pugio

The methods for the P. pugio LD^,, tests were similar to those
mentioned above for L vannainei. with a few exceptions. Because
P. pugio were smaller (2.1 to 3.4 cm and weighing from 0.2 to 0.4
g). only 5 jjlL of a saline containing bacteria was injected, and the
shrimp were held in smaller test chambers with 400 mL of ASW.
Water in all experimental jars was changed once every 24 hours.
Animals were fed Marine Tetra Flakes (as above) every 12 hours.
The P. pugio LD50 test was repeated two times with different
bacterial concentrations for each (ranging from 2.25 x 10' to 2.25
X 10' CFU/shrimp for test 1, and ranging from 5 x 10' to 5 x 10^
CPU/shrimp for test 2).

Challenge Test Design

It was not possible to maintain appropriate levels of oxygen in
the jars used for the L. vaimamei and P. pugio LDjo tests by
directly using Wosthoff gas mixing pumps and individually aer-
ating the jars. This was because of the low output of the pump, the
variability in aeration to each jar. and the high oxygen demand of
the shrimp. Therefore, a new experimental design was employed
for the hypoxic challenge tests for both species. Ten-gallon aquaria
were divided into four chambers of equal size to hold the shrimp
and one smaller chamber to hold a circulating pump (see below)
using Plexiglas drilled with holes to allow water to flow freely
among the chambers. Nine L of 30 ppt filtered (0.45 |jim) artificial
seawater (Crystal Sea marine mix) was added to each tank. A
small, submersible pump (Penguin 550) in each experimental tank
circulated water among the compartments. Normoxia was main-
tained by vigorous aeration. Hypoxia was maintained by control-
ling aeration. The consumption of oxygen by the shrimp lowered
the oxygen pressure in the water. Oxygen pressure in the water was
monitored using an oxygen electrode and meter (YSI Model 58).
Output from the oxygen meter was monitored by a Sable System
data acquisition system, which was used to control tank aeration by
an air stone at a user-defined setpoint. To control water CO, pres-
sures, a Wosthoff gas mixing pump delivered mixtures of CO, and
nitrogen continuously. The CO, and N, gas mixture also served to
lower the oxygen pressure. At steady state, this system maintained
constant oxygen and CO, pressures (Fig. I ). Oxygen levels in the
chambers remained within I torr of the set value.

L. vannamei Challenge Tests at 45 torr O, -Normocapnia Hypoxia

L vannamei challenge tests were performed under two levels of
hypoxia mimicking two different environmental conditions (see
Table 1 ). The first set of tests compared disease susceptibility
between animals held under normoxia and animals held under
normocapnic (i.e.. very low CO, pressure) hypoxia with no added

CO, (treatment 1 ). This test evaluated the effect of low oxygen
only. For these tests, ASW was made hypoxic by bubbling pure N,
into the tlrst chamber. Gassing the water with N, drove off excess
CO,, keeping pH and CO, at normoxic levels, with pH 7.8-8.1 and
0.03% CO,. Normoxia was maintained by bubbling ambient air
into the tanks through three air stones.

Using the method described above, juvenile shrimp ranging
from 5.7 cm to 9.0 cm (1.0 g to 4.5 g) were injected intramuscu-
larly with 50 ijlL of bacterial suspension or with 2.5% NaCl buff-
ered with 20 mmol/L HEPES. The bacterial numbers for these tests
ranged from 1.8 x 10"^ CFU/shrimp to 2.25 x 10* CFU/shrimp,
which were greater than the previously determined average LD^,,
but were within the 95% confidence interval (Table 2). Nine
shrimp with or without injected bacteria were placed in each of the
four chambers of the appropriate tanks for a total of 36 animals per
tank. Animals were placed randomly in either the hypoxic or nor-
moxic tanks. All four treatments (one per tank) were conducted
simultaneously and were counted as one replicate. Mortality was
recorded, and dead or moribund animals were removed at 2, 4, 8,
12, 16, 20, 24, 28, 32, 36. 40, 44, and 48 hours after injection
challenge. Water was changed in all tanks at 12, 24, and 36 hours,
and then whenever necessary in individual tanks. The replacement
water for the normocapnic hypoxia treatment was gassed ahead of
time to appropriate treatment pressures to avoid a change in oxy-
gen pressure. Animals were fed commercial shrimp food every 12
hours. This challenge test was repeated three times.

L. vannamei Challenge Tests at 30 torr O, + 15.2 torr
CO r-Hypercapnic Hypoxia

The second set of tests (treatment 2) compared disease suscep-
tibility between animals held under normoxia and animals held
under hypercapnic hypoxia at 30 torr oxygen and 15.2 torr CO, (4
and 2%, respectively). These tests were conducted as described
above (treatment 1 ): however, the CO, and pH were adjusted to
mimic hypoxic levels (Table 1 ). The resulting pH of the hypoxic
water was 6.8-7.0. The bacterial concentration used in these tests
was 1.125 x 10*, which was within the 95% confidence interval
previously determined in the LD,,, tests. This challenge test was
repeated three times.

P. pugio Challenge Tests

Both of the grass shrimp challenge tests compared disease sus-
ceptibility between animals held under normoxia and animals held
under hypercapnic hypoxia. These challenge tests were pertormed
under two levels of hypercapnic hypoxia: 45 torr O, -1- 15.2 torr
(2%) CO2 and 30 torr O, 4- 15.2 torr (2%) CO,. Hypercapnic
hypoxia was achieved as de.scribed above for both treatments with
only the set point for the data acquisition system differing between
the two levels of hypoxia (Table 1).

The methods for the P. pugio challenge treatments were similar
to those mentioned above for the L. vannamei treatments, with a
few exceptions. For the grass shrimp tests, only 5 [jlL of a saline
containing bacteria was injected, and 10 animals were placed in a
chamber for a total of 40 animals per tank. The bacterial concen-
trations for these tests ranged from 9.10 x 10"* to 1.25 x 10^'
CFU/shrimp. These values were outside of the confidence interval
previously determined in the LD5,, tests because of an experimen-
tal error in the original calculation of the confidence intervals.
However, because the same bacterial concentration was used for
both treatments in a test, the results were unaffected. These chal-
lenge tests were each repeated four times.

304

MiKULSKI ET AL.

Water
Pump

Relay controls air pump

Figure 1. Schematic drawing of the tanks used to hold animals under different gas pressures. Oxygen pressure was measured with an oxygen
electrode and adjusted using an air pump controlled hy a computer. The infusion of air was opposed by gassing with mixtures of nitrogen and
carbon dioxide (depending on the experiment). The tank was divided into compartments, and the water was circulated using a submersible
pump.

.Stall slical Analysis for Challenge Tests

Challenge tests were performed using a I'liII factorial design
with bacteria and oxygen as the effect variables. Eacfi cliallcnge
test produced four survival curves: normoxia without bacteria.
normocapnic hypoxia (hypoxia with very low CO-,) or hypercapnic
hypoxia (hypoxia with elevated CO-,) without bacteria (depending
on the treatment in question), normoxia plus bacteria and nor-
mocapnic hypoxia or hypercapnic hypoxia plus bacteria. Using the
statistical program JMP IN (SAS Institute. Inc.), a quadratic poly-
nomial was fitted to each curve to obtain an intercept, response
coefficienl. and response coefficient" for each line. The combined
coefficient and coefficient" parameters were then analyzed as the
response variables in a multivariate analysis of variance
(MANOVA) with bacteria, oxygen, and the interaction of bacteria
and oxygen (bacteria*oxygen) as the x values. Differences in the
coefficient and coefficient" revealed differences in the survival
rate of shrimp among Ircalmenls. The intercepts were not ana-

lyzed, because differences in the intercept were artifacts of fitting
a quadratic polynomial to a survival curve and did not reveal
information about the rate of survival. Four MANOVA tests were
run, one for each suite of tests: L. vaniuinwi at 45 torr oxygen and
<1 torr CO,, L vannamei at 30 torr oxygen with 15.2 torr CO,. P.
piiftio at 45 torr oxygen with 15.2 torr CO,, and P. pugio at .''O ton-
oxygen with 15.2 torr CO,. Results of the MANOVA tests re-
vealed if oxygen level/CO, Irealnienl (normoxia \s. hypoxia), bac-
teria (absence vs. presence) and/or the interaction of the two
(bacteria*oxygen) had a significant effect on (P < 0.05) shrimp
survival following bacterial challenge. Univariate analysis of vari-
ance tests (ANOVAs) were then run to see if the significance
found in the MANOVA was attributable to coefficienl. coeffi-
cient", or both.

Total Hemocyte Count

The impact of hypercapnic hypoxia at .30 torr oxygen. 15.2 torr
CO, and pH 6.9-7.1 on total hemocyte counl/mL heniolymph in

Survival of Shrimp Challenged with Vibrio

305

TABLE 1.
Water quality variables used in the challenge tests and total hemocyte count (THC) assay.

Vibrio Challenge
Tests

o.

CO2

torr

% air sat.

mg/L

torr

%

pH

Liropcinieus vannamei

Normoxia (control)

150-155

21

7.29

0.23

0.03

7.6-8.0

Normocapnic hypoxia
treatment 1

45

6

2.12

0.23

0.03

7.8-8.1

Hypercapnic hypoxia
treatment 2

30

4

1.41

15.2

2

6.8-7.0

Palaemonetes pugio
Normoxia (control)

155

21

7.29

0.23

0.03

8.0-8.2

Hypercapnic hypoxia
treatment 1

45

6

2.12

15.2

2

6.9-7.0

Hypercapnic hypoxia
treatment 2

30

4

1.41

15.2

2

6.9-7.0

Total hemocyte count
Normoxia (control)

150-155

21

7.29

0.23

0,03

8.0-8.2

Hypercapnic hypoxia
Ranges of variables
observed in nature

30
0-285

4
0-38.6

1.41
0-14

15.2

0.23-35.6

2
0,03^.7

6.9-7.1
6..5-8.3

Ot and COt are presented several ways for comparison with water quality data in the literature. The following references were used to report the
environmental ranges listed for O,. CO,, and pH: Breitburg. 1990. Winn and Knott, 1992; Rabalais et al., 1994; Cochran and Burnett, 1996.

Litopenaeus vaniuimei was measured over the 48 hours to replicate
the time period of the challenge tests. At time zero, shrimp were
placed randomly in normoxic or hypercapnic hypoxia tanks.
Hemolymph from individual adult L. vannamei was withdrawn
from the ventral sinus at the base of the fourth or fifth walking leg
at a specified time point (4, 8, 16, 24, or 48 hours) into a 1.0 mL
syringe with a 26-gauge needle. Hemolymph was diluted with an
anticoagulant solution (AS) described by Lee et al. (1995): 207^
filtered seawater, 30 mmol/L trisodium citrate, 0. 1 mmol/L glu-
cose, 26 mmol/L citric acid, 10 mmol/L EDTA at pH 4.6. Total
hemocyte counts were performed using a hemocytometer, taking
into account the dilution of the hemolymph with AS during bleed-
ing. Twenty shrimp were bled at 4, 16. 24, and 48 h ( 10 each from
normoxia and hypercapnic hypoxia); 22 shrimp were bled at 8 h
(II each from normoxia and hypercapnic hypoxia). Individual
shrimp were used only once. Mortality was monitored throughout
the experiment.

TABLE 2.

48-Hour LD,,, values for Litopenaeus vannamei and Palaemonetes
pugio for Vibrio parahaemolyticus.

Test

48-hour LD,,

95% Confidence Interval

Litopenaeus vannamei

1 6.04X lO-'CFU/shrimp

2 1.37X 10''CFU/shrimp

3 5.89X lO'CFU/shrimp
Mean 8.54 x lO' CFU/shrimp

(3.06 X lOVg wet weight)
Palaemonetes pugio

1 1.46X lO'CFU/shrimp

2 2.16X lO-'CFU/shrimp
Mean 1.81 x lO-'tCFU/shrimp

(6.O81X 10''/g wet weight)

2.69 X lU'-1.36x 10'*
7.09 X 10'-2.64 x 10''
2.90 X 10^-1.19 X 10"

6.22 X 10'-3.44x lO^*
2.16 x 10''-3.88 X lO-"

The LD;,) values are presented as colony forming units (CFU) per shrimp
and per gram shrimp wet weight.

A two-way ANOVA test was performed using time, oxygen,
and the interaction of time times (*) oxygen as the x values and
total hemocyte count (THC) as the response (y) value (a = 0.05).
An a posteriori /-test was used to compare means at 48 hours.

RESULTS

LD,„ Tests

Vibrio parahaemolyticus has dose-response pathogenicity to
both Litopenaeus vannamei and Palaemonetes pugio. Dead or
moribund shrimp exhibited signs of Vibriosis, including opaque-
ness of the abdominal muscle, lethargy, expansion of the chro-
matophores, and abdominal flexure that peaked at the third ab-
dominal segment (Lightner 1988). LD^,, values for L. vannamei
ranged from 5.89 x 10^^ to 1.37 x 10" CFU per shrimp with an
average value of 8.54 x lO'^ CFU/shrimp (Table 2, n = 3). The
95<7r confidence interval ranged from 2.69 x 10^ to 2.64 x lO"
CFU/shrimp. Concentrations of bacteria used for the challenge
tests remained within these confidence intervals.

LD5f, values for P. pugio were 1 .46 x 1 0"" CFU/shrimp and 2. 1 6
x lO"* CFU/shrimp. with an average value of 1.81 x 10"" CFU/
shrimp (Table 2, n = 2). The 95% confidence interval ranged from
6.22 x 10' to 3.88 x lO^* CFU/shrimp.

Challenge Tests

Control survival

L. vannamei control survival was greater than 77.8, 88.9, and
86.1% in the experiments testing normoxia, normocapnic hypoxia
at 45 torr oxygen, and hypercapnic hypoxia at 30 torr oxygen,
respectively. P. pugio control survival was greater than 92.5, 97.5.
and 95% in the experiments testing normoxia, hypercapnic hyp-
oxia at 45 torr oxygen, and hypercapnic hypoxia at 30 torr oxygen,
respectively (Fig. 2 and 3). These results show that the levels of
hypoxia used were not lethal to either organism.

306

MiKULSKI ET AL.

100-

ra 80

>

E

40

20

0

100

a 80

>

E

5 60

40

20

0

Litopenaeus vannamei

No Bacteria

Normocapnic
Hypoxia O
Normoxia D

Bacteria
Normoxia ■

Normocapnic
Hypoxia #

Palaemonetes pugio

B

No Bacteria
-C3— Q Hypercapnic
Hypoxia O
Normoxia D

+ Bacteria
I I Normoxia ■

# — • • — • Hypercapnic

-'--'- -"-I Hypoxia «

0 4 8 12 16 20 24 28 32 36 40 44 48

Time (hours)

Figure 2. A. Litopenaeus vannamei survivai I'oliowing bacterial chal-
lenge under normoxia (Poj = 150-155 torr, Pco, = 0.23 torr, pH 7.6-
8.0) and normocapnic hypoxia (Po, = 45 torr. Pco, = 0.23 torr, pH
7.8-8.1). Shrimp were injected intramuscularly with 50 pL of Vibrio
parahaemolyticus bacterial suspension (1.8 x lO" CFU/shrimp) or with
HEPES buffered 2.5% NaCI for controls. There were 36 shrimp per
treatment. Values at each time point are the mean (h = 3 experiments):
standard errors are indicated except where the error is small and falls
within the width of the datapoint. The effects of oxygen/CO, treatment
and the interaction of bacteria*oxygen/CO, treatment on disease sus-
ceptibility were not significant as determined by a MANOVA (/" =
0.6478 and P - (1.3594). B. Palaemonetes pugio survival following bac-
terial challenge under normoxia (Po, = 150-155 torr. Pco, = 0.23 torr,
pH 8.0-8.2) and hypercapnic hypoxia (Poj = 45 torr, Pco, = 15.2 torr,
pH 6.9-7.0). Shrimp were injected intramuscularly with 5 pL of Vibrio
parahaemolyticus bacterial suspension (1.0 x lO" CFU/shrimp) or with
HEPES buffered 2.5% NaC'l for controls. There were 40 shrimp per
treatment. Values at each time point are the mean (;i = 4 experiments);
standard errors are indicated except where the error is small and falls
within the width of the datapoint. The elTects of oxygen/C'O, treatment
and the interacticm of bacteria*oxygen/CO, treatment on disease sus-
ceptibility were not signincant as determined by a MANOVA (P =
0.7379 and P = 0.7412).

L vannamei — normocapnic hypoxia at 45 torr oxygen

These ehiillcnge tests examined (he et'teet of moderate hypoxia
only (normocapiiie hypoxia) without added Co, (hypercapnia) on
L vannamei survival (Fig. 2A). The effects of oxygen and the
interaetion of baeteria*oxygen were not significant at Po, = 45
toiT as determined by a MANOVA (f = 0.6478 and P = 0.3594,
respectively). The effect of bacteria alone was significant {P
sO.OOOl ). Subsequent ANOVA tests revealed that the significance
was due to differences in the coefficient and coefficient" (P <
0.0001 for both). These resiihs intlicalcd thai differences in /..

100

a 80

E

5 60 H

40

20

0

100

ra 80

>

E

W 60i
40
20-
0-

Litopenaeus vannamei

No Bacteria

Normocapnia

5 — S Hypoxia o

Normoxia n

jT T T T T Normocapnic

Hypoxia

-I I I I L.

Palaemonetes pugio

B

No Bacteria
Hypercapnic
Hypoxia O
Normoxia D

* Bacteria
9 — m Normoxia ■
Hypercapnic
)4ypoxi8 ^

0 4 8 12 16 20 24 28 32 36 40 44 48

Time (hours)

Figure 3. A. Litopenaeus vannamei survival following bacterial chal-
lenge under normoxia (Po, = 150-155 torr, Pco, = 0.23 torr. pH 7.6-
8.0) and hypercapnic hypoxia (Po, = M) torr, Pco, = 15.2 torr. pH
6.8-7.0). Shrimp were injected intramuscularly with 50 pL of Vibrio
parahaemolyticus bacterial suspension (1.125 x 10'' CFU/shrimp) or
with HEPES buffered 2.5% NaCl for controls. There were 36 shrimp
per treatment. Values at each time point are the mean [n = i experi-
ments); standard errors are indicated except where the error is small
and falls within the width of the datapoint. The effects of oxygen/CO,
treatment and the interaction of bacteria*oxygen/CO, treatment on
survival were significant as determined by a MANOVA (/" = 0.0009
and P = 0.0493). .\nimals held under this level of hypercapnic hypoxia
were more susceptible to Vil>rio challenge than those held under nor-
moxia. B. Palaemonetes pugio survival following bacterial challenge
under normoxia (Po, = 150-155 torr, Pco, = 0.23 torr, pH 8.0-8.2) and
hypercapnic hypoxia (Po, = 30 torr, Pco, = 15.2 torr, pH 6.9-7.0).
Shrimp were injected intramuscularly with 5 pi, of Vibrio para-
haemolyticus bacterial suspension (9.10 x 10^ CFl/shrimp) or with
HEPES bulTered 2.5% NaCl (controls). There were 40 shrimp per
treatment. Values at each time point are the mean {n = 4 experiments);
standard errors are indicated except where the error is small and falls
within the width of the datapoint. The effects of oxygen/CO, treatment
and the interaction of bacteria*oxygen/CO, treatment on survival
were significant as determined by a MANOV.\ (/' = 0.01 13 and P =
0.0095). .\ninials held under this level of hypercapnic hypoxia were
more susceptible to Vibrio challenge than those held under normoxia.

viinihiniei sur\i\al were altribulahle lo the injection of bacteria
over the injection of sahne, and not lo differences in oxygen levels
ol the water.

/'. pugio — hypercapnic hypoxia at 45 torr oxygen +
1.5.2 torr (2%) COj

These Vihno challenges tested the effect of hypercapnic hyp-
oxia on P. jtiigid sur\ i\al at a moderate level of hypoxia (45 torr

Survival of Shrimp Challenged with Vibrio

307

or 6% O^) (Fig. 2B). The effects of oxygeiVCO, treatment and the
interaction of bacteria*oxygen/CO, treatment were not significant
{P = 0.7379 and/' = 0.7412. respectively). The effect of bacteria
was significant {P < 0.0001 ) and was attributable to differences in
the coefficient and coefficient" (P < 0.0001 for both. ANOVA).
These results show that there was no additional disease suscepti-
bility in P. /)»,?/o held under this level of hypercapnic hypoxia than
those held in normoxic water.

L vannamei — hypercapnic hypoxia at 30 torr oxygen + 15.2 torr
(2%) CO,

These challenge tests investigated the effects of hypercapnic
hypoxia on L. vannamei at a more severe level of hypoxia (30 torr
or 4% O2) (Fig. 3A). The effects of oxygen/CO, treatment, bac-
teria, and the interaction of bacteria*oxygen/C02 treatment were
significant [P = 0.0009. P < 0.0001 and P = 0.0493. respec-
tively) and were attributable to differences in the coefficient and
coefficient". These results show that L vannamei held under this
level of hypercapnic hypoxia experienced a higher rate of mortal-
ity from Vibrio challenge than shrimp held under normoxic con-
ditions. Average survival at 48 h for animals in normoxia was 28.7
+ 2.4% standard error (SE) versus 15.7 + 4.6% SE for those in
hypercapnic hypoxia.

P. piigio — hypercapnic hypoxia at 30 torr oxygen -1-
15.2 torr (2%) CO,

These Vibrio challenges tested the effect of hypercapnic hyp-
oxia on P. pugio at a more severe level of hypoxia (30 torr or 4%
O,) (Fig. 3B). The effects of oxygen/CO^ treatment, bacteria, and
the interaction of bacteria*oxygen/C02 treatment were significant
(P = 0.0113. P< 0.0001. and P = 0.0095. respectively) and were
attributable to differences in the coefficient and coefficient". These
results show that P. pugio held under this level of hypercapnic
hypoxia experienced higher mortality rates from bacterial chal-
lenge than animals held under normoxic conditions. Average sur-
vival at 48 h for normoxia was 29.4 ± 6.4% SE versus 3.1 ± 2.4%
SE for hypercapnic hypoxia.

Total Hemocyte Count

Total hemocyte count significantly decreased in adult L van-
namei held under hypercapnic hypoxia when compared to animals
held under normoxia over 48 hours. THC/mL was reduced in
hypercapnic hypoxia by 60.7. 34.1. 34.3. 40.4 and 16.7% at 4. 8.
16. 24, and 48 hours, respectively, in relation to the normoxia
value at the same time point (Fig. 4). A two-way ANOVA indi-
cated that there was a significant effect of oxygen level/CO, treat-
ment {P < 0.0001) on THC/mL; however, there was no significant
effect of time (P = 0.2907) or the interaction between time and
oxygen/CO, treatment (P = 0.2276). An a posteriori r-test used to
compare means between oxygen levels at 48 hours revealed that
although oxygen level/CO, treatment was significant in the two-
way ANOVA. THC/mL was not significantly different between
the two treatments at 48 hours (P = 0.3207. Fig. 4).

DISCUSSION

Estuarine organisms routinely encounter fluctuations in oxy-
gen, carbon dioxide, and pH that may affect their ability to defend
against infections. Previous research has linked poor water quality,
particularly hypoxia, with increased incidence of infectious disease
(Snieszko 1974. Hargis et al. 1989. Landsberg et al. 1998). For

40

E

35

0

X

30

^J

c

3
0

25

0

20

0

0

15

X

S

10

.0

Normoxia

'"n

— •/<

II M-

~~~-|

\t

*

..

*

;

yr

*

i-

--']

y^

1

Y

Hypercapnic Hypoxia

16
Time (hours)

24

Figure 4. Total hemocyte counts (THC) per mL in Lilopenaeiis van-
namei over 48 hours. Shrimp were held under normoxic conditions
(closed squares, Po, = 150-155 torr, Pco, = 0.23 torr, pH 8.0-8.2) or
under hypercapnic hypoxic conditions (open circles, Po, = 30 torr,
Pco, = 15.2 torr, pH 6.9-7.1). Individual shrimp were used for each
time point and for each test condition (h = 10 shrimp for normoxia and
hypercapnic hypoxia at 4. 16, 24, and 48 h, ;/ = 1 1 shrimp for normoxia
and hypercapnic hypoxia at 8 h). A two-way ANOVA indicated that
there was a significant effect of oxygen level/CO, treatment {P =
<0.0001 ) on THC/mL; however, there was no significant effect of time
(P = 0.2907) or the interaction between time and oxygen/CO, treat-
ment (P = 0.2276). An a posteriori (-test revealed that THC/mL was not
significantly different between normoxia and hypercapnic hypoxia at
48 hours (P = 0.3207). Values are mean ± standard error (a = 0.05).

example. Landsberg et al. (1998) found that the occurrence of
opportunistic protist infections increased in fish subjected to low
oxygen conditions. Noga et al. (1994) reported that blue crabs
Callinecres sapidus collected in areas of the estuary where hypoxia
is common, have low serum bacteriostatic activity. This decreased
antibacterial activity was correlated with an increase in shell dis-
ease. Haley et al. (1967) attributed infections by Aeromonas liq-
uefaciens in the threadfin shad Dorosoma petenense and the
American shad Alosa sapidissima to low dissolved oxygen. How-
ever, in these field studies, pollutants or other physical factors,
such as temperature, may have exacerbated the effects of hypoxia.
There have been relatively few studies on the direct effects of
hypoxia, hypercapnia. and low pH on disease susceptibility. In the
present study, survival following challenge with Vibrio para-
haemolyticus was depressed in hypercapnic and hypoxic water
(Po, = 30 torr. Pco, = 15.2 torr and pH = 6.8-7.0) in the
penaeid shrimp Lilopenaeus vannamei and in the grass shrimp
Palaemonetes pugio (Fig. 3A and B). In addition, the THC/niL in
L. vannamei was reduced under the same conditions (Fig. 4).

The present study used a known pathogenic strain isolated from
shrimp with vibriosis. LD5,, values were reproducible using the
same strain and produced consistent mortalities when used in the
challenge assays. These are the first reported LD51, values for L
vannamei and P. pugio using V. parahaemolyticus.

The 48-h LD50 of V. parahaemolyticus for L. vannamei re-
ported in the present .study (Table 2) is similar to the LD,,, of the
same bacterial species for P. monodon (3.16 x 10"^. 95% C.I. 9.60
X I0'*to 1.03 X lO^'CFU/shrimp). The latter values were calculated
from data in Alapide-Tendencia and Dureza (1997) using the
trimmed Spearmann-Karber program. Arume (1989) reported
LD,,, values of Vilvio isolates to Lilopenaeus stylirostris ranging
from 4.0 x 10" to 3.3 x lO^^ CFU/g, which is lower than the value

308

MiKULSKI ET AL.

of 3.06 X lOVg wet weight calculated for L. vcinnamei (Table 2).
However, the species of Vibrio used was not reported. In contrast.
V. paniluiemolyticiis had a much higher LD^,, value tor M. japoni-
cus juveniles of 4.27 x 10^ CFU/shrimp (Vera et al. 1992). This
inconsistency in LD^„ values may be attributable to host specific-
ity and the differences in the size of the animals (Vera et al. 1992.
Lee et al. 1996). In addition, virulence of bacteria can vary among
strains (Arume 1989, Thune et al. 1993, Wong et al. 1996).

L. vaunainei and P. pugio were more susceptible to V. para-
haemotyticus when held under hypercapnic hypoxia at 30 ton-
oxygen (1.41 mg/L), 15.2 torr (2%) CO. and a pH of 6.8 to 7.0
than under normoxia at 150-155 torr oxygen (7.29 mg/L). approxi-
mately 0.23 torr CO, (0.03%) and a pH of 7.6-8.2 (Figs. 3A and
B). This decrease in disease resistance was not attributable to
enhanced bacterial growth under these conditions (data not
shown). Le Moullac et al. (1999) also found that mortality under
hypoxia at 1 mg 0,/L (48%) was significantly greater than control
(well-aerated water) mortality (32%) when L. stylirostris was chal-
lenged with V. alginolyticiis. However, the levels of CO, and the
resultant hypoxic pH were not controlled or reported by the in-
vestigators. As a result, it is unclear if the animals were subject to
hypercapnic hypoxia or to normocapnic hypoxia. In the present
study, there was no significant effect on disease susceptibility in L.
vannainei of normocapnic hypoxia at 45 torr oxygen with less than
I torr CO, or in P. pugio under hypercapnic hypoxia at 45 torr
oxygen and 15.2 torr CO, (Figs. 2A and B).

The level of hypoxia at which disease susceptibility increased
(Po, = 30 torr) over normoxia in both species may be explained,
in part, by the shrimps' critical oxygen tension. The critical oxygen
tension for an organism is the oxygen tension below which an
organism is unable to maintain its rate of oxygen uptake. Below
the critical oxygen tension, organisms may be unable to sustain an
internal oxygen level sufficient to defend against infection. Co-
chran and Burnett (1996) reported a critical Po, for P. pugio be-
tween 30 and 35 torr, which may partly explain differences in
susceptibility at 30 torr (1.41 mg 0,/L) and 45 torr oxygen (2.12
mg 0,/L) observed in grass shrimp in this study (Figs. 2B and 3B).
On the other hand. Hutcheson et al. ( 1985) reported a much higher
critical Po, (approximately 95 ton) for the same species. Nielsen
and Hagerman (1998) reported critical Po,s for Palaemonetes
varians and Palaemon adspersiis of 2.4 mg 0,/L (approximately
46 torr) and 2.87 mg 0,/L (approximately 55 torr). respectively,
which are both above the highest level of oxygen used in the
present experiments. Villarreal et al. (1994) identified a critical
Po, of 1.3 mg Oi/L (approximately 34 torr) in L. vcinnamei. This
value is similar to the value of 1.41 mg 0,/L (30 torr) found to be
significant to disease resistance in L. vcinnamei in the present
research. In contrast. Rosas et al. (1999) found that juvenile Lilo-
penaeiis setifenis were oxyregulators down to 4 mg 0,/L (approxi-
mately 92 torr), but were oxyconformers between 3 and 2 mg 0,/L
(approximately 69 and 46 torr) suggesting that the critical Po, lies
between those two values. The variability in published critical
oxygen pressures may be attributable to many factors including
temperature, salinity, activity, molt cycle, size, and experimental
technique that can affect the critical Po, of a species (Herrcid
1980, Dall 1986. Cochran and Burnett 1996).

It is intportani to note that the oxygen tensions used in the
present study were well above the lethal limits reported for these
and similar species. Hopkins ct al. (1991) reported an oxygen
lethal limit of I mg 0,/L (approximately 22 torr) for L vcmmimei.
Allan and Maguirc ( 1991 ) calculated 9S-h and 24-h oxygen LC^i.s

for juvenile P. mcmodon of 0.9 mg 0,/L and 0.6 mg 0,/L (ap-
proximately 21 and 14 torr). respectively, demonstrating that the
duration of the hypoxia also has an effect. Stickle et al. (1989)
showed that F. aztecus were much more sensitive to low oxygen
than P. pugio. The 28-day LC50 values were 123 torr (5.94 mg
Oj/L) for F. aztecus and 46 torr (2.22 mg 0,/L) for P. pugio.
Differences in disease susceptibility between the two species used
in the present study (L. vanncunei and P. pugio) could not be
compared statistically because of differences in the size of the
shrimp and the bacterial challenge dose; however, they exhibited
similar responses to the two levels of oxygen tested (Figs. 2 and 3).

As mentioned previously, studies that investigate the effects of
hypoxia on estuarine organisms often do not take into account
hypercapnia and the low pH that accompanies it (Hutcheson et al.
1985. Seidman and Lawrence 1985. Allan and Maguire 1991.
Charmantier et al. 1994. Direkbusarakom and Danayadol 1998.
Nielsen and Hagerman 1998. Le Moullac et al. 1999). Neverthe-
less, these variables may have contributed, in combination or in-
dependently, to the decreased disease resistance observed in the
present work. Martinez et al. (1998) reported that the lethal dis-
solved oxygen concentrations for postlarval and juvenile L. se-
tiferus are higher under low pH (pH = 6) than under high pH (pH
= 8). In addition, McCulloch ( 1990) found that low pH raised the
critical oxygen concentration from 1 .54 mg 0,/L at pH 9.0 to 2.08
mg 0,/L at pH 6.5 for Palaen\onetcs l<acliakensis. Cochran and
Burnett ( 1996) demonstrated that oxygen uptake was significantly
higher at high CO, than at low CO, in the spot Leiostcmuts xcm-
thurus. Cruz-Neto and Steffensen ( 1997) reported that hypercapnia
increased the critical oxygen concentration from 25 torr to 40—45
torr in the European eel Angitilla anguilla. These studies show that
hypercapnia can adversely affect hypoxia tolerance.

Total hemocyte count was significantly reduced in L. vannamei
held under hypercapnic hypoxia (Po, = 30 torr. Pco, = 15.2 torr.
pH 6.8-7.0) when compared to shrimp held under normoxia (Po,
= 150-155 torr. Pco, = 0.23 ton-. pH 7.6-8.0) at 4. 8, 16, and 24
h (Fig. 4). Similarly. Le Moullac et al. (1999) found that THC/mL
decreased in L. stylirostris exposed to hypoxia at I mg 0,/L for 24
hours. Alvarez et al. (1989) also noted a reduction in hemocyte
concentration in oysters held under two levels of hypoxia (9 and 80
torr oxygen) for 3 days.

Although not measured in the present study, injection of whole
bacteria or isolated cell wall components of bacteria and yeast can
trigger a decrease in THC/mL in crustaceans (Hauton et al. 1997.
Smith et al. 1983. Lorenzon et al. 1999). Using bacterial lipopoly-
saccharide to suppress circulating THC. Lorenzon et al. (1999)
reported threshold lethal limits for THC of 28.9, 32.9. and 15.3%
of the initial circulating cells for P. elegans. C. crangon, and
Sc/uilla mcuuis, respectively. These observations suggest that L.
vainiainei and P. pugio in the present study thai were challenged
with bacteria while being held under hypercapnic hypoxia may
ha\ c experienced a greater decrease in THC than the unchallenged
adults held under hypercapnic hypoxia alone. The average total
hemocyte count in L. vannamei in the present study was reduced
10 39.3''f of the normoxic average 4 hours after placement in
hvpcrcapnic hypoxia (Fig. 4). The combined effects of hypercap-
nic hypo.xia anil bacterial injection could reduce cell density in
shrimp lo a level below the minimum necessary for survival. This
is a possible explanation for the increase in mortality in shrimp
challenged with bacteria and exposed to hypercapnic hypoxia as
compared to animals challenged with bacteria and maintained un-
der normoxia in this study. Il is iiiiporlaiil to note, however, that

Survival of Shrimp Challenged with Vibrio

309

dead or moribund animals exliibited signs of vibriosis and that low
hemocyte number was not the sole cause of mortality. More likely,
the low cell numbers in animals held under hypercapnic hypoxia
were insufficient to defend against the bacterial challenge.

The decrease in THC/mL observed in this study may contribute
to the increase in mortality rate following bacterial challenge under
hypercapnic hypoxia, but many other factors also may play a role.
Le Moullac et al. (1999) found that respiratory burst activity, as
measured by NBT reduction, decreased in L stylirostris subjected
to hypoxia (1 mg 0-,/L), but that phenoloxidase (POl activity
increased significantly because of a lower amount of inhibitors
regulating the prophenoloxidase system. Direkbusarakom and
Danayadol (1998) demonstrated that phagocytosis and bacterial
clearance efficiency v\ere reduced in P. numodon exposed to 1 .8-
2.0 mg OVL. Although Alvarez et al. (1992) reported that phago-
cytosis by hemocytes of the eastern oyster C. virginica was not
affected by hypoxia, Boyd and Burnett (1999) demonstrated that
reactive oxygen intermediate (ROI) production by hemocytes was
significantly depressed under hypoxia in the same species. Boleza
( 1999) found that ROI production and bactericidal activity of ph-
agocytes in the munimichog Fundidus hewroclitis were sup-
pressed under hypercapnic hypoxia. Comparable studies of cellular
and acellular bactericidal factors in P. vannamei and P. piigio
under relevant water quality conditions may clarify the defense
mechanisms that are sensitive to dissolved gasses and pH.

The results of the present study show that hypercapnic hypoxia
at 30 torr O,, 15.2 torr CO, and a pH range of 6.8 to 7.0 decreases
survival following bacterial challenge in both L. rannamei and P.
piigio and decreases total hemocyte count in L. vannmnei (Fig. 3A
and B and 4). This has implications regarding the health of these

organisms in both the natural environment and in aquaculture.
Diaz and Rosenberg (1995) reported that the occurrence of hyp-
oxia in shallow coastal and estuarine systems is increasing world-
wide. Dissolved oxygen monitoring by Summers et al. (1997) in
the mid-Atlantic and Gulf of Mexico regions suggests that the
extent of hypoxia is often substantially underestimated. Thus, con-
ditions that suppress disease resistance may become more preva-
lent, which could affect the penaeid shrimp fishery and reduce the
density of the ecologically important grass shrimp. In addition, a
decrease in THC/rnL under hypercapnic hypoxia, as observed in
this research (Fig. 4), could result in a decrease in immune func-
tion and possibly a reduction of the effectiveness of immunostimu-
lants used in aquaculture to prevent outbreaks of disease (Sung et
al. 1991. Sung et al. 1996, Itami et al. 1998, Devaraja et al. 1998,
Teunissen et al. 1998). Taken together with the well-recognized
importance of water oxygen, dissolved COj and concomitant
changes in pH that accompany naturally occurring hypoxia should
be carefully monitored and regulated to sustain the wild shrimp
fishery and optimize farm production.

ACKNOWLEDGMENTS

The authors thank Dr. Craig Browdy, John Ravenel, and Bill
Cox for their help in obtaining juvenile and adult Litopenaeus
vannamei. Dr. John Fauth helped with statistical analyses: K.
Boleza, J. Early, L. Farrelly, and C. Milardo provided technical
support. This research was supported by the EPA (Agreement
R-826399), Sigma Xi Grants in Aid of Research, the Slocum-Lunz
Foundation, and the University of Charleston's Deep Water Fund.
This paper is contribution 170 of the Grice Marine Laboratory.

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virulence of three species of Vibrio bacteria in Penaeus japoniciis {Bale Wong, H. C., C. C. Liu, C. M. Yu & Y. S. Lee. 1996. Utilization of iron

1881) juveniles. AgHoni/fHrc 107:119-123. sources and its possible roles in the pathogenesis of Vibrio para-

Villarreal, H., P. Hinojosa & J. Naranjo. 1994. Effect of temperature and liaemolyticus. Microbiol. Immunol. 40:791-798.

Jounuil i>f Shellfish Research. Vol. 19. No. 1. 313-319, 2000.

PARALYTIC SHELLFISH TOXINS IN GEODUCK CLAMS (PANOPE ABRUPTA):
VARIABILITY, ANATOMICAL DISTRIBUTION, AND COMPARISON OF TWO TOXIN

DETECTION METHODS

KELLY M. CURTIS,' VERA L. TRAINER,^ AND
SANDRA E. SHUMWAY'

Jamestown S'Klallam Tribe,
Department of Natural Resources,
Sequlm. WA 98382

'National Oceanic and Atmospheric Administration Northwest Fisheries
Science Center,

Environmentcd Consen'ation Division,
Seattle, Washington 98112
Natural Science Division,
Southampton College,
Long Island University,
Southampton, New York 11968

ABSTRACT The geoduck clam. Panupe uhniptu. is a valuable economic resource in Washington State. Prior to the mid 1970s, the
levels of paralytic shellfish poisoning (PSPl toxins in Washington State geoducks were not considered by the Washington State
Department of Health (WDOH) to be a risk to public health because the viscera were presumed to be discarded. Recent monitoring
information indicates that geoducks accumulate high levels of toxins, primarily in the viscera. The purposes of this study were to
determine; ( I ) the seasonal concentration of paralytic shellfish toxins in geoduck clams at two sites and at two depths within each site;
(2) the variability of PSP toxin levels among individual clams within each site; (3) the anatomical distribution of toxins; and (4) the
correlation between two methods for estimating PSP toxins. From the summer of 1997 through the winter of 1998, 12-24 geoducks
were collected biweekly from a shallow (7 ml and a deep (17 m) location in each of two tracts in Puget Sound. Washington;
Quartermaster Harbor (QH) and Agate Pass (AP). Geoducks. dissected into siphon, mantle, and visceral portions, were assayed
separately using the mouse bioassay (MBA), while only the visceral portions were assayed using the receptor-binding assay (RBA).
Results indicated that toxin variability between individual clams was high in the shallow areas, with coefficients of variation (CVs)
ranging from 20-98'7r. and lower in the deep areas (CV = 18-62%). In QH. only geoducks from the shallow water had toxin levels
greater than the regulatory level of 80 (ig saxitoxinequivalents (STX eq) • 100 g shellfish tissue"', while all geoducks from AP
contained toxin above the regulatory level, with clams from shallow water considerably more toxic than those from deep water.
Anatomically, the highest concentrations of PSP toxins were localized in the viscera of geoducks. There was a significant positive
correlation between toxin levels measured by the MBA compared to values obtained using the RBA (r = 0.83). The large differences
in toxicity between geoducks sampled at different depths and harvest tracts indicate that careful management plans must be designed
in order to ensure public health.

INTRODUCTION whole, live geoducks: the market for shucked or frozen product is

General Background ''^'^ *"^^"- ^'^'''^ geoducks are one of many species of bivalves

known to filter and accumulate toxic dinoflagellates, few data exist

Toxins that cause paralytic shellfish poisoning (PSP) are accu- jj,^, ^^^^^-^^^ pSP ^^^-^^ -^ ^^^-^ organism (Shumway 1990; Bricelj

mulated by filter-feeding bivalve mollusks when they ingest toxic ^^^ shumway 1998). The risk of PSP to consumers is therefore

dinoflagellates from the genus Ale.xandrium. These algae produce j^.-reased. which may lead to devaluation of the geoduck as a food

the toxins naturally, and the PSP syndrome results trom the human ^^^^^ j-^^ ^^^^^ consumption if toxic product reaches the market,
consumption of toxic bivalves. Large-scale problems with PSP

stem from the extreme difficulty in predicting the timing and ex- Washington's Geoduck Fishery

tent of dinoflagellate blooms, in turn making it difficult to monitor Prior to the mid 1970s, PSP toxin levels in Washington State

toxicity in shellfish efficiently (Boesch et al. 1996. Homer et al. geoducks were not considered by the Washington State Depart-

1997). Difficulties also arise because each species of shellfish is ment of Health (WDOH) to be a risk to public health because the

unique in the kinetics of uptake and elimination of toxins. In geoduck viscera were presumed to be discarded. However, we now

addition, shellfish toxicities do not always coincide with observed know that the viscera are consumed by some members of tribal and

toxic algal blooms (Cembella and Shumway 1993. Bricelj and immigrant communities, who use them in soup (K. Chew, Univer-

Shumway 1998). sity of Washington pers. comm.. 1996. M. Antee. WDOH pers.

The geoduck clam. Panope abrupta. is a valuable economic comm., 1997). In addition, toxic algal blooms are extending into
resource in Washington State, with revenues ranging from $5-7 previously benign areas of central and southern Puget Sound
million annually (Washington State Department of Natural Re- (Nishitani and Chew 1988, F. Cox. WDOH pers. comm., 1997),
sources (WDNR). unpublished data, 1997). Recently, the demand which is leading to unprecedented high levels of PSP toxicity in
from newly developed large markets both domestically and over- geoducks and toxicity that lasts well into the winter months, re-
seas (e.g.. Hong Kong. Japan, and Singapore) has sent the price of suiting in thousands of dollars of an unharvestable resource. The
geoducks up from $1.50 per pound in the late 1980s to a current recent increased demand for geoduck meat is resulting in new
average price range of $12-14 per pound. Public demand is for tribal and state commercial tracts being opened in some areas of

313

314

Curtis et al.

central and northern Puget Sound where PSP is known to occur
(F. Cox. WDOH pers. comm.. 1997).

Little information exists regarding PSP toxicity in geoducks.
However, recent monitoring programs indicate considerable inter-
and intrapopulation variability (F. Cox pers. comm.. 1997).

An understanding of the reasons for toxin variability is crucial
in designing a regional monitoring and sampling program. The
current method used by the WDOH in monitoring and testing for
PSP in the geoduck does not account for individual variability in
the clams because composite viscera from three clams are tested
for toxicity as one sample. In the absence of variability and ana-
tomical distribution information, it is difficult to assess the effec-
tiveness of the current Washington State geoduck monitoring pro-
gram in protecting public health. This study describes toxin vari-
ability in geoducks in relation to water depth and geographical
location, thereby providing basic information that can be inte-
grated into future monitoring efforts by the WDOH.

MATERIALS AND METHODS

Sampling

Quartermaster Harbor

Quartermaster Harbor (QH). located between the southern tips
of Vashon and Maury Islands (Fig. I ), is currently a prohibited
harvest area due to consistent levels of PSP toxicity > 80 (j.g of
saxitoxin equivalents (STXeq)/100 g of tissue (all toxicities are
given in micrograms of STXeq/100 g of shellfish tissue) (Nishitani
and Chew 1984) and pollution problems resulting from failing
septic systems (Washington Department of Fish and Wildlife
1997). There are two tracts in QH. and tract number 10300 was
randomly chosen as the study site (Washington Department of Fish
and Wildlife 1997).

A shallow and a deep sampling location within this tract were
randomly selected. The depth of the shallow location averaged 7

m, adjusted to mean lower low water, and the deep location aver-
aged 1 7m (mean lower low water). A diver collected 6-14 geo-
ducks within a circular area approximately 27 m in diameter from
both depth locations, at 2-wk intervals from June through October
1997.

Agate Pass

Tract number 0700 in Agate Pass ( AP), located north of Arrow
Point on the west side of Bainbridge Island (Fig. 1), is currently a
WDOH-approved harvest tract (Washington Department of Fish
and Wildlife 1997). A shallow and a deep sampling location were
randomly selected in the same manner as in QH. Divers collected
geoducks at 2-wk intervals from August 1997 through January
1998. All of the geoducks from deep water consistently came from
the same sampling location. In the shallow zone, however, the lack
of sufficient numbers of geoducks necessitated a constant lateral
shift in collection sites, but all of the shallow sites were within an
approximately 300-m section along the shoreline.

Laboratory Determinations

Geoducks were dissected, and toxicities of the siphon, mantle,
and visceral portions of individual geoducks were determined by
mouse bioassay (MBA) (Association of Official Analytical Chem-
ists 1965). All of the visceral tissue, except the gills, was combined
and tested. The gills were saved for future testing, time and fund-
ing permitting. Additionally, the visceral portions were tested us-
ing the receptor-binding assay (RBA) (Davio and Fontelo 1983.
Doucette et al. 1997, Trainer and Poll, 2000). In this assay,
nerve terminal membrane from the rat brain, containing sodium
channel receptors (STX binding sites) is used to test for the pres-
ence of STXeq in a sample. Toxin in the sample displaces radio-
actively labeled STX from its specific receptor sites, thereby re-
ducing the level of radioactivity in the shellfish sample. Geoduck
samples analyzed using this method had toxin levels ranging from
40-1.800 (jLg (determined by MBA). No samples below the detec-
tion limit of the MBA were used.

RESULTS

Anatomical Distrihiilion

The actual toxin levels (given in micrograms of STXeq per 100
g of shellfish tissue) in each of the dissected tissues (siphon,
mantle, and viscera) from all clams collected from QH and AP (;i
= 361), are shown in Figure 2. In QH samples, delectable levels
of toxins were found in the mantle portion of three individual
clams, but the values were well below the fishery closure level (80
|jLg) at 46, 47, and 51 jj-g (Fig. 2B). In AP samples, detectable
levels of toxin were found in the mantle portion of se\en indi-
vidual clams and in the siphon portion of nine individual clams
(Fig. 2B), however, the values were again well below the fishery
closure level. At no time during the study period did the siphon
portion from any geoduck show detectable levels of toxicity. All
toxicities above the fishery closure level were in the visceral por-
tion only.

(Juarlermaster Harbor
.SliiilloM Water

hiyiirc. I. M;.i) i.f c.aMal Washington and Puget Sound showing the Toxin levels above the fishery closure level were detected on

study collection sites at AP and QH. all eight sampling dates from June through October, except July 27

Paralytic Shellfish Toxins in Geoduck Clams

315

A: viscera

Fishen closure level ofSOMgSTXeq/IOOg lissue

bidhiduats overtime (June 1998 through Januarj' 1999)

180 -
160 .
140
120
100

80

60

40

20 .
0

Q- ■ Mantle g Siphon

Fishery closure level ofSOligSTXeq/IOOg tissue

Individuals over time (June 1998 through January 1999)

Figure. 2. Toxicity levels in each of the dissected tissues (A = viscera;
B = mantle and siphon). Each bar represents an individual geoduck.
All geoducks collected from QH and AP during the study are included
(« = 361) and are shown in chronological order of collection. The
mantle portions of 10 geoducks and the siphon portion of 9 geoducks
had detectable levels of toxin but were still below the fishery closure
level.

{Fig. 3A). When toxicity was above closure levels, there was a
large variation in toxin levels among individual clams. On July 27.
variability was low and toxicity levels ranged from 0-61 jjtg. The
largest variation occurred on October 5, with toxin levels ranging
from 38-998|jig (Table I).

Deep Water

In QH deep water, toxicity was consistently below the closure
level, and values were considerably lower than those observed in
the shallow location (Fig. 3B). Toxin levels ranged from nonde-
tectable to 38 \x.g on all collection dates except October 20. when
toxin levels ranged from 0-67 (ig. Variability between individuals
was low on all sampling dates.

AP

Shallow Water

Toxicities were consistently above the fishery closure level on
all 12 collection dates from August through January with the ex-
ception of three individual clams, one each on November 12 and
25. and January 6 (Fig. 4A). There was a large variation in toxin
levels among individuals on all sampling dates.

Collection date

ISO
160 --

B

Bdeep

- fishery closure level of 80IJgSTXcq/l OOg lissue

Collection date

Figure. 3. Toxicity of the viscera in geoducks from the QH collection
areas taken from June through October 1997. Each bar represents an
individual geoduck. The dashed line indicates the regulatory closure
level of 80 fig STXeq/100 g of shellfish tissue. A = shallow; B = deep.

Deep Water

Toxicities were consistently above the fishery closure level of
80 |xg on most of the 1 1 collection dates, except December 10
when 509^ were below and 50% were above the closure level of 80
jjig (Fig. 4B). There was a large variation in toxin levels among
individuals on all sampling dates. The largest variation occurred on
January 20 with toxicities ranging from 107-545 |jig.

Comparison of PSP Detection Methods

Correlations between the MBA and the RBA methods are shown
in Figure 5. Figure 5A illustrates the relationship between all samples
tested, which ranged from 60-1,700 |jLg (by MBA). A comparison
of the two methods showed a significant positive correlation (/- =
0.83). Figure 58 illustrates the relationship between samples with
toxicities < 85 \x.g (by MBA) and demonstrated a significant posi-
tive correlation between the two methods (/" = 0.55).

DISCUSSION

Variability

The high degree of toxin variability observed among individual
geoducks (Figs. 3, 4) is not surprising and has been seen in many
other shellfish species. For example, Atlantic surfclams (Spisida
solidissima) taken off the coast of Maine showed an average co-
efficient of variation (CV) of 48.6%, and ocean quahogs (Arctica
islaiulica) showed a mean CV of 56% (White et al. 1993). Soft-

316

Curtis et al.

TABLE 1.

Summary of the variation in levels of PSP toxins among individual geoducks collected from QH and AP during each 1-d collection period,

with clams separated by depth.

Geoducks with

CV

Mean CV

Area

Sampling Date

;i

>80 ng of Toxin

Range"

Mean ± SD"

Pooled SD

(%)

(%)

QH shallow

June:i

9

9

113-460

298.8+ 119

40

July 27

10'

0

0-61

53 ± 7

13

August 3

11

6

39-179

95 ±43

44

August 14

11

7

38-475

158 ± 130

82

August 28

10

8

44-365

173 ±103

60

September 19

10

10

116-845

474 ± 237

50

October 5

10

9

38-998

305 ± 274

90

October 20

10

5

46-146

81 ±36

153

44

53

QH deep

June 21

6

0

0-38

N/A''

N/A"

August 3

6

0

0-38

N/A"

N/A"

August 14

14

0

0

N/A"

N/A"

October 5

10

0

0-38

N/A"

N/A"

October 20

11"

0

0-67

51 ±9

N/A

18

N/A

AP shallow

August 19

12

12

892-1,937

1,272 ±335

27

September 2

10

10

530-1,413

885 ± 258

29

September 17

9

9

290-692

476 ± 160

34

October 7

9

9

203-666

334 ± 140

42

October 14

10

10

102-577

289 ± 143

49

October 28

5

5

172-1,521

649 ±621

96

November 12

6

5

49-318

202 ± 88

44

November 25

10

9

38-835

398 ±219

55

December 9

14

14

138-1,314

408 ± 292

72

December 23

13

13

224-1,113

606 ± 288

48

January 6

11

10

61-813

346 ± 195

57

January 20

10

10

98-966

431 ±318

271

74

52

AP deep

August 19

10

10

359-958

717±164

23

September 2

11

11

342-930

546 ±166

30

September 17

12

12

212-544

409 ± 107

26

October 7

11

9

38^41

247 ±115

46

October 14

11

11

183-357

271 ±61

23

October 28

10

9

116-195

144 ±29

20

November 12

10

10

81-304

151 ±74

49

November 25

10

10

106-521

278 ± 150

54

December 9

12

6

47-183

92 ±44

48

December 23

10

9

65-386

252 ± 94

37

January 20

10

10

107-545

233 ± 144

115

62

38

■' Values given as micrograms of STXeq/100 g of shellfish tissue,

" N/A = not applicable. These values were below detection level and could not be determined.

••' Only three geoducks had toxicities >38 jjtg. These values were used to calculate mean. SD, and CV.

'' Only six geoducks had toxicities >38 (ig. These values were used to calculate mean, SD. and CV.

shell clams {Myci arenaria) from the Bay of Fundy shov\'ed an
average CV of 49% (Medeof et al. 1947). Prior to the present
study, the only variability information available for geoducks was
from an unpublished study in Alaska, where the mean CV for 10
sets of geoducks was 41% (Ketchikan Puhlic Health Laboratories,
unpublished data, 1981 ).

Some variability in P.SP toxin levels among individtial geo-
ducks can be accounted for by the variability (± 20%) in the MBA
test (McFarren 1962). The mean CVs for each set of geoducks
(defined by collection area and depth) were close to or greater than
twice that in the MBA (.38%., 52%', and 3.3% ). However, within
sets of geoducks, the CV reached 96% (Table I ), indicating that
there was considerable variability between individual geoducks
that was not due to an error in the MBA.

Matiy factors have been suggested to accotnil for variations
between individual shellfish, includiiit; dilferences in feedinsj

rates, availability of food due to vertical and horizontal depth
gradients, reproductive condition, individual sensitivity to P.SP
toxins, and variation in body mass (Prakash and Medeof 1962.
Nishitani and Chew 1984, Bricelj et al. 1991. Bricelj and Laby
1996, Mackenzie et al. 1996). Much of the variation between
individual geoducks within one depth may be attributable to dif-
ferences in feeding rales (D. Williams, WDNR pers. comm..
1997). At any given time, geoducks are expected to have a 70%
"show factoi," meaning that only 70% of the population will have
their siphons protruding out of the sand but will not necessarily be
feeding. This show factor varies with the time of year and could be
attributed to changes in water temperature or localized distur-
bances (e.g., the presence of divers, crabs, siphon-nipping fish, or
marine mammals), causing the geodticks to retract their siphons.
The availability o\ food, often directly related to the behavior of
algal cells, is very likely to be the reason for the high degree of

Paralytic Shellfish Toxins in Geoduck Clams

317

Collection date

Figure. 4. Toxicity of the viscera in geoducks from the AP collection
areas taken from August 1997 through January 1998. Each bar rep-
resents an individual geoduck. The dashed line indicates the regulatory
closure level of 80 (ig STXeq/100 g of shellfish tissue. A = shallow; B =
deep.

variability between depths (shallow and deep). The toxic Ale.xaii-
driiim cells have been found to undergo diel vertical migrations,
reaching a maximum depth of 8 m ( Nishitani and Chew 1 984 ). The
depth of the dinoflagellates also depends on currents and winds,
which mix them deeper into the water column. This may explain
why the geoducks from the deep water of QH, a shallow, quiet bay
without strong currents or vertical mixing, were never over the
toxicity closure level, while the ones from the shallow water ex-
hibited high toxin levels (Fig. 3). It is likely that the geoducks from
the shallow water were exposed to the toxic dinoflagellates more
frequently than the ones from the deep water, thus increasing their
overall toxicity. Because AP experiences mixing due to strong and
variable currents, with speeds ranging from 0.3-6.6 knots (U.S.
Department of Commerce 1973), cells are mixed to greater depth
within the water column, making them available for uptake by the
geoducks in deeper areas. However, it is likely that the geoducks
from the shallow water were exposed to toxic cells more fre-
quently, accounting for their higher overall toxicity.

The difference between collection depths has some implica-
tions for the geoduck industry. Currently, harvesting for the market
and collection for PSP monitoring occurs primarily in the shal-
lower depths of a harvest tract (D. Winfrey. Puyallup Tribe pers.
comm., 1997, D. Williams, WDNR pers. comm., 1998). Geoducks
are easier to find, and the divers can collect more clams in a shorter
amount of time. In order to accommodate the toxicity differences.

A

y^

1800

•

y^

1600

•

^ m

1400

• /

/^

•

1200 \

^/»

1000

•

•

800 .

• 5'-»

m

600 -

• #«^

•

400

\

•

•

r'=0.83

200

,

m

f$

0 -

1 p

..-„,

,

, ,

0 200 400 600 800 1000 1200 1400 1600 1800 2000

RBA (HE STXeq/lOOg tissue)

B

^

180 H

y^

160

y"^

140

X

120

y^

100 -

y^

80 ^
60

X

• •
• • •

•

40 -

y^»

•

20
0 -

y^

r= = 0.55

0 20 40 60 80 100 120 140 160 180 200

RBA (Mg STXeq/IOOg tissue)

Figure. 5. Correlation between the MBA and the RBA. A = all samples
analyzed (h = 73): B = samples with toxin levels between 32 and 85 pg
(by MBA, n = 12). The diagonal line represents perfect correlation (r"
= 1). There was a significant correlation between the two methods: r"
= 0.83 for all samples and r^ = 0.55 for samples < 85 ng.

harvests could be limited to certain depths during periods of high
toxicity (typically in the summer through early winter months). For
example, as toxicity levels increase, harvest depths could also be
required to increase.

The large difference in overall toxicities between AP and QH is
difficult to explain (Figs. 3, 4). It is not unreasonable to suspect
that QH would have higher toxicities since it is a "breeding bay"
for A. catenella (Nishitani and Chew 1984). Breeding bays are
defined as certain shallow, protected bays in which strong thermal
stratification occurs relatively frequently. In these bays, dense
populations of A. catenella can develop and become available to
the shellfish. However, the AP study area had toxicity levels up to
five times (Table 1 ) those seen in the QH study area, even though
AP is an area of strong currents and very little thermal stratifica-
tion. One explanation for the higher toxicities in AP is the possi-
bility of dinoflagellate cysts in this area, which can be more toxic
than motile cells (Dale et al. 1978). Since A. catenella forms
dormant cysts, it is possible that the geoduck harvesters or the
strong currents in AP are stirring up the toxic dormant cysts in the
sediments, making them available for uptake by the geoduck and.
thereby, accounting for the higher overall toxicities in that area. In
addition, QH is a closed harvest area with no diver activity and
slow currents, further supporting the explanation for lower overall
toxicities.

318

Curtis et al.

An additional explanation for higher toxicities in AP could be
that motile toxic cells are potentially being exported from a nearby
breeding bay. Pugel Sound, a tjord with a long, deep main channel,
has numerous relatively shallow and often poorly flushed bays,
where blooms of A. catenella could potentially originate (Horner et
al. 1997). The toxic cells, if exported horizontally during periods
of reduced turbulence, could cause toxicity levels in shellfish in a
nearby area to be several times greater than in the breeding bay
itself (Nishitani and Chew 1984).

Individual shellfish within the same sampling population are
known to exhibit differential sensitivities to PS? toxins (Bricelj
and Laby 1996). This has not been studied in geoducks. Variations
in body mass also may have effects on individual toxin accumu-
lation rates. Smaller individual clams can reach equal or higher
toxicities than larger individuals collected from the same location
(Medcof et al. 1947. Aalvik and Framstad 1981). In this study.
there was no relationship between geoduck weight and toxicity
level on any date or in any collection area.

Anatomical Distribution

In this study, all PSP toxin levels above closure level were
concentrated in the visceral ball in geoducks from all collection
sites (Fig. 2). Only the siphon and mantle portions contained an
amount of toxin that was below regulatory levels, and. therefore.
these were the only portions that would be considered safe to
consume during periods of PSP intoxication. This raises the im-
portant question of: ""How do we protect the public health from the
dangers of consuming toxic geoduck viscera?"

The current program used by the WDOH in monitoring for PSP
in geoducks could be modified to better protect public health and
will be discussed in the next section. Second, geoducks could be
shucked and eviscerated prior to being sent to the market. How-
ever, 80% of the current market, both domestic and overseas, is for
whole, live geoduck, where consumers often pay $12 or more per
pound. There is not a large demand for processed geoduck meat (J.
Lo, Evergreen International Food Stuff, pers. comm., 1999 L. El-
liott, E.C. Phillips and Son, pers. comm.. 1999). Typical prices for
shucked body meat range from $3-5 per pound, and for neck meat,
from $12-24 per pound. These prices depend mainly on the
economy and on the availability of whole, live product. In addi-
tion, the volume is so small that even the high prices for neck meat
do not make up for the overall value of live product. A third
solution to the question of how to protect public health is to in-
crease public awareness and education on the dangers of consum-
ing toxic geoduck viscera.

PSP Monitoring

The large difference in toxicity levels between depths and be-
tween tracts has implications for the industry and the WDOH.
Each harvest area will have lo be treated separately when deter-
mining sample si/.e and PSP monitoring effort. Perhaps a larger
number of samples could be taken in the shallow areas, since most
harvest activity occurs in those zones. Because sites vary widely in
wind patterns, bathymetry, tidal currents, and turbulence, the ex-
tent to which toxicity differences will actually occur may also be
expected to vary considerably and can best be tested on a sile-by-
site basis. It must be noted that the information gained from ibis
study is only applicable to the specific study sites. However, gen-
eralizations, such as high variability between individual geoducks,
can he niaile lo nllicr populations.

Assay Comparison

In this study, the RBA overestimated MBA results by an av-
erage of 22.8%. Doucette et al. (1997) found that the RBA agreed
very closely with MBA results from one laboratory but tended to
overestimate those originating from a second source. The reasons
for overestimation are unclear. Differences are expected, given
that the RBA is performed on a static system and the MBA is
performed on a dynamic system (live mice). Resulting toxicities
can be affected by metabolic changes in the mice. Other work has
shown that the MBA is known to underestimate actual toxicity by
as much as 60% at lower toxicity levels (McFarren 1957, Park et
al. 1986). Therefore, the lower levels of toxicity obtained by MBA
in this study could have been underestimated by as much as 60%,
accounting for most of the overestimation by the RBA. Variability
at low toxicity levels in the MBA is affected by many factors,
including salt content during sample preparation, pH, and storage
(McFarren 1957, Park et al. 1986). Last, after initial sample prepa-
ration, some degradation of the low-toxicity compounds Bl and
B2 (N-sulfocarbamoyI toxins) to the nonsulfated carbamate toxins,
STX and neosaxitoxin, could have occurred, resulting in increased
toxicity by the RBA (Cembella et al. 1993).

The overestimation of toxicity levels by the RBA has implica-
tions for the industry and the WDOH if this were chosen as the
approved method of toxin detection. At very low levels of toxicity
(near the regulatory level of 80 p.g), the geoduck fishery would be
closed to harvest more often. However, the RBA could prove to be
a useful tool in prescreening shellfish for PSP toxins. It also may
have applications as a diagnostic tool in suspected cases of STX
poisoning in humans and marine animals. Overall, the two meth-
ods were in very good agreement, as confirmed by a significant
correlation coefficient (r = 0.83 for all samples, r = 0.55 for
samples < 85 p,g). The assay warrants consideration as a rapid,
reliable, and cost-effective alternative to the MBA.

CONCLUSIONS

1. Geoducks collected at shallow depths in both tracts were
more variable in levels of toxicity and were more toxic than
geoducks from the deeper waters.

2. Toxicity levels in the shallow AP area were about two times
those in the shallow QH area. Toxicities in the deep AP area
were about five times tho.se in the deep QH area. In the deep
AP area, toxicity levels were almost always well above the
closure level, while those in the deep QH area were always
below closure level.

3. Results indicate substantial variability in toxicity levels
among individual geoducks within a small population. It
appears that the overall variability among geoducks in both
shallow areas can be generally characterized as having a CV
of about 539( . and in the AP deep area having a CV of 38%.
In the QH deep area, the CV could not be measured because
of an insufficient numbers ol geoducks with detectable lev-
els of toxicity.

4. All toxin levels recorded above the regulatory closure level
(80 |xg STXeq/100 g of tissue) were in the viscera only.

5. At low levels of toxicity (< 85 (xg STXeq/100 g of tissue),
the RBA overestimated the MBA. However, most of the
overestimation can be accounted for by the inherent vari-
ability in the MBA and its tendency to underestimate low
levels ol loxicilv bv as much as 60%. Overall, the two

Paralytic Shellfish Toxins in Geoduck Clams

319

methods had a high degree of correlation {r = 0.83 for all

samples. /' = 0.55 for samples < 85 jj-g STXeq/100 g of

tissue).

The results of this research have implications for the geo-
duck industry and public health agencies. The following recom-
mendations can be implemented to improve geoduck sampling and
analysis.

1. Due to the toxicity differences in harvest depth, the col-
lection of geoducks during the PSP season could be limited
to the deeper areas of a harvest tract to avoid fishery clo-
sures.

2. Farmers interested in culturing subtidal geoducks should
consider doing so in deeper areas to avoid the high toxicities
found in the shallow areas.

3. From a risk-management standpoint, a larger number of
samples collected from shallow areas would have to be ana-
lyzed to reduce the risk of PSP intoxication in consumers.

4. The toxicity difference between tracts implies that the physi-
cal aspects of each tract may have to be considered when
sampling and monitoring for PSP in geoducks.

5. Geoducks should be tested for PSP on an individual basis

rather than as a composite of three samples, to account for
the high degree of individual variability seen in this study.
6. The viscera could be immediately removed and discarded
prior to consumption of the siphon and mantle portions,
which have been shown to be safe to consume even during
times when viscera are highly toxic.

ACKNOWLEDGMENTS

We are grateful to Dr. K. Chew at the University of Washing-
ton, and F. Cox, L. Hanson. M. Antee. J. Tebaldi. M. Guichard. J.
Jernigan. G. Hilton. M. Panoke. and D. Nguyen, at the Washington
State Department of Health (WDOH) for their help and support in
this research. Thanks also go to S. Jennison, J. Markert, M. Cheva-
lier, and D. Williams at the WDNR, and to D. Winfrey with the
Puyallup Tribe for all of their support in this research. Thanks go
to J. Wekell and B. Conrad for statistical advice, and to R. Homer
who offered critical comments on the manuscript. Geoducks.
divers, boat operators, and boat time were provided by the WDNR
and the Puyallup Tribe. Funding for this research was provided by
the Washington Sea Grant Program, WDNR, WDOH, the Tulalip
Tribe, and the University of Washington School of Fisheries.

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Bricelj. v., J. Lee & A. Cembella. 1991. Influence of dinoflagellate cell
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Bricelj, V. & D. Laby. 1996. Differential sensitivity and PSP toxin accu-
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Bricelj. V. & S. Shumway. 1998. Paralytic shellfish toxins in bivalve
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Davio. S. & P. Fontelo. 1983. A competitive displacement assay to detect
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Doucette. G.. M. Logan. J. Ramsdell & F. Van Dolah. 1997. Development
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Homer. R.. D. Garrison & F. G. Plumley. 1997. Harmful algal bkxims and red
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Mackenzie. L., D. White & J. Adamson. 1996. Temporal variation and
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McFarren. E. 1957. Chemical determination of paralytic shellfish poison in

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Joiimat ofShelljhh Research. Vol. 19, No. I, 321-324. 200U.

VIABILITY OF THE TOXIC DINOFLAGELLATE PROROCENTRUM LIMA FOLLOWING
INGESTION AND GUT PASSAGE IN THE BAY SCALLOP ARGOPECTEN IRRADIANS

ANDREW G. BAUDER AND ALLAN D. CEMBELLA

Ncilional Research Council
Institute for Marine Biosciences
Halifax. Nova Scotia
Canada B3H 3Z1

ABSTRACT Bay scallops Argopecten irradians were fed cells of the epibenthic dinoflagellate Prorocentnim lima, a known producer
of diarrhetic shellfish poisoning (DSP) toxins, in controlled microcosms in the laboratory. Examination of scallop fecal ribbons
revealed that ingested P. lima cells were capable of survival and cell division following passage through the scallop gut. This implies
that viable P. lima cells released via fecal deposition from transferred stock may be capable of long-term survival and growth. The
ability of this dinoflagellate to adapt and survive in a wide variety of benthic environments coupled with circumstantial evidence of
its involvement in toxic events, suggests that release of viable cells poses a risk of increasing the geographical range of this species,
with negative consequences for the shellfish industry.

KEY WORDS: Argopecten. bay scallop. Prorocentnim, dinoflagellate. DSP toxins

INTRODUCTION

Contamination of bivalve mollusks by diarrhetic shellfish poi-
soning (DSP) toxins poses an economic threat for shellfish har-
vesters in many parts of the world (Shumway 1990). Dinotlagel-
lates recognized as producers of DSP toxins include several plank-
tonic Dinophysis species (Reguera et ai. 1993. Yasumoto 1990)
and a few benthic/epibenthic species of Prorocentnim (McLachlan
et al. 1994). Although species of Dinophysis are implicated most
frequently as the causative organisms of DSP events, there is
growing evidence that Prorocentnim lima (Ehrenberg) Dodge is
the cause of DSP toxicity at aquaculture sites in eastern Nova
Scotia (Jackson et al. 1993. Lawrence et al. 1998).

The possibility of toxigenic dinoflagellate cells surviving pas-
sage through the digestive tracts of shellfish is an area of special
concern for growers. After contamination by toxic dinoflagellates,
shellfish stocks may be transferred to "clean" waters to depurate
(Haamer et al. 1990, Silvert and Cembella 1995). If live di-
noflagellate cells are released into pristine waters via fecal depo-
sition from newly transferred stock, they may divide and populate
the transfer site. Furthermore, even in the absence of measurable
to.xieity. there is a possibility that viable cells could be inadver-
tently transferred to new sites via the expanding trade in cultured
shellfish, especially when the apparent increase in the frequency
and variety of harmful algal blooms is considered (Hallegraeff
1993). Studies demonstrating the survival of vegetative cells and
pellicular cysts of the dinoflagellate Alexandrium tamarense
(Scarratt et al. 1993), A. fundyense (Bricelj et al. 1993), and A.
miniitiim (Laabir and Gentien 1999), producers of paralytic shell-
fish poisoning (PSP) toxins, in mussel (Mytihis edidis) and oyster
(Crassosirea gigas) feces have warned that such a process could
serve as a potential seed source for subsequent dinoflagellate
blooms.

Recent studies have demonstrated that DSP toxins are accumu-
lated in tissues of the mussel M. ediilis (Pillet et al. 1995) and the
bay scallop Argopecten irradians (Bauder et al. 1996) when toxic
P. lima cells are ingested. However, the fate of undigested cells is
also of interest, because no studies have examined whether di-
noflagellates known to produce DSP toxins can survive gut pas-
sage in bivalves. The objective of the present study was to inves-

tigate the likelihood of survival of P. lima cells following ingestion
by A. irradians.

METHODS

Bay scallops Argopecten irradians (mean shell height = 38 ±
4 mm), were exposed to toxigenic Prorocentnim lima (strain Pa)
at a constant density of 10'^ cells L"' for 13 d in an 80-L aquarium
at 17 °C (Bauder 1997). Cells were kept in suspension using two
recirculating pumps mounted on the aquarium. Scallops were sub-
sequently transferred to another aquarium and depurated for 1
week on a nontoxic diet of the diatom Thalassiosira weissflogii
(ACTIN, CCMP #1336). During the depuration period, the entire
volume of the aquarium was replaced each day with 1 |jim-filtered
seawater to reduce the possibility of scallops re-ingesting fecal
ribbon contents.

Fecal ribbons produced by scallops after 12 days of exposure to
P. lima cells were gently removed from the aquarium by a Pasteur
pipette and rinsed by allowing the feces to settle in 20 mL scin-
tillation vials containing \-\i.m filtered seawater. Feces were then
transferred to another vial containing filtered seawater and kept on
ice.

Viability of P. lima cells within fecal ribbons was determined
by inoculating triplicate samples of intact or disrupted (vortex-
mixed for 30s) fecal ribbons in flasks containing 200 mL of K-
niedium (Keller et al. 1985). A 5 mL sample of P. lima stock
culture was also inoculated in triplicate as a control for comparison
of cell division rates. Cultures were grown on a 14:10 L:D pho-
tocycle at a photon flux density of 90 ixmol m"" s"' at 17 °C. Cell
concentrations in each of the flasks were determined every 7days
over a 4-week period by enumerating 5 mL subsamples in a 0.1
mL Palmer-Maloney chamber under phase-contrast microscopy
( lOOx). Division rates were calculated according to the formula of
Guillard (1973).

Fecal ribbons produced by scallops during exposure to P. lima
cells and during the depuration period were photographed at up to
lOOOx magnification under Nomarsky interference microscopy.
Prorocentnim lima cells in the feces were examined for such ob-
vious characteristics as thecal integrity and cell motility.

321

322

Bauder and Cembella

" '«!.

Evidence that some P. lima cells were digested during passage
through the scallop gut was provided by the presence of P. lima
thecal fragments and free starch granules within the feces. A
greater proportion of digested P. lima cells within fecal ribbons
was observed at the beginning of the exposure period than during
the latter portion (Fig. 1 ).

Live P. lima cells were observed by microscopy in fecal rib-
bons produced by scallops until the fourth day of depuration (Fig
Ic). Pigments and thecal fragments derived from digested Thalas-
siosira weissflogii cells were abundant in fecal ribbons; however,
unlike P. lima cells, very few of these diatom cells were intact.

Mean division rates (k) calculated over exponential growth
phase of P. lima cells contained in fecal ribbons were 0.26 d~' (SD
= 0.03) and 0.21 d"' (SD = 0.04) for disrupted and undisrupted
fecal ribbons, respectively, indicating that the cells were viable and
able to divide at rates comparable (ANOVA, P > 0.05) to those of
P. lima cultures (k = 0.24 d"', SD = 0.01 ) when inoculated into
growth medium (Fig. 2). Similar division rates for cells inoculated
from intact and dispersed fecal ribbons show that P. lima cells
were capable of freeing themselves from the fecal ribbons.

DISCUSSION

The present study is the first to demonstrate the survival of
DSP-toxigenic algal cells in bivalve fecal ribbons. Although Mac-
kenzie ( 1 998 ) found large numbers of intact thecae of another DSP
toxin producer, Dinaplnsis acuta, in fecal pellets deposited by the
mussel Perna canaliculus, viable cells were not observed. It was
further noted that mussel stomach fluids rapidly lysed D. acuta
cells.

In a study demonstrating that PSP toxin-producing Ale.xan-
drium cells could survive and reproduce following passage

%

#•

ai

Figure 1. Numarsky interferi'nce photomicrographs of fecal ribbons
produced hy Argopeclvii irnulians after (a) 2 days and (b) 12 days
exposure to I'rorocviilriim lima, and after (c) .' days of depuration on
a diet of Thalassioisra weissflogii. Scale bars = 20 (iiii la, b) and 100 )jm

(c).

RESULTS

Microscopic examiiuilion of tccal samples rcscalcd that iiuaci
and motile Piomccnirum lima cells were abundant in scallop Iccal
ribbons throughout the exposure period. Cells were frequently
swimming by flagellae within the fecal ribbons, as well as along
the exterior margins of the ribbons, suggesting that these cells had
recently migrated from the feces into the surrounding medium.

E
o

c
o
o

CD
.S

A control culture

□ from disrupted feces

O from undisturbed feces

10

—] —
15

— 1 —
20

25

30

Time (days)

Figure 2. (Jrowth i}{ Pronuciilnim lima cells in K-mcdium. Inoculated
from either /'. limn culture or fecal ribbons of Argnpecleii irradians
l'olli>»ing ingestion of /'. lima cells (undisturbed and disrupted fecal
ribbons). Krror bars: ± 1 standard de\iation, n = 3.

Viability of the Toxic Dinoflagellate Prorocentrvm lima

323

through the gut of Mvriliis edtilis. Scarratt et al. (1993) reported
thai after 3.9 h of depuration, almost all of the fecal ribbons were
free of Alexandhum cells. These authors suggested that 12 h of
purging should be sufficient to rid mussels of toxic cells before
transferring stock to new waters. Passage of Prorocentniin lima
cells in bay scallop guts occurred over a much longer time scale,
as demonstrated by the presence of live cells in fecal ribbons even
after 3 days of depuration, during which the scallops were con-
tinually ingesting Thalassiosira weissflogii cells.

Bomber et al. ( 1988) reported that live P. lima cells have been
found attached to the viscera of tropical fish and argued that the
ability of these cells to survive in fish viscera for extended periods
represents an important system of dispersal for this species. Simi-
larh. the slow passage of P. lima cells in scallop guts represents a
potential mechanism of cell dispersal to new aquaculture and wild
harvest sites via shellfish stock transfer.

Although P. lima cells released in scallop feces have been
shown by this study to be viable in culture, the analogous situation
at a shellfish aquaculture site must be addressed. In the absence of
a strong vertical mixing component, bivalve fecal ribbons contain-
ing P. lima cells would rapidly sink out of the water column to the
benthic environment below the site. For many species of photo-
synthetic microalgae. this environment would be unfavorable for
survival, because it is characterized by low irradiance and tem-
peratures as well as enhanced levels of ammonium and organic
detritus derived from shellfish waste products. However, observa-
tions consistently indicate that P. lima is a very robust species,
well suited to a wide variety of benthic environments.

Bomber et al. (1985) observed survival of P. lima among
benthic detritus in unaltered .seawater for up to 6 months. Simi-
larly, McLachian et al. (1994) reported that P. lima cultures can be
kept in the same culture medium for over 6 months and that the
cells are capable of long-term survival at temperatures of 0 °C. The
cellulose theca of P. lima is extremely resistant to physical dis-
ruption; overwintering stages are primarily in the form of vegeta-
tive cells, rather than cysts. This demonstrates the potential for this
species to be introduced and established at sites that experience
low winter water temperatures. Exposure to high concentrations of
ammonium and other organic nutrients from shellfish excretory
products may even be a benefit to P. lima populations. Carlson and
Tindall (1985) noted that P. lima is associated with natural habitats
characterized by high nitrogen concentrations. Bauder et al. (un-
publ. data) observed that batch cultures of P. lima grew well in
enriched natural seawater (K- medium) with augmented ammo-
nium concentrations as high as 300 (jlM.

The evidence provided in this study of P. lima viability fol-
lowing gut passage in scallops, coupled with the broad ecophysi-
ological tolerances of this dinoflagellate strongly suggest that P.
lima cells released into the waters of a shellfish aquaculture site via
fecal deposition from transferred stock would have a high prob-
ability of long-term survival and growth.

ACKNOWLEDGMENTS

We thank Dr. V. M. Bricelj and Dr. J. Grant for helpful advice
and N. Lewis for technical support. Support for this study was
provided in part by an NSERC scholarship to AB.

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Yasumoto. T. 1990. Marine microrganisms toxins: an overview, pp. 3-8.
In: E. Graneli, B. Sundstrom, L. Edler, and D. M. Anderson (eds).
Toxic Marine Phytoplankton. Elsevier, New York.

Joiirmil of Shellfish Research. Vol. 19. No. 1. 325-331. 2000.

IMMUNOLOGICAL RECOGNITION OF MARINE BIVALVE LARVAE FROM

PLANKTON SAMPLES

ALAIN PAUGAM,' - MARCEL LE PENNECr AND
ANDRE-FONTAINE GENEVIEVE'

^ Labomtoiie de Patlwlogie-Infectieuse
Ecole Nationale Veterinaire de Nantes BP 40706
44307 Nantes cedex 03. France

'Institiit Universitaire Europeen de la Mer
UMR CNRS 6539. Universite de Bretagne Occidentale
Place Nicolas Copernic. 29280 Plouzane. France

ABSTRACT This study concerns the development and use of an immunological method for rapid identification of bivalve larvae in
plankton samples. Protein extracts from larvae and adults of various bivalves were prepared and analyzed according to molecular
biology techniques (electrophoresis and transfer) for the purpose of species identification. Protein extracts of larvae and adults of
Pecten ma.ximiis induced the production of polyclonal rabbit antibody directed against this scallop. The specificity of these sera was
tested by an immunological transfer method (Western blotting i. The antibodies selected were used directly on plankton samples. The
antigen-antibody pair and. thus, the larvae of P. muxinnis were revealed by secondary markers (colorimetric, fluorescent, and
magnetic). The use of such markers is considered for routine work, especially in shellfish farming and marine ecology.

KEY WORDS: Bivalve larvae, antibody, protein recognition
INTRODUCTION

Most benthic marine invertebrates (e.g., the scallop Pecten
maximus) have a life cycle divided into two distinct stages, benthic
for adults and planktonic for larvae, which are closely related in
terms of shellfish fanning and management. AUhough most mac-
roscopic species of marine invertebrates have been thoroughly
studied, their microscopic stages have often been ignored, particu-
larly because of a lack of suitable "tools" and materials to study
organisms under 250 |i.m in size living in three-dimensional (3-D)
space.

Several methods have been used to identify bivalve larvae from
plankton samples. One, based on analysis of prodissoconch shape,
is applicable only to such families as Anomiidae and Ostreidae
(Chanley and Andrews 1971. Le Pennec 1978, Loosanoff et al.
1966). Another limited to the study of hinge characteristics (Rees
1950) was subsequently used with scanning electron microscopy
(SEM), enabling different species of bivalves to be reared in the
laboratory, particularly during larval and postlarval periods (Le
Pennec 1978, Lutz et al. 1982). Unfortunately, this method is
time-consuming and difficult to implement, especially for inves-
tigation of young stages of bivalve larvae in plankton. Herrera and
Cordoba ( 1981 ) used immunochemistry to identify bivalve species
and suggested that this technique could be applied to taxonomic
classification of these organisms in their early stages of develop-
ment.

In general, immunological techniques allow precise recognition
of certain antigens, so that the identification of antigens specific
for a given species is possible. This method has not only been
applied classically to the medical diagnosis of such pathogenic
agents as bacteria and viruses, but also in ecology to determine the
diets of different marine vertebrates (Pierce et al. 1990). In fact, the
suggestion of Herrera and Cordoba ( 1981 ) was not further pursued
until the beginning of the 1990s, when Hu et al. (1992) identified
bivalve larvae (Crassostrea gigas) using protein separation by
electrophoresis. A year later. Demers et al. (1993) developed an
immunological technique based on monoclonal antibodies to de-
tect the larvae of Pectinidae [Placopecten magellanicus) in the sea.
Unfortunately, the data provided by these authors were imprecise.

which led us to design a new immunological protocol, but not
based on monoclonal antibodies. Our basic notion is that molecular
modifications probably occur during the different stages of larval
life and may block the binding of monoclonal antibodies to their
epitope. To avoid this phenomenon, it is preferable to multiply the
number of epitope recognized. Thus, our purpose was to develop
an iminunolabeling process based on the use of polyclonal anti-
bodies that could recognize bivalve larvae in the wild.

MATERIALS AND METHODS

Larval Samples from Hatcheries

The preparation of monospecific antigens to produce "poly-
clonal" antibodies required the use of batches of larvae of the same
species produced by stockbreeding. Several species from three
different families (Pectinidae, Ostreidae, and Veneridae) were
available. The larvae were collected directly in rearing tanks,
washed three times with phosphate-buffered saline (PBS), freeze-
dried for 3 h in a Maxi-Dry Coldfinger (FTS ® System Inc. Model
FD-4.5-90) and then stored at - 80 °C. P. maximus larvae were
obtained from a private fishermen's cooperative at Tinduff (Brit-
tany, France) and from the IFREMER experimental bivalve hatch-
ery in Argenton (Brittany, France); Ostrea edulis larvae from the
IFREMER experimental genetics laboratory at La Tremblade
(Charente-Maritime, France); C. gigas larvae from the IFREMER
hatchery in Argenton and from SATMAR (Socete Atlantique de
Mariculture, Gatteville-Phare. Normandy, France); and Tapes de-
cussatus larvae from SATMAR and from Tinamenor SA Cant-
abrica (Spain). All larval stages were represented, from the D-
shaped one to pediveliger.

Plankton Samples

Plankton samples were collected in the Bay of Brest using a
high-discharge submersible pump, as described by Tremblay and
Sinclair (1990). Seawater flowed downward through a 25-cm di-
ameter sieve composed of two nets (the upper one with 300-(j.m
mesh to remove the largest elements, and the lower one with

325

326

Paugam and Le Pennec

Pecten maximus larvae

other larvae

1

1

Crushing and extraction

4f"

Purification

Specific antibodies

1

Larvae recognition

Figure 1. Protocol.

50-(jLm mesh to retain the plankton fraction likely to contain bi-
valve larvae). The mesh sizes were suitable for trapping most of
the largest bivalve larvae during pelagic development in experi-
mental conditions. Once collected, the plankton was quickly
washed with distilled water and stored at 4 °C in 50 mL of 70 °
alcohol.

Adult Bivalves

Thirty or more 3-year old individuals of P. imixinni.s and C.
gii^as were collected by scuba diving in the Bay of Brest for
subsequent protein extraction.

Protein Extraction

Protein extracts were prepared for electrophoretic analysis and
immunisation of New 7.ealand rabbits. Except for the crushing
method, the same protocol was applied to larvae and adults. Larvae
were either sonicated or processed with a French press (Bioblock
Scientific G 43221 ); whereas, adults required a precrushing step to
break fibres before sonication or French press extraction.

Protocol for l.arvai'

/. Ullrasonic i:\inuiion. Larvae were progressively thawed
from - XO ' C in a cold room to 4 C" and then covered willi 10 ml.

of phenol and 10 mL of distilled water before sonication at 50 Hz
with a Branson sonifier. To avoid heating, they were subjected to
10 1 -second sonication sequences and then placed momentarily (a
few seconds) in an ice bath before the next sonication series. This
procedure was repeated 1 2 times to crush most of the shells and
suspend the organic matter. The entire solution was stirred for 15
min in a water bath maintained at 68 °C and then cooled to below
10 °C in an ice bath. After centrifugation for 45 min at 4.000 x g
(4 °C), the pellet was suspended in 4 mL of water at 0.2'7f in
sodium dodecyl sulphate (SDS) containing 1 mmol of phenyl-
methane sulphonylfluoride (PMSF) and ethylenediamine tetraac-
etate (EDTA) (proteolysis inhibitors). This solution was dialysed
for 2 days in PBS 7,2 containing 0,02% sodium azide. homog-
enized with a vortex in 8 mL of cold methanol at 0 °C. 4 mL of
chloroform and 3 mL of distilled water and then centrifuged for 15
min, at 4,000 x g. Once the aqueous phase had been removed, the
protein interface was collected and rinsed with 2 mL of distilled
water before centrifugation for 15 min, at 4,000 °x g. The resulting
pellet was dissolved in 2 mL of water before the next centrifuga-
tion. This procedure was repeated three times. The final pellet was
dissolved in 5 mL of distilled water with 0,2'7r SDS and I mmol
PMSF and EDTA to constitute the larval protein extract,

2. French press extraction. The French press was used only to
prepare larval extracts. Two millilitres of freeze-dried larvae were
suspended in 15 mL of a chloroform:methanol mixture (2 v:l v),
to which 5 mL of distilled water were added before the final
solution was poured into the previously cooled 50-mL cylinder of
the French press. After 16 metric tons of pressure were applied by
a hydraulic press for at least 2 min, the liquid was stirred for 2 min
at room temperature and centrifuged for 15 min at 4.000 x g at 4
°C, As noted above, the protein extract was contained in the in-
terface layer and required further washing with water.

Protocol for Adults

Because of the large amount of organic matter studied in adult
as compared to larval tissues, the tools used for preparation of
extracts differed. The tissues were mixed for 2 min at full speed
with a kitchen mixer (Moulinex), and the resulting homogenates
were treated as for larvae. In the case of scallops, some specific
extracts (muscle, mantle, gill, gonad, and digestive gland) were
also prepared.

Protein Titration

Protein concentration was determined by a protein-dye binding
assay with bovine serum albumin (BSA) as standard (Protein As-
say and DC Protein Assay kits. Biorad, ref 500-0001 and 500-
0111. respectively).

Electrophoresis and Blotting

The various protein extracts were partitioned by SDS-
polyacrylamide gel electrophoresis (PAGE) (Laemmli 1970). The

blotting technique applied was initially designed by Towbin et al,
( 1979), improved by Kyhse-Andcrsen ( 1984), v\ht) used a semidry
system, and finally modified b> Gitton et al. 11992),

Kleclrophuresis

Aliquots (5 fxg) of bivalve proteins were diluted in sample
buffer (1:4. v:v) containing 5,59^ SDS. S.SVr glycerol, and l,7'/f
bromophenol blue in 0,5 M Tris. pH 6,8. and then heated for 3 min
al 95 C and subjected lo SDS-PAGE in PROTEAN 11-type tanks

Recognition of Marine Bivalve Larvae from Plantkon Samples

327

(Biorad) using the discontinuous buffer system of Laemmli (1970).
An optimal acrylamide concentration of 12.5% was selected for
the gel. A discontinuous buffer system under electric feed moni-
tored by a Biorad generator (Power-Pac 3000, software version
3.27) was used. Power was constant (17 W). maximum voltage
never exceeded 400 V, and the ammeter was set at 100 mA. The
gels were stained with Coomassie brilliant blue R-250 O'Farrell.
Determination of molecular mass. The molecular mass of the
different proteins was estimated from known standards (Biorad).
The method chosen corresponded to a degree 3 polynomial regres-
sion model designed from Molecular Analyst software. This model
was efficient in describing protein migration in a gel of constant
concentration.

Blotting

Electrophoretic profiles were transferred onto nitrocellulose
sheets (0.45 \i.m pore size. Biorad) with a .semidry system (Trans-
Blot SD Biorad) using Tris buffer (pH 9.2. 48 mmol Tris. 39 mmol
glycine. 1.3 mmol SDS, 20% methanol). The nitrocellulose was
rinsed out for 5 min in Tris-buffered saline (TBS, pH 7.5) and then
blocked for 30 min in TBS-3% gelatine (w:v). Further rinsing with
TBS and Tween 20 (TTBS) at 0.05% was necessary, and the
nitrocellulose was subjected overnight to the action of rabbit serum
at the appropriate dilution ( 1:2,000) in TTBS at 1% gelatine (w:v).
Two additional rinsing in TTBS and one in TBS were necessary to
remove unbound antibodies before the marked antiglobulin was
labeled with alkaline phosphatase. The blot was then incubated
with anti-rabbit IgG antibodies previously conjugated with alka-
line phosphatase at a 1:3,000 dilution (v:v) in TTBS- 1 % gelatine
(w:v) and maintained for 30 min in the previous solution. The
nitrocellulose was steeped twice in TTBS (5 min) and once in TBS
(5 min) and then soaked in Biorad buffer containing nitroblue
tetrazolium and 5-bromo-4-ch!oro-3-indolyl phosphate. The sheet
was finally steeped for 10 min in distilled water. All these steps
were carried out at room temperature on an orbital shaker.

Antibody Preparation

Two "primary polyclonal" sera were prepared: one antilarval
serum (LPm) and one antiadult serum. (Apm). Rabbit antisera
were directed against P. maximus extracts. Each serum was pre-
pared using two immunized rabbits. Five immunizations per ani-
mal were performed by the subcutaneous route in the back of the
rabbit. Each 1-mL dose consisted of one volume of protein solu-
tion (900 |j.g of protein antigens in 500 jjlL of extract) plus one
volume of Freund adjuvant. The first immunization was enhanced
by complete adjuvant and the following ones by incomplete adju-
vant. The second immunization was performed 2 weeks after the
first, and the last three at 1-week intervals. The final serum was
collected 5 days after the last injection.

Antibody Purification

The entire antiscallop larval serum (LPm) was consumed in 1
day under stirring at 4 °C against an extract of adult C. gigas. The
purified serum or LPm^„„,„^^,j obtained in supernatant after 45-
min centrifugation of LPm-C gigas extract at 4.000 x g at 4 °C.
Compared to the dilution ratio used for blotting, LPm^.„„,,„,„;,j was
less diluted (1:400).

Larval Recognition by Antibodies

All organisms contained in plankton samples were washed
twice for 5 min in PBS under soft stirring, incubated for 1 h with

1% glutaraldehyde in PBS. washed twice for 5 min in PBS, incu-
bated 2 to 15 min in 0.2% Triton X-IOO in PBS, and washed again
for 5 min with PBS. To reduce nonspecific staining, organisms
were incubated for 30 min with 3% hydrolyzed gelatine in PBS
(w:v). After two washes in PBS (5 min), larvae were incubated

overnight in LPm^

, (diluted 1 :400 in PBS), rinsed twice with

PBS and incubated for 30 min with goat antirabbit IgG labeled: ( 1 )
with alkaline phosphatase (Biorad); or (2) fluorescein-
isothiocyanate (Interterchim); or (3) with magnetic beads (Dyna-
beads M 280-Dynal®, 2.8 ixm diameter). Once washed with PBS,
all organisms were ready for observation, except those tagged with
alkaline phosphatase (this stain required a substrate to induce blue
color: i.e., nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate in Tris buffer. pH 9.0).

All organisms were observed with a universal epifluorescent
microscope (Zeiss Orthoplan model) using a 200-W mercury lamp
with an appropriate FITC filter. Photos of plankton samples were
taken with a mix of transmitted and epifluorescent light. The mer-
cury lamp was turned off to observe the two nonfluorescent-
stained samples.

RESULTS

Protein Titration

The protein-dye binding assay kit ( Bradford 1976, Lowry et al.
1951 ) was used to evaluate extraction yield. The concentrations in
larval protein measured in P. maximus from Argenton and Tinduff
were 5 and I mg/mL^' respectively; whereas, in Ostreidae, they
accounted for 1 mg/mL~' in C. gigas and 0.4 mg/mL~' in O.
edulis. The level in T. decussalus was 1.7 mg/mL~'.

Results were similar, regardless of the extraction method used
(ultrasound or the French press), but the French press protocol was
shorter and, thus, time-saving.

Electrophoresis

After protein determination, the different extracts (larvae and
adults from P. maxinuis and C. gigas: larvae from O. edulis and T.
decussatus) were subjected to 12.5% SDS-PAGE with Coomassie
brilliant blue staining. Adult extracts displayed several bands with
similarities indicative of a common profile for the tissues of these
two species (P. maximus and C. gigas). However, the sequence
and thickness of the different bands depended on each species and
were characteristic of them. Although common strips were found
between larval and adult extracts, there were some significant
differences within a given species.

Comparative analysis of electrophoretic profiles from the
whole set of extracts highlighted several bands, each characteristic
of a single bivalve species. The 87 kilodalton (kDa) band corre-
sponded to adult P. maximus the 30 kDa one to P. maximus larvae,
and the 70 and 32 kDa bands, respectively, to Ostreidae and T.
decussatus. However, the protein bands, though specific, were not
necessarily immunogenic.

Blotting

Rabbit antibodies on blots, which corresponded to all the elec-
trophoretic profiles after transfer, revealed the immunological re-
sponse. Serum LPm (Fig. 2A) and APm (Fig. 2B) were applied,
respectively, onto the blot at a dilution of 1:2,000.

After full immunization, rabbit antibodies were able to recog-
nize numerous protein antigens. LPm. like APm (both produced

328

Paugam and Le Pennec

123456 789

99kDa
66kDa

45kDa

32kDa

21.5 kDa

14.4 kDa

B

12 3 4 5 6

99 kDa
66 kDa

-H

45 kDa
32 kDa

HI

21.5 kDa

.•»

14.4 kDa

1

i ■

7 8 9

n

1 2 3 4 5 6

7a 7 8 9

Figure 2. Western blotting of proteins obtained from /'. maximus, C.
gigas, R. dcciissaliis, (). ediilis. (,\) revealed bv APni. (B) revealed by
I,Pm. (Cl revealed by i.Pni consumed. I.anes : I to 6 extracts of P.
maximus. I. Adult muscle: 2. Adult mantle; 3. Adult gill: 4, Adult
gonad: 5, Adult digestive gland: A, Larvae. 7a, Adult ('. gigas; 7, Lar-
vae C. gigas; 8, Larvae R. decussatiis; 9, Larvae (>. ct/u/i's.Mulecular
weight Is indicated as kilo-Dalton for each lane.

Irom /'. iiHixiniKs cxiracis). rc;i(.lcd with llic other bivalve species
investigated. Polyelonal rabbit sera allowed us to detect antigenic
proteins in scallop extracts by inmunioblotting and also high-
lighted several antigenic bands shared in common by scallops on
extracts from other species. Each electrophoretic profile was com-
pared with the two imniunoblots, allowing identification of the
antigenic bands on the electrophoretic profile and especially (.le-

tection of those found only in the extract from the chosen species:
that is. P. inaxiiinis larvae.

Immunostaining intensity was correlated with antiserum sensi-
tivity; whereas. Coomassie blue staining was related to protein
abundance.

Immunological Recognition of P. maximus Extracts

Investigation of the immunological recognition of P. maximus
extracts showed that LPm serum reacted more specifically with
extracts from P. maximus larvae than with those from adults;
whereas, the reaction with APm was stronger against adults than
laiA'ae. The effect of two antilarva and antiadult sera on an adult
extract of scallops differed significantly, especially for antigens
below 21.5 kDa. This molecular mass constituted a limit below
which homology was no longer apparent. For each blot, homolo-
gous reaction was always greater than heterologous reaction.

Immunological Recognition of all Other Bivalve Extracts

The antiadult serum (Apm) reacted preferably with C. gigas
adults The response against lairal extracts was evenly distributed
along the strips, but did not allow clear identification of the dif-
ferent bands, except for the lOO-kDa band, which occurred in
all bivalve extracts. This serum did not distinguish between the
different extracts. For each of the four extracts; that is, C. gigas
larvae and adults, O. ediilis larvae and T. decussatus larvae, the
antilarval serum (LPm) did not "react" below 16 kDa. Once again,
the resulting immunoblolting profiles were characteristic of the
species represented. However. LPm serum recognized bands prob-
ably shared by all species investigated. A comparison of profiles
with that corresponding to the larval extract from P. maximus
enabled us to identify two specific bands at 48 and 2,^ kDa respec-
tively.

For the two sera, analysis of the sequences of all bands con-
stituting each profile indicated that each protein extract was spe-
cific and that the sera used to recognize them had a strong affinity
for the extract, which had induced their elaboration. However, the
polymorphism of these sera was too great to allow their direct use
as a recognition marker.

Because the antigens were generally common to several species
and especially to the extract from adult C. gigas. antibodies of
LPm antiserum were adsorbed on a mixture of C. gigas tissues to
remove all the antibodies shared in common by C. gigas adults and
P. maximus larvae. The resulting consumed serum. LPm eonsumcd-
(Fig. 2C), did not react with the extract of C. gigas adults. This
procedure allowed the capture of antibodies common to this spe-
cies of bivalve. Among the larval extracts, only that of P. maximus
was identified by the adsorbed serum. This purified serum con-
firmed the presence of specific P. maximus larval bands at 48 and
2.^ kDa and identified a new one at 19 kDa.

Ill \'ivo Labeling Process

Indirect antibody staining was performed on the LPn\„„.,,„„„|
serum (Fig. ?>) using three different types of anti-antibodies labeled
with alkaline phosphatase (ALPi (Fig. }A). FITC (Fig. .^B, D) or
magnetic beads (Dynal®)(Fig. .^C).

With the first staining procedure, labeling was especially con-
centrated around the shell on the mantle edge. The FITC antiglobu-
lin allowed us to localize a scallop veliger from a plankton sample

Recognition of Marine Bivalve Larvae from Plantkon Samples

329

Figure 3. Larval immunostaining. (A) Scallops larvae revealed with Alkaline Phosphatase: (B) Scallops larvae revealed by FITC; (C) Scallops
larvae revealed by magnetic beads; (D) FITC labeled scallop larva revealed in a plankton sample. Scale bar = 100 urn.

under epifluorescent microscopy, using mixed fluorescence wave-
lengths (Fig. 3D) and visible ranges, as suggested by Demers et al.
(1993). LPm^.„„,„^^j recognized both D larvae (Fig. 3B) and the
pediveliger. The immunomagnetic marker (Dynabeads M-280)
formed a complex with LPm^,„j,.,„„,^.j to stain scallop larvae, but did
not magnetize them sufficiently to allow total collection by mag-
netic power. Nevertheless, many larvae were captured in the mag-
netic field (in Fig. 3C, each black point is an aggregate of many
magnetic beads).

DISCUSSION

Only monoclonal or polyclonal antibodies can be used to stain
bi\alve larvae by an immunological technique. The formers ha\e
high specificity dependent on selective binding to only one
epitope. Demers et al. (1993) tested this technique and found vari-
ability in stain intensity within the same batch of larvae. One
possible explanation for these results was a differential degrada-
tion of antigenic structures attributable to transport and conserva-
tion conditions. A second possibility was that antigen expression
changed naturally as a result of physical and (mainly) physiologi-
cal conditions during larval development, as previously reported
by Boreham and Ohiagu ( 1978). Feller (1986). Wang et al. (1992).
Westin (1972). etc. To decrease the variability in staining intensity.
Demers etal. ( 1993) used a pool of monoclonal antibodies selected
among three specific lines.

Instead of applying a pool of monoclonal antibodies, we used
the rabbit for preparation of a polyclonal serum, which processes
several antibodies against all the antigens in a preparation and.
unlike monoclonal antibodies, produces stable multivalent inter-

actions conducive to high affinity. Among substances likely to be
immunogenic, proteins are most often used, and our extraction
procedures sought to obtain a good protein immunogen. Two
grinding methods were tested (results not shown). The French
press proved not only more efficient for protein extraction from
bivalve larvae, but also saved time by avoiding a first extraction
with a mixture of water and phenol. In addition, the simplicity and
efficiency of this method reduced solvent consumption and larval
cost while lowering inhalation risk in a small enclosure. In any
event, the electrophoretic profiles were similar with both methods,
and analysis identified the bands characteristic of the proteins con-
tained in each extract and thus in each bivalve species.

Extraction yields were always evaluated using the protein-dye-
binding assay kit. Protein amounts varied with larvae batches, but
were not correlated with the investigated species. Adult extracts
presented no difficulties. Regardless of the extraction yields, elec-
trophoretic profiles were characteristic of a single species. When
protein levels were below 1 mg/mL~' at the end of extraction for
larvae, only the readability of the profile was affected (blurred
bands), suggesting a possible correlation between low protein con-
tent and blurred profiles. As extractions were always carried out in
the same environmental conditions and electrophoresis was per-
formed on the same amount of proteins, the only possible expla-
nation for this yield reduction related to the larvae themselves. In
fact, all came from stock farming. Although their shell morphol-
ogy criteria were fine, their true health conditions remained un-
known. Unfortunately, no information was available on the settling
capacity of each batch of larvae, which might have allowed the
quality of the electrophoretic profile to be related to postlarval
settlement (the only true index of larval physiological health).

330

Paugam and Le Pennec

As noted above, any protein extract from a given bivalve spe-
cies was liable to produce a polyclonal serum with a specificity
sufficient to differentiate one extract from another by immunob-
lotting. LPm and APm enabled us to distinguish the immunologi-
cal bands characteristic of a particular P. maximus extract, and
molecular analysis software allowed us to highlight the succession
of bands characteristic of scallops. Extracts from adults and larvae
shared several bands. However, this approach neglected the im-
portance of using larval extract as antigens to induce specific "anti-
larval" antibodies. Although the specificity of our LPm serum was
sufficient to recognize scallop larval extract among several others,
it was unable to recognize scallop larvae in plankton (unpublished
data).

Two possible procedures were considered to enhance the speci-
ficity of our first sera: the capture of specific antibodies by chro-
matography and the removal of nonspecific antibodies by deple-
tion. The second solution proved easier to implement. To limit
potential cross reaction, an attempt was made to purify LPm before
subjection to heterologous molecules. The recognized antigen al-
lowed the formation of a complex with antibodies that settled
down. After centrifugation, the pellet was easily removed and
discarded. Thanks to this technique, a new version of the LPm
serum (LPmj.„„.,^„„^.j) was obtained, which in immunoblotting tests
recognized scallop extracts (mainly from larvae). There were no
further cross reactions with larval extracts from other species. The
absence of a heterologous reaction allowed us to use this serum for
larva labeling. Antibody binding was revealed by three different
techniques. First, staining with alkaline phosphatase localized the
area of antibody binding near the mantle. Like Demers et al.
( 1993), we found that no staining disorder was apparent because of
shell closure. Alkaline phosphatase was not selective, because it
revealed the enzyme bound to the antibody as well as the endog-
enous enzyme. Revelation of the latter caused background noise in
plankton samples (some controls were made on batches of larvae
with negative antibodies or with direct incubation in AP substrate),
which affected the interpretation of bivalve larval detection. For-
tunately, this inconvenience can be avoided if revelation time for
the enzyme does not exceed \5 min.

Fluorescent staining gave better results (Fig. 3B, D), allowing
scallop larvae to be identified in plankton samples. Because the
wavelength used to induce antibody fluorescence sometimes
makes phytoplankton fluorescent, some adjustments will be re-
quired before a How cytometer can be used to automate the count-

ing of bivalve larvae. Staining could be improved by replacing the
fluorescent marker with another one in order to change the exci-
tation wavelength.

Magnetic beads have already been used in marine biology for
isolation of the toxic dinotlagellate Alexandriwn fwidyense from
preserved seawater (Aguilera et al. 1996). In our study, beads were
found within the larvae. Although their magnetization was too
weak for efficient separation of all scallop larvae from plankton,
bigger bright-colored magnetic beads could allow the separation
and recognition of scallop larva even by an inexperienced re-
searcher. All three of these staining methods allowed rapid iden-
tification of bivalve larvae among the whole plankton community.

In conclusion, this first approach to recognition of bivalve lar-
vae indicates the possible benefit of the methods considered here.
Electrophoresis revealed immunogenic and specific proteins in
several bivalve species. Once purified, such proteins will be able to
induce new polyclonal sera endowed with higher capabilities, as
recommended by Knudsen (1985) and Diano et al. (1987).

This approach reduces the time required for sorting out and
identifying scallop larvae in plankton samples. The use of poly-
clonal antibodies to stain the larvae extracted from our samples
seems promising, and it is likely that this method, after a few
adjustments, can be applied in ecological programs or for shellfish
farming.

The major interest of this technique is species-specific identi-
fication to distinguish close species such as Pecten maximus.
Aequipecten operciilaris and Chlamys varia in the Bay of Brest, or
Placopecten magellaniciis and Chlamys islandica in the Gulf of St.
Lawrence in Canada. However, many more families could be
treated as well. The use of an immunoassay for identification of
marine larval species could save precious time and allow auto-
mated identification and measurement of larvae. All these im-
provements could be useful in shellfish farming to monitor the
settlement of postlarvae and determine the best period for setting
up a collector.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Andre-Fontaine and all members
of the Laboratory of Infectious Pathology for their comments,
suggestions, and technical support. This study was supported by
the PNDR (Programme National sur le Determinisme et du Recr-
utement).

Aguilera. A.. S. Gon/.alcs-Gil. B. A. Kcaler & D. M

Imnninomagnetic separation (if cells ot the toxic dinotlagellate Alex-

andnuin fuiulycnse trom natural plankton samples. Mur. Ecol. Prof;.

Scr. 14.^:255-269.
Borehani. P & C. Ohiagu. 1978. The use of serology in evaluating inver-
tebrate prey-predator relationships: a review. Bull. Enlomol. Rc:\. 6X:

171-194.
Bradford. M. M. I')76. A rapid and sensitive melhoil lor llie i.|iianlilMllon ol

microgram quanlilics of protein utilizing the principle of proloin-dyc

binding. .Anal. Hii>tlu'iii. 72: 24S-254.
Chantey. P. E. & J. D. Andrews. 1971. Aids for identilieulion of hi\al\c

larvae of Virginia. Malmolugiu 1 1: 45-1 19.
Demers, A., Y. Lagadeuc. J. J. Dodson & R. Lcmieux. IW3 hiinuiiionu-

oresccnce identification of early life history stages of scallops (Pcc-

linidael. Mar. Ecol. Prof;. Scr 97:8.VX9.
Diano, M.. A. Le Bivie & M. Him. 19X7. A method for the production ol

highly specific polyclonal anlibodies. Anal. Hiochem. 166:224-229.

LITERATURE CITED

Anderson. 1996. Feller. R. J. 1986. Immuno:

ical detection ol Mcrccnaha tncrccnaria. a
prey predator, and a preparation of si/e-class specific antibodies. Vc-
//,i;('/-28:.M 1-347.

Ginon. X., G. Andre-Fontaine. F. Andre & J. P. Ganiere. 1992. Immuno-
blotting study of the antigenic relationships among eight serogroups of
Lcptosfyira. Vet. Microbiol. 32:29.3-30.V

Herrera. E. M.. F. Cordoba. 1981. Identificacion y classificacion immuno-
quiniica de bivalvos (almejas). Inj. Gen. Labores Cent. Invest. Biol.
Baja Calif.: 321-331.

Hu, Y. P.. R. A. Lut/ & R. C. Vrijenhoek. 1992. Electrophoretic identifi-
cation and genetic analysis of bivalve larvae. Mar. Biol. I 13:227-230.

Knudsen, K. A. 1985. Proteins transferred to nitrocellulose for use as
imniunogens. Anal. Biochem. 147:285-288.

Kyhse-Andersen. J. 1984. Electroblotting of multiple gels: a simple appa-
ratus without buffer tank for rapid transfer of proteins from polyacryl-
amide to nitrocellulose. J. Biochem. Biophy.s. Melli. 10:203-209.

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Laemmli. U. K. 1970. Cleavage of structural proteins during the assembly methods to identification of fish prey in diets of seals and dolphins. /

of the head of bacteriophage T4. Naliire 227:680-685. Exp. Mm: Biol. Ecol. 137: 123-140.

Le Pennec, M. 1978. Genese de la coquille larvaire et post-Iarvaire chez Rees. C. B. 1950. The identification and classification of lamellihranch

divers bivalves marins. Doctoral thesis. Universite de Bretagne Occi- larvae. Hull. Bull. Mar. Ecol. 3:73-104.

dentale. 229 pp.. 108 pi. Towbin, H.. T. Staehelin & J. Gordon. 1979. Electrophoretic transfer of

Loosanoff, V. L.. H. C. Davis & P. E. Chanley. 1966. Dimensions and proteins from acrylamide gels to nitrocellulose sheets: procedure and

shapes of some marine bivalve mollusks. Malacologia 4:351^35. some applications. Proc. Nail. Acad. Sci. USA. 76:4350-4354.

Lowry. O. H.. N. J. Rosehrough. L. A. Farr & R. J. Randall. 1951. Protein Tremblay. M. J. & M. Sinclair. 1990. Diel vertical migration of sea scallop

measurement with the Folin phenol reagent. J. Biol. Chem. 193. 265- larvae Placopeclen inam'llanicus in shallow embayement. Mar. Ecol.

275. Prog. Ser. 67: 19-25.

Lull. R.. J. Goodsell. M. Castagnan. S. Chapman. C. Newell, H. Hidu. R. Wang. L.. Y. Feng & J. L. Denburg. 1992. A multifunctional cell surface

Mann, D. Jablonski, V. Kennedy. S. Sidall, R. Goldberg. H. Beattie. C. developmental stage-specific antigen in the cockroach embryo: in-

Falmagne, A. Chestnut & A. Partrige. 1982. Preliminary observations volvement in pathfinding by CNS pioneer axons. J. Cell. Biol. 118:

on the usefulness of hinge structure for identification of bivalve larvae. 163-176.

J. Shellfish Res. 2:65-70. Westin. M. 1972. The occurrence of stage-specific antigens during eariy

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Journal of Shellfish Research. Vol. 19. No. 1. 333-+91. 2000.

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ResTOwnon

TECHNICAL PAPERS

Presented at

INTERNATIONAL CONFERENCE ON
SHELLFISH RESTORATION

Hilton Head Island, South Carolina

November 18-21. 1998

333

Journut of Shellfish Research. Vol. 19, No. 1. 334. 2000.

SELECTED PAPERS FROM THE

1998 INTERNATIONAL CONFERENCE ON SHELLFISH RESTORATION:

"FORGING PARTNERSHIPS TO IMPROVE THE HEALTH OF COASTAL ECOSYSTEMS

THROUGH SHELLFISH RESTORATION"

Throughout the world there is a growing commitment to the
restoration of degraded coastal ecosystems. Political pressure by
shareholders in the future of the world's coastal areas has resulted
in renewed interest in preserving and enhancing coastal resources
at all levels of government. At the local level many volunteer
organizations have developed successful programs to identify
problem areas, recommend improvements and monitor progress.

The Second International Conference on Shellfish Restoration
(ICSR) was held on Hilton Head Island, South Carolina, U.S.A. on
November 18-21, 1998. ICSR provides an opportunity for gov-
ernment officials, resource managers, users, and residents to dis-
cuss approaches to restore coastal shellfish ecosystems through
remediation and pollution abatement, habitat restoration and stock
enhancement. Case studies of successful projects are presented,
with opportunities for roundtable discussions.

The first ICSR event, held in 1996 was extremely successful.
More than 200 participants from ten countries joined together to
learn about and discuss innovative management, ecological, and
social approaches to restore degraded shellfish habitat and improve
coastal ecosystem health. ICSR has been so successful that a Eu-

ropean version of ICSR was bom — ICSR'99 was organized and
held last year in Cork, Ireland.

A unique feature of ICSR is the diversity of individuals who
participate. The opportunity for internationally recognized resto-
ration experts to interact with local town planning officials does
not occur often enough. ICSR provides that forum and also the
building blocks for such interactions to occur in the future.

The papers that follow are representative of the diversity of the
topics and individuals that participated in ICSR"98. We are grate-
ful to the many conference sponsors for their support, the members
of the ICSR Steering and Program Committees for their time and
energy, and to the Journal of Shellfish Research and its editor,
Sandy Shumway. for publishing these contributions. We also wish
to thank Anne B. Miller for serving as our Technical Editor. Thank
you all.

From the ICSR'98 Co-Chairs: Dorothy Leonard, NOAA-
NMFS, M. Richard DeVoe. Elaine L. Knight, and Linda Black-
well, S.C. Sea Grant Consortium, and William Rickards, Virginia
Sea Grant College Program.

334

JoKimil of Shellfish Research. Vol. 19. No. 1, .^35-339. 2000.

RESTORING THE OYSTER REEF COMMUNITIES IN THE CHESAPEAKE BAY:

A COMMENTARY

ROGER MANN

Virginia Institute of Marine Science
College of William and Mary
P. O. Box 1346
Gloucester Point, Virginia 23062

ABSTRACT Restoration of the oyster Crassostrea virginica resource to the Chesapeake Bay is a widely supported goal. This
manuscript explores the questions of why. how. and in what time frame this should be attempted. Restoration goals based simply on
support of a coniinercial fishery fail to address the role of the oyster as a cornerstone species within the Chesapeake Bay and should
only be considered in the context of a long-term sustainable fishery exploitation. The argument is proffered that a restored resource
sustaining a fishery at the historical harvest level is unrealistic, because: ( 1 ) harvest probably exceeded biological production for much
of the recorded history of exploitation; and (2) maximum production, a desired end for fishery support, occurs at approximately half
the maximum (virgin, unexploited) biomass. and, thus, can only be achieved with disruption of the virgin complex community
structure. Thus, the direct harvest economic value of a fishery based on a restored resource will not reach historical levels if there is
an accompanying goal of long-term community development that is self-sustaining in the absence of restoration effort. The role of the
oyster as a cornerstone organism and the pivotal link in benthic-pelagic coupling is examined in the context of current and projected
watershed management problems, including agricultural and urban development with associated nutrient and sediment erosion issues,
in the entire Chesapeake Bay watershed. Restoration efforts to date have focused on rebuilding three-dimensional reef structures, often
with subsequent oyster broodstock enhancement, in predominantly small estuaries with retentive circulation to provide demonstration
of increased resultant recruitment. Such examples are used to increase public awareness of the success of restoration processes and
increase long-term participation in such programs by schools, nonprofit and civic organizations, and commercial and recreational
fishing groups.

KEY WORDS: oysters, Crassoslreci virginica. Chesapeake Bay, reefs, restoration, watershed, management, benthic-pelagic coupling

DEFINING THE PROBLEM, PART 1: BIOLOGY. ECONOMICS,
PERCEPTION, AND TIME FRAMES

The Chesapeake Bay has a history related to the eastern oyster
Crassostrea virginica. Much of the biology of the bay over the past
10,000 years is arguably dependent on the reef-forming habit of
this cornerstone species. Oysters were an important food source to
pre-Colonial native populations, were quickly recognized for their
value after Colonial settlement, became the center of a national and
international trade before the end of the 19th century, and re-
mained a substantial component of the Middle Atlantic economy
through the first si.x decades of the 20th century. The past four
decades have been marked by the appearance and continued de-
structive effects of two disease vectors, Haplosporidium nelsoni.
commonly known as MSX, and Perkinsiis marinus. commonly
known as Dermo, in the higher salinity regions of the bay.

When considered together with the cumulative effect of many
decades of overfishing and environmental decay, the result is a
sadly depleted oyster resource in the Chesapeake Bay. Although
consensus is growing that attempted restoration of this resource is
a noble and worthwhile cause, the task before us is to ask why,
how, and in what time frame this should be attempted.

Given that the oyster has long supported a commercial fishery
in the Chesapeake Bay, a logical first question is "Should the
revitalization of the oyster fishery be the prime motivation for
restoration of the oyster populations in the bay?" Such a question
has a number of inherent qualifiers. Fisheries utilize a biological
resource to optimize or maximize economic or societal return.
Restoration of the resource for this prime purpose would be in a
form that optimizes harvest over a defined time frame — a form that
may not, as is discussed later, be considered best for optimizing
ecological complexity and stability. Economies have time horizons

of importance, thus any restoration effort must respect and be
responsive to this time frame. The societal component must be
equally addressed in that restoration to enhance an economic con-
tribution to a thriving economy must be responsive in a politically
expedient time frame; that is, efforts must create a strong public
perception of improvement in the face of multiple competing
needs for public funding. Economy, perception, and time frame, in
addition to biology, become important factors in setting fishery
restoration goals.

What might be reasonable goals for a fishery-driven restoration
program? The recent and current oyster fishery in the Chesapeake
Bay has several components. These must be distinguished from the
oyster industry, which includes processing of oysters originating in
regions other than the bay. In Maryland, there currently exists an
active public fishery prosecuted by watermen who purchase li-
censes to harvest oysters from resources in regions held in public
trust by the State of Maryland. The harvest from this fishery typi-
cally exceeds its Virginia counterpart by a very substantial amount.
However, this harvest is "underwritten" both by substantial public
funds and by the continuing effort by the Maryland Department of
Natural Resources to plant shell substrate in selected regions in
advance of seasonal oyster settlement (spatfall) and to move the
resultant "seed" to regions for optimal growth in the face of po-
tential disease pressure. This program is arguably very responsive
to a fishery need; the long-term issue of resource restoration is not
a prime mission of the program. A similar program of shell de-
ployment and subsequent "seed" movement on grounds retained in
public trust has also been pursued in Virginia. The incremental
impacts of disease have reduced the effectiveness of the Virginia
program in supporting a continuing industry, and current landings
from the fishery are at an all-time low. As in Maryland, the focus
of this "plant and move seed" program has been short term, with

335

336

Mann

no statement on long-term restoration. Virginia also allows leasing
of "suboptimal"' bottom adjacent to public grounds. These regions
sustain a very substantial fishery harvest essentially in grow-out of
"seed" oysters but are operated on a put-and-take basis with a 2-3
year growth period. Again, these are strictly for-profit operations
by private individuals or corporations with no restoration goal (see
Haven et al. 1981a,b). Such efforts have all but disappeared in the
past decade as a result of the continued incidence of disease. The
losses accompanying the fishery's decline since the major onset of
disease have had a subtle societal impact that has generated con-
siderable public debate and. in some instances, sympathy. Com-
mercial fishermen are among the few remaining "hunter-
gatherers" in modern society, and their visible demise in the
Chesapeake Bay oyster fisheries is viewed (perhaps unrealisti-
cally) as a loss of individuals who operate with large amounts of
personal freedom in a society that pays little attention to that same
personal freedom. A reasonable goal from an economic position
would be the restoration of a fishery resource to support a predis-
ease level of harvest, typically several millions of bushels per year
for Maryland and Virginia combined, with some enhancement of
the societal role supported by the fishery.

Is a fishery-driven restoration to sustain a predisease level of
harvest a reasonable goal for ecological restoration? Arguably, no.
An examination of the historical fishery harvest finds that the
harvest was much greater before to the turn of the century. The
combined harvest of oysters in 1865 by Maryland and Virginia
alone was approximately 17 million bushels (Hargis and Haven
1988) — enough oysters to bury a football field to the depth of 656
feet! This is an astonishing amount given the primitive dredging
and longing techniques employed, but it illustrates simply the level
of fishing pressure employed in the latter half of the 19th century.
We know from the works of Ingersoll (18811. Brookes (1891 ). and
others that a century ago strong concerns about overfishing and its
eventual impact were expressed to regulatory bodies. Although
these concerns stimulated a limited regulatory effort, and the sur-
veys of Winslow in Maryland and Baylor (1894) in Virginia to
define the extent of the public resource, the comments did little to
abate the revisiting of the "tragedy of the commons." The impor-
tant issue to this commentary, however, is that the enormous re-
movals of oysters had proportionate impact on the biology of the
oyster in the bay. Neither as part of the process of oyster harvesting
nor as part of the discussion (minimal for much of the time) on
resource management was a formal assessment of stock size or
estimate of productivity ever made. However, the fundamental
understanding of the importance of these processes was already
central to the discussion of marine finfish slocks on both sides of
the Allanlic before the turn of the century, as demonstrated by the
work of Spencer Baird, G.O. Sars, and their peers. Although very
large and obviously old oysters were still abundant in the bay
during the heyday (1860s) of harvest (de Broca 1865), we also
know from navigation charts prepared by the U.S. Navy before the
turn of the century, that three-dimensional oyster reef structures
were exposed only at low tide in many regions ol the bay. These
reefs gradually became permanently sublidal with conlinued
wholesale mining of the resource lor both food and industrial
(chicken gril to limestone to road surfacing material) purposes.
indeed, gradual submergence of the reefs could not be ascribed to
sea level rise!

Proceeding further back in time, we move from the period of
highest harvest in the latter half of the 19th century to the period
of Colonial settlement, when inlertidal ovster reefs were abundant

and notable features of the bay. It is this presettlement era that
illustrates the most defensible target for restoration goals.
Throughout the preceding discussion there has been frequent men-
tion of fishery harvest, but purposely not of biological production.
In a well-managed, economically exploited resource, the harvest
does not exceed production. Given the lack of assessment and
productivity data, a definitive temporal analysis of the post-
Colonial settlement harvest in excess of productivity is not pos-
sible. However, we do know that the cumulative result has been
the removal in less than 400 years of complex reefs that developed
over a 10.000-year period, beginning with the inundation of the
bay in the current postglacial warming period.

Acceptance of the tenet that cumulative harvest was clearly in
excess of cumulative production places the question of restoration
for fishery harvest in a new light. The projection of a restored
resource being able to sustain a fishery at the historical harvest
level is unrealistic because: ( 1 ) harvest probably exceeded biologi-
cal production for inuch of the recorded history of exploitation;
and (1) maximum production, a desired end for fishery support,
occurs at approximately half the maximum (virgin unexploited)
biomass (as defined in Applegate et al. 1998. Restrepo et al. 1998)
and. thus, can only be achieved with disruption of the virgin com-
plex community structure. Indeed, the direct harvest economic
value of a fishery based on a restored resource will not reach
historical levels unless there is an accompanying goal of long-term
community development that is self-sustaining in the absence of
restoration effort. It is. therefore, unreasonable to consider a res-
toration effort for oyster fishery support purposes alone. This con-
clusion prompts the question. "If the goal is not just the fishery
harvest, what end point should restoration goals seek to achieve?"
I argue that oyster restoration should be viewed as the re-
establishment of (one of several) cornerstones in an ecosystem.

DEFINING THE PROBLEM, PART 2: A CORNERSTONE IN
THE ECOSYSTEM

The reason oysters are the focus of shellfish restoration in
Chesapeake Bay is their value as a cornerstone species in the bay.
Oysters are a major benthic-pelagic coupler; one that supports a
diverse food web in higher trophic levels and. as an added bonus,
is the basis of a commercial fishery of secondary importance to the
food web structure.

How big is the baywide problem? Enormous. The Chesapeake
Bay is 298 km long ( 185 miles), has a surface area of 8.484 km"
(3277 .sq. miles), and has a volume of 71.5 x 10'' m' (Cronin and
Pritchard 1975). Within this context the biology of the oyster
deserves attention. Oysters are gregarious and long-lived (there-
fore, large) in a pristine environment. Spawning efficiency is
maximized by simultaneous gamete release in these dense aggre-
gations (see studies by Levitan 1991. Levitan et al. 1991. 1992 for
sessile benthic organisms, sea mchins. in spawning and fertili/a-
tion efficiency). Individual fecundity increases with size (Thomp-
son et al. 1996 using data from Cox and Mann 1992), so dense
aggregations of large animals should be a goal of restoration,
because they help prcnide long-term stability through provision of
larval forms. Dense aggregations grow in the third dimension (up)
in the presence of adequate food. Multigeneration aggregate settle-
ment creates three-dimensional structure as older animals die but
remain as substrate for new recruits to the benthos. Three-
dimensional structure would, therefore, seem to be a further de-
I'ensible uoal of restoralise efforts.

Restoration of Oyster Reefs

337

The trophic role of oysters in the Chesapeake Bay and other
similar systems has been well studied: therefore, quantitative ar-
guments can be proffered: ( 1 ) to support the level of restoration:
and (2) to estimate the trophic impact on both nutrient reduction
through grazing and higher trophic production through support of
enhanced food chains (see Newell 1988. Baird and Ulanowicz
1989, Ulanowicz and Tuttle 1992, for examples). An examination
of these contributions underscores the need to consider oyster res-
toration not as a singular goal but as a component of a holistic
approach to watershed management that includes land use prac-
tices and the subsequent impact of riverine input to water column
processes throughout the bay and its subestuaries. Water column
processes are then to be considered in the context of local habitat
and benthos (including oyster reefs), progressing to include resi-
dent and seasonally migratory transient macrofauna. The complex-
ity and size of the problem has. fortunately, received much atten-
tion. The NOAA and EPA Chesapeake Bay Program databases in
addition to those of the U.S. Geological Survey (most of these are
now available through the World Wide Web) are replete with
useful information to guide the restoration plan. To reiterate, a
restoration process must be placed in a time context. The changes
in the original watershed from forested to a mix of urban, agricul-
ture, and forest occurred over the period from Colonial times to the
present: the projected population growth through 2020 within the
watershed, especially the coastal regions of Maryland and Vir-
ginia, exceeds projected national growth rates. Increases in the
human population within the watershed from the current 14 mil-
lion to 16-18 million are within reason in this time period. At-
tempts to plan and control growth within the watershed are and
will continue to be both politically charged and difficult to resolve.
Unfortunately, historical precedent illustrates a general lack of
resolve in this country to limit growth and exploitation effectively.
Therefore, land use and runoff issues associated with these pro-
jections will raise discussion of freshwater diversion, use. re-use,
discharge, groundwater use and contamination, and saltwater in-
trusion. Every item on this list directly affects nutrient and sedi-
ment inputs to the bay and will tax the capabilities of recent ame-
lioration strategies to reduce negative effects.

The biological consequences of increased inputs of nutrient and
particulate material to the bay watershed are well understood. Nu-
trients stimulate productivity in excess of the grazing capacity of
the resident filter feeders, notably the benthic filter feeders. Sedi-
ment loads that inhibit the filtering process exacerbate the situa-
tion. With limited grazing, eutrophication is inevitable. Sediment
loads similariy inhibit extension of submerged aquatic vegetation
(SAV) by limiting light penetration of the water column. The
complex nature of the restoration problem is well illustrated by
consideration of a two-species interaction: that of oysters with
SAV. On a riverwide scale the presence of multiple reef systems
with vertical relief in otherwise open bodies of water, like much of
the Chesapeake Bay. reduces fetch and. hence, wind-driven resus-
pension of particulate material in the water column. The presence
of fringing reefs reduces sediment input from shoreline erosion. At
a smaller scale, filter feeding by oysters reduces water column
loads of sediment and plankton: thereby, increasing light penetra-
tion and increasing SAV growth. Bottom stabilization by SAV
increases water quality: thereby, encouraging a positive feedback
loop to oyster growth. There is nonlinearity in this feedback: when
the suspended sediment load increases above a certain level. SAV
growth essentially ceases, and the response of the oyster filter-
feeding rate to sediment load approximates a parabola. Thus, al-

though publicly stated goals of 40% nutrient reduction in nutrient
input are laudable, they must be accompanied by a critical reduc-
tion in sediment load to allow SAV growth and the oyster-SAV
positive feedback interaction to develop. This multifaceted prob-
lem of both elevated nutrients and sediments is notable in areas
that once supported abundant oyster populations — the James,
York, and Rappahannock rivers, and Pocomoke-Tangier Sound —
and are given critical status on current Chesapeake Bay Program
and EPA World Wide Web sites. Proceeding above a "simple"
two-species interaction. Lenihan and Peterson (1998) underscore
the sensitivity of the multispecies interaction on reefs to multiple
environmental factors.

The enormousness of the potential restoration effort and its
primary goal is easily recognized. Is there a logical recovery pro-
tocol? I argue, yes. The unique aspects of the biology of the oy.ster
in the bay that must be exploited to facilitate restoration are
known: high density and a three-dimensional structure in a loca-
tion where filter feeding will not be overwhelmed initially by local
water quality conditions. In Virginia, these aspects have been used
to guide the choice of location for early restoration efforts. A
critical issue from both the biological and political view is the
choice of sites. Sites must be selected such that the impact of the
effort is visible in a short (months to a small number of year) time
frame: that is. the signal from the restoration effort must be "vis-
ible" above the natural variability or "noise" in the target system.
Thus, there is a need to match scale of effort with goals. Attempt-
ing wholesale restoration of large river systems at the outset is
clearly not viable for either cost or biological considerations, but
there are many smaller parts of candidate systems that are attrac-
tive. Using such resources as the Baylor ground maps (1894).
natural reef "footprints" have been identified that can be cleaned of
remaining oysters and used as a base to build three-dimensional
structure.

Under the guidance of the Shellfish Replenishment Program at
the Virginia Marine Resources Commission, a reef-based restora-
tion effort was initiated in the Piankatank River in 1993 with
construction of a single reef at Palace Bar. No broodstock addition
was effected at the site. Construction is described in Bartol and
Mann (1997). Since its construction, this site has been studied
intensively in terms of oyster recruitment and growth (Bartol and
Mann 1997. in press. Mann and Wesson unpublished data), disease
progression in recruited oysters (Volety et al. 2000. this issue), and
development of associated fish and benthic communities (Harding
1999, Harding and Mann 1999, 2000). A contrasting approach was
employed in the Great Wicomico River in 1996 (Southworth and
Mann 1998). The success of this effort wanants description as a
model for restoration programs. The Great Wicomico River is a
small, trap-type estuary on the western shore of the Chesapeake
Bay that once supported substantial oyster populations. The com-
bined effects of Tropical Storm Agnes in 1972 and subsequent
disease mortalities related to Pevkinsus mavinus and Haplospo-
ridium nelsoni essentially eliminated these populations. Oyster
broodstock enhancement was initiated in June 1996 by the con-
struction of a three-dimensional intertidal reef with oyster shell,
followed by "seeding" of the reef in December 1996 with high
densities of large oysters from disease-challenged populations in
Pocomoke and Tangier Sound. (In these donor locations, the extant
oyster population density is too low to effect reasonable probabil-
ity of fertilization success and subsequent recruitment.) Calcula-
tions of estimated fecundity of the resultant reef population sug-
gested that oyster egg production from this source were within an

338

Mann

order of magnitude of total egg production in the Great Wicomico
River before Tropical Storm Agnes. Field studies in 1997 indicated
spawning by reef oysters from July through September; whereas,
plankton tows recorded oyster larval concentrations as high as
37,362 ± 4,380 larvae/m' (on June 23)! Such values are orders of
magnitude higher than those typically recorded for Virginia sub-
estuaries of Chesapeake Bay in the past three decades and strongly
endorse a premise of aggregating large oysters to increase fertil-
ization efficiency. Drifter studies suggest strong local retention of
larvae, a suggestion reinforced by marked increases in local oyster
spalfall on both shell string collectors and bottom substrate in
comparison to years before 1997. Although disease was evident
in the population — Perkiusus prevalence increased from 32% in
June to 100% in July, and intensity increased from June to Sep-
tember— the effort demonstrated that choosing locations where
local circulation promotes larval retention combined with reef con-
struction and broodstock enhancement may provide an accelerated
method for oyster population restoration. Following the above ob-
servation in the Great Wicomico, two reef sites in the Piankatank
have been added as part of the broodstock enhancement program
using large oysters collected from high salinity regions of the bay
where disease pressure remains high. Similar efforts are underway
in two small tributaries of the Potomac River (the Coan and Yeo-
comico), the Elizabeth River, Pungoteague Creek on the bay side
of the Eastern Shore of Virginia, and Lynnhaven Bay on the south
shore of the Chesapeake Bay mouth. In addition, reefs of various
substrate types have been constructed at Fisherman's Island at the
southern tip of the Eastern Shore of Virginia and are the site of
continuing intense study by Luckenbach and collaborators based at
the Virginia Institute of Marine Science Wachapreague Labora-
tory.

Although there is a clear generic component to these individual
efforts of small reefs in small systems, each site is unique along a
salinity cline within Virginia waters. They represent a mosaic of
habitat types with differing environmental values in both biology
and physical structure. Such unique aspects of each reef system are
examined further by Breitburg et al. (2000, this volume). Provision
of complex physical habitat structure provides opportunity for re-
cruitment by species other than oysters as demonstrated by Breit-
burg et al. (1995), Breitburg (1999). Harding and Mann (1999,
2000), Nestlerode and Luckenbach (in press), and Coen and Luck-
enbach (in press). To date, the progression of increasing species
richness and complexity in relation to presence or absence of
"seeded" oyster broodstock has not been investigated, although it
is reasonable to suggest that the presence of the latter accelerates
development of the multitrophic community on and around the
reefs.

The problem tor proponents of reef restoration as a central
mechanism lo restt)re oyster resources is not so much the demon-
stration of biological recruitment in the field as the social and
political recruitment of citizens to support such efforts on a long-
term basis. Demonstration of "success"' in field programs, such as
the recruitment event associated with reef construction and brood-
stock "seeding" in the Great Wicomico River in 1997, provide a
vehicle to educate the public and foster vested interest groups. The
target audience here is broad, as demonstrated by success lo date
in developing parlncrships, which is illustraled by Ihc following
examples. Established environmental nonprofit groups, such as the
Chesapeake Bay Foundation, use their considerable resources and
infrastructure to support reef efforts on a regional basis. In stark
contrast to ihe "iioi in mv back vard" menlalilv associaleil wilh

environmentally adverse programs, reefs are environmentally at-
tractive structures that are desired "in my back yard." Conse-
quently, local citizens groups sponsor reefs in their own "back
yards" and school groups grow oysters to seed local reefs as part
of the restoration effort. Currently lacking from this team is strong
endorsement of both the commercial and recreational fishing com-
munities in the bay. This is surprising, given the obvious long-term
advantage to both, but probably reflects the immediacy of benefit
that is required to attract these groups. Education is the avenue to
forge this relationship, as demonstrated by the active support en-
joyed by SAV restoration efforts from the fishing community. An
integral part of this education must be the demonstration of the
economic value of an ecological asset not just in terms of the
commercial and recreational end product. It must be evident that
that there is a cumulative positive impact of restored ecosystems in
nutrient processing that is preferable to the current "single-
payment option" exercised by some point-source nutrient abate-
ment policies. The challenge remains to enjoin a broad citizen base
in supporting ecological restoration on a broad base, understanding
that they have vested interest as long-term investors in the water-
shed in which they communally reside with the Chesapeake Bay
flora and fauna.

ACKNOWLEDGMENTS

This manuscript was presented as a plenary presentation com-
mentary at the Second International Conference on Shellfish Res-
toration, convened at Hilton Head. South Carolina, on November
19-21, 1998. This work of the author and collaborators described
in this article was supported in part by the EPA Chesapeake Bay
Program; the Commonwealth of Virginia, Department of Environ-
mental Quality. Chesapeake Bay and Coastal Programs, and op-
erating funds of the Virginia Institute of Marine Science. Partial
support to the author during the period of manuscript preparation
was provided by National Science Foundation Grant OCE-
9810624. Support to present the manuscript al the Second Inter-
national Conference on Shellfish Restoration was provided by the
National Oceanic and Atmospheric Administration. These sources
of support are gratefully acknowledged. I thank my colleagues,
James Wesson, Mark L. Luckenbach, Ian Bartol, Juliana Harding,
Melissa Southworth, Janet Nestlerode, Francis O'Beirn, and Wil-
liam J. Hargis. Jr., for many interesting discussions on reef biology
and the Chesapeake Bay. This contribution is dedicated to William
J. Hargis, Jr., who during both his years as director of the Virginia
Institute of Marine Science and since retirement has argued tire-
lessly for oyster restoration in the Chesapeake Bay. Contribution
nmnber 231 1 from the Virginia Institute of Marine Science.

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Joiinml of Shellfish Research. Vol. 19, No. 1, 341-347, 2000.

PROGRESSION OF DISEASES CAUSED BY THE OYSTER PARASITES, PERKINSUS MARINVS
AND HAPLOSPORIDIUM NELSONI, IN CRASSOSTREA VIRGINICA ON CONSTRUCTED

INTERTIDAL REEFS

ASWANI K. VOLETY' * FRANK O. PERKINS,"
ROGER MANN,' AND PAUL R. HERSHBERG^

^National Research Council

U.S. Environmental Protection Agency

Gulf Ecology Division

I Sabine Island Drive

Gulf Breeze. Florida 32561
^University of Hawaii at Manoa

105 Bachmann Hall

2444 Dole Street

Honolulu, Hawaii 96822
' School of Marine Science

Virginia Institute of Marine Science

College of Willicnn and Mary

P. O. Bo.x 1346

Gloucester Point. Virginia 23062
'^Meteorology Department

Florida State University

Tallahassee, Florida 32306

ABSTRACT The progression of diseases caused by the oyster parasites Perkinsus marinus and Haplosporidhim nelson! were
evaluated hy periodic sampling (May 1994-December 1995) of eastern oysters Crassostrea virginica on an artificial reef located in
the Piankatank River. Virginia. The infections observed were recorded as a function of (1) prevalence and intensity. (2) oyster size
and age; and (3) depth below mean low water at which the host oyster was found on the reef Only a very small number of oysters
were infected with the two species of pathogens on the oyster reef during the first 1 1 months of life. In the second year of oyster life,
epizootiological patterns of disease development followed temperature and salinity trends. Oysters at residence depths £45 cm below
mean low water exhibited significantly (P < 0 .0001 ) lower prevalence and intensity of infections than oysters at depths >90 cm. In
contrast, oysters at residence depths £90 cm had significantly higher growth rates (P < 0.05) than those at S45 cm. However, size
differences were not significant {P > 0.05) at the end of the study. Results from this study may be used in managing oyster fisheries
on natural or artificial reefs.

KEY WORDS: Crassosrrea virginica. Perkinsus marinus. Haplosporidhim nelsoni. artificial reefs, disease progression, growth

INTRODUCTION lack of three-dimensional complexity observed in natural intertidal

communities. Little is known about the colonization and ecology
of C. virginica on inteilidal reefs. In addition, the advantages of
oyster settlement on constnjcted reefs are not well understood.
Therefore, this study was conducted to determine: ( 1 ) whether
residence depth influenced the extent or intensity of disease infec-
tion; and (2) the size and age at which oysters became infected and
the depths that resuhed in significant infections. Observations from
the present study are of interest to individuals responsible for
constructing artificial reefs and individuals who must determine
when to harvest oysters to avoid excess losses.

Eastern oysters Crassostrea virginica were an economic and
ecological resource in the Chesapeake Bay until the early 1900s
(Hargis and Haven 1999). Years of overharvesting, diseases
caused by the pathogens Perkinsus marinus and Haplosporidiwn
nelsoni, environmental degradation, and poor resource manage-
ment have led to a dramatic decline in oyster populations in the
Chesapeake Bay (Andrews 1988. Haskin and Andrews 1988). To-
day, Virginia's oyster population is less than 19!- of what it was just
35 years ago. (Wesson et al. 1999). Various efforts have been
initiated to rejuvenate dwindling local oyster populations. These
include spreading of hatchery-reared juvenile oysters on natural METHODS AND MATERIALS

oyster beds in estuaries, spreading oyster shell in an attempt to
increase hard substrate for settlement of oyster larvae, and con- Perkinsus marinus Infections

struction of artificial reefs. Rejuvenation efforts, such as spreading ^ „ • • u

.... r , ,. 1 J . ■ u .. f 1 1 Oysters were assayed for the presence ot P. marinus usmg the

thin layers of shell over coastal and estuarine bottom lor larval „ , „ ,, ,. , . „ ,„^, ,, ■

, , . .,. . . T-u- u J ■ _.. .u Ray s fluid thioglycollate medium technique (Ray 1954). Samples

attachment have had limited success. This may be due in part to the ,•'. . , , ,. ,, , ■

ot gill and digestive gland were incubated in the medium. Perkins

(unpublished data) determined that these organs can more fre-

*Current address: Florida Gulf Coast University. 1 0501 FGCU Blvd. South, quently detect the presence of the parasite when infections are very
Fort Myers, FL 33965; email: avolety@fgcu.edu light or light than when mantle or rectal samples are used. The

341

342

VOLETY ET AL.

intensity of infections was recorded using a modification of the
Mackin scale (Mackin 1962) in whicii 0 = no infection, 1 = very
light, 2 = light. 3 = light-moderate, 4 = moderate, 5 = mod-
erate-heavy, and 6 = heavy.

Haplosporidium nelsoni Infections

Haplosporidium nelsoni was detected using histological, par-
affin-embedded sections stained in hematoxylin and eosin. The
scale of Burreson et al. (1988) was employed to record intensities
of infections where 0 = no infection, 1 = cells were rare, 2 =
fewer than two cells per field of view (40x objective), 3 = two-
five cells per field of view, and 4 = more than five cells per field
of view.

Oyster Sampling

From May 5, 1994 to December 14, 1995, the progression of
diseases caused by the oyster parasites P. inariniis and H. nelsoni
were evaluated by periodic sampling of oysters that had naturally
set on the artificial reef located in the Piankatank River, Virginia,
in August 1993. The reef was constructed by the Virginia Marine
Resource Commission using aged oyster shells. Details of reef
construction are described elsewhere (Bartol and Mann 1997).
Sampling of oysters was conducted once every 2 to 4 weeks during
the study period. Oysters were obtained by hand or by using oyster
tongs, depending on the depth. Six samples of 25 oysters each
were obtained for each sample time at two locations on the reef.
The base of the reef was 2-3 m below mean low water. Prevalence
and intensity (weighted prevalence) of P. marinus, and H. nelsoni
infections, oyster size and age, and depth below mean low water at
which the host oyster was found on the reef were measured. The
total number of reef oysters sampled was 3,908. With respect to
depth, the data were analyzed according to the oyster's residence
depth on the reef: £45 cm and s90 cm mean low water. The
observations are expressed in terms of number of weeks after
setting. Most of the set in 1993 occurred from August 5 to August
12. To facilitate the handling of the data, August 12 was selected
as the date of set.

The progression of infections in the reef-set oysters were com-
pared with the progression through a population of adult oysters.
Uninfected, adult oysters (350) were obtained from the upper
James River seed beds (Horsehead rock), a region known to have
low P. marinus and H. nelsoni infections in oysters (Burreson and
Ragone-Calvo 1996). These adult oysters were placed in plastic
mesh bags and then were placed on the Piankatank River reef near
the sample sites for the reef oysters: placement was June 16. 1994.
the time of the third sampling of the reef oysters. The depth of
placement was about midway between the top and bottom of the
reef (ca. 100 cm below mean low water). At the time iif placement,
a sample of 25 oysters was analyzed for the presence of the two
parasites, using techniques described previously. To confirm that
the parasite detection methodology was being properly applied and
to check for patchiness in distribution of the parasites, 350 James
River oysters from the same population used on the Piankatank
River reef were placed in plastic mesh bags in the York River
behind the Virginia Institute of Marine Science (VIMS), an area in
which both diseases are commonly present al high levels. Treat-
ment of the 350 oysters held at VIMS was the same as described
for the 350 adult oysters deployed in the Piankatank River. Each
batch of 350 oysters was sampled (25 oysters per sampling time)
simultaneously with the reef oysters until none remained. In ihc

following spring (April 14, 1995), another 350 oysters from the
same James River site were placed on the reef and 350 at VIMS as
in the previous year and sampled until none remained.

Statistical Analyses

The effects of depth and sampling time (age) of oyster on
disease susceptibility (prevalence and intensity of infection) to P.
marinus and H. nelsoni were examined using logistic regression
analysis (Agresti 1990). The differences in growth of oysters
sampled at <45 cm and >90 cm depths during different sampling
times was assessed using a two-way analysis of variance
(ANOVA)

RESULTS

Temperature and Salinity

Temperatures and salinities during the study period showed
typical seasonal patterns, higher temperatures during summer
months and lower during winter months (Fig. 1 ). Salinity remained
fairly constant during the study period. Temperature and salinity
ranges during the study period were 2-30 °C and 10-20 ppt. The
conversion between oyster age and .sampling dates is presented in
Table 1.

Perkinsus marinus Infections

No P. marinus or H. nelsoni cells were found in the 25 adult
oysters sampled before the deployment of the oysters at Pianka-
tank River and VIMS. P. marinus prevalence (Fig. 2a) and inten-
sity (Fig. 2b) in oysters set on the Piankatank reef indicate that
infections did not appear until 14 weeks into the study when the
oysters were 1 year old (Table 1). For the next 44 weeks or until
the oysters became 1 year and 10 months old, the number of
infected oysters ranged between 15 and 35'/f: in the ensuing 2
months, the prevalence ro.se rapidly to 100%. Oysters then exhib-
ited high prevalence (nearly 1009^) until the end of the study, when
the oysters were almost 2.5 years old. The infection intensities
during the plateau phase remained mostly below very light until
the end of the plateau (I year. 10 months old), rose rapidly to
moderate to moderate-heavy at 2+ years old, followed by a decline
to light and light-moderate at the end of the study.

Perkinsus nuirinus prevalence was significantly higher (P <

Temperature
Salinity

30 40 50 60 70 80 90 100 110 120
Oyster age (weeks)

Figure 1. Temperatures and salinities at the reef site during the study
as a functiiin (if oyster age. The conversion of oyster age to correspond
to sampling dates is presented in Talile t. Discontinuities in the curves
denote lack of data.

Dermo and MSX in Oysters on Artificial Reefs

343

TABLE 1.

Time scale for sampling times used in the study. The oyster ages are

estimated assuming a setting time of August 12, 1993.

NA = Not available.

a 100

Sampling

Oyster

Time

Sampling

Age

Elapsed

Temperature

Salinity

Date

(weeks)

(weeks)

(X)

(ppt)

May 5. 1994

38

0

NA

NA

May 26

41

3

20

10

June 16

44

6

27.8

12

June 30

46

8

26

17

July 15

48

10

29.8

16

July 28

50

12

27

16

August 12

52

14

27.5

16

August 26

54

16

26

16

September 8

56

18

22.8

16

September 23

58

20

21

16

October 5

60

22

19

NA

October 20

62

24

NA

NA

November 1 1

65

27

NA

NA

December 8

69

31

11

16

January 12, 1995

74

36

7

14

February 10

78

40

2

16

March 13

83

45

8

12

April 14

87

49

14

14

May 11

91

53

19

14

June 15

96

58

23

16

June 30

98

60

27.8

18

July 13

100

62

29

19

July 31

103

65

31

19

August 24

106

68

27.2

19

September 18

110

72

23.5

20

October 24

115

77

18

20

December 14

122

84

NA

NA

0.0001) in oysters collected from depths s90 cm compared to
those from s45 cm (Fig. 2a). Prevalence significantly increased (P
< 0.0001 ) in oysters from all depths with increasing age of oysters,
indicating that continued exposure to P. marinus or increasing age
of oysters results in increased infection. Similar results were ob-
served when P. marinus infection was expressed as weighted
prevalence. Although the difference in infection intensity was not
as great as the prevalence, it was significantly higher at the greater
depths {P < 0.01 ) and significantly increased in oysters from both
depths with age (P < 0.0001).

Perkinsiis marinus infection prevalence was the same in adult
oysters held in plastic mesh bags at the York River and at the
Piankatank River reef during 1994. In 1995, infection was ex-
pressed earlier at the Piankatank River and reached \009c 15
weeks before those held in the York River (Fig. 3a,b).

Haplosporidium nelsoni Infections

Prevalence of W. nelsoni was markedly different from that of P.
marinus in reef oysters (Fig. 4a). With the exception of one lightly
infected, 50- week-old oyster, the onset of H. nelsoni infections did
not occur until the oysters were over 1 .5 years old, as opposed to
the appearance of P. marinus in 1-year-old oysters. Thereafter, the
infection prevalence of H. nelsoni rose rapidly, reaching a maxi-
mum of 45% when the oysters were 21 months old. The infections

<45cm
>90cm

<45cm
>90cm

140

Oyster age (weeks)

Figure 2. Prevalences (a) and intensities (weighted incidences) (b) of
Perkinsus marinus infections in Piankatank River reef oysters that set
in August 1993 are presented as a function of oyster age and depth of
residence below mean low water (<4S cm and >90 cm).

then declined precipitously to almost 0% when the oysters were
over 2 years old, followed by a slight increase, which remained
below 10% for the final 4 months of the study. The intensities of
infections peaked at the age of nearly 2 years, which was 1 month
before the prevalence peak was reached, and declined almost as
rapidly as did the prevalence (Fig. 4b).

Similar to P. marinus infections, oysters collected from >90
cm depth had a significantly higher prevalence and intensity of H.
nelsoni infections compared to those from £45 cm depth (P <
0.0001) (Fig. 4a,b). In addition, H. nelsoni prevalence and inten-
sity increased with increasing oyster age (P < 0.0001).

Haplosporidium nelsoni infections (Fig. 5a,b) were nearly non-
existent in the Piankatank River reef oysters during 1994; whereas,
in the York River stock, infections were above a prevalence of
60% during the summer and fall of 1994.

Growth and Size

The sampling time and the residence depth of oysters signifi-
cantly influenced the growth of oysters (P < O.OOI) (Fig 6). Oys-
ters at both depths grew with increasing age (P < 0.001) and
sampling time (P 0.001). Oysters at depths a90 cm had signifi-
cantly greater growth than oysters at depths <45 cm. Although the
differences in size were pronounced during age 56-100 weeks,
they were less pronounced during oyster ages 103-122 weeks, and
insignificant (P > 0.05) at the end of the .study. The rate of oyster
growth, as estimated from size measurements, decreased 4 weeks

344

VOLETY ET AL.

100

PR
YR

10 20 30 40 50 60 70 80 90

PR
YR

0 10 20 30 40 50 60 70 80 90

Sampling time (weeks)

Figure 3. Prevalence (a) and intensity Iweiglited incidences) (b)

of

Perkinsus marimis for adult oysters imported from the upper James
River and placed on the Piankatank River reef and in the York River
behind the Virginia Institute of Marine Science. Oysters (350 at each
site) Here twice placed at the sites and assayed until the populations
were depleted by sampling and natural mortalities. The disease organ-
ism data are expressed as a function of site and sampling time in the
studv.

before the first P. inarinus infections (10 weeks and 14 weeks into
the study) (Fig. 6).

DISCUSSION

Intei^jretation of epizootiological data such as those generated
in this study is confounded by many factors that dictate disease
prevalence and intensity. These include temperature, salinity, wa-
ter quality, density of oysters, patterns of water movement, oyster
age and/or size, genetic strains, physiological condition, food
availability (density and species composition of planktonic food
organisms present), and numbers and levels of other parasitic spe-
cies causing stress on the oysters. A further complication is the fact
that the reservoir of H. iielsoni infective cells is unknown, and
transmission of infections is not from oyster-to-oyster as with P.
marinus. Despite these complicating factors, patterns of disease
progression of both the diseases and oyster growth at different
residence depths were apparent. The residence depth of the oysters
relative to mean low water is of considerable interest, because the
premise behind construction of artificial reefs is that the survival of
oysters in the presence of P. marinus and H. nelsoni will be en-
hanced if they are grown in the more natural environment of an
oyster shell reef off the bottom of ihe estuary. In fact, this study

a 60

,2 40

<45cm
>90cm

30 40 50 60 70 80 90 100 110 120

b 2

s 1

A

1^ ■T**

< 45 cm
> 90 cm

30 40 50 60 70 80 90 100 110 120
Oyster age (weeks)

Figure 4. Prevalences (a) and intensities (weighted incidences) (b) of
Haplusporidium nelsoni infections in Piankatank River reef oysters
that set in August 1993 as a function of oyster age and depth of resi-
dence below mean low water (<45 cm and >90 cm).

clearly indicates that residence depth of oysters significantly in-
fluences prevalence and intensity of P. marinus and H. nelsoni
infections.

Oysters that are growing at s43 cm depth can be expected to
have lower prevalences and intensities of infections of both patho-
gens compared to those living at >90 cm depth (Figs. 2a,b. and
4a,b). The prevalence and intensity of P. marinus in reef oysters
from the current study, while agreeing with those of Mackin
(1962). differ from studies by Quick and Mackin (1971) in the
Atlantic and Gulf of Mexico coasts of Florida. Weighted inci-
dences (intensities) in their study showed a decrease with increas-
ing depth and no effect of depth on prevalences from intertidal to
3 m below mean low water. Similarly, Burrell et al. ( 1984) found
higher prevalences and intensities of P. marinus in intertidal oys-
ters than in subtidal oysters. Mackin (1962) speculated that lower
infection prevalences and intensities in intertidal oysters may be
because these oysters are not exposed to as many infective cells as
subtidal oysters by virtue of the increased amount of time they are
closed and not feeding.

Growth of oysters at both the depths {^45 cm and a9() cm)
decreased from age 74 weeks, and coincided with increased /'.
marinus incidence in oysters. The decrease in growth of oysters
upon acquisition of P. marinus infections support the findings of
Paynter and Burreson (1991) who observed a decrease in the
growth rate of juvenile and adult oysters immediately after or just
before infection. Haplosporidium nelsoni did not seem for more
than 7 months alter the change in arowth rate, and it is concluded

Dermo and MSX in Oysters on Artificial Reefs

345

a 100

80 -

ft-

1 \

4

i

1 k

■

1

bU -

1

\:.

40 -

1

\\l \

' r ^

20 -
0 -

»•*

1 * *^ 1 — *; i

70

10 20 30 40 50 60 70 80 90

f 3

1 -

PR
YR

10

20 30 40 50 60 70
Sampling time (weeks)

Figure 5. Prevalence (a) and intensity (weighted incidences) (b) of
Haplosporidium nelsoni for adult oysters imported from the upper
James River and placed on the Piankatank River reef and in the Vork
River behind the Virginia Institute of Marine Science. Oysters (350 at
each site) were twice placed at the sites and assayed until the popula-
tions were depleted by sampling and natural mortalities. The disease
organism data are expressed as a function of site and sampling time in
the study.

that P. marinus was responsible for the decreased growth rate. The
decline in oyster sizes after the age of 2 years (65 weeks into the
study) is believed to be atributable to death of the larger oysters
from infections of the two pathogens.

The higher growth of oysters residing at depths >90 cm than
those at S45 cm is surprising. Given the higher infection preva-
lence and intensities of both P. marinus and H. nelsoni in oysters
at depths >90 cm. we would expect that these oysters would grow
less because of disease pressure. The biological, chemical, and
physical processes associated with the bottom waters may influ-
ence the physiological and defense responses of organisms inhab-
iting these areas. Organic matter near the bottom of the reef close
to the sediment has been speculated (Dahlback and Gunnarsson
1981) to have contributed to the increased growth in oysters from
>90 cm. However, at the end of the study, the differences in sizes
of oysters from the two depths were not significant.

The present dataset is unique, because it is the first time a
population of naturally set oysters of known age has been assayed
in situ for progression of infections by P. marinus and H. nelsoni
over an extended period of time. Other epizootiological studies
have involved placing naturally set or hatchery set oysters of
known age in containers in an endemic area or placing adult oys-
ters of unknown ages from nonendemic or marginally endemic

20

10

PR

YR 60-

■

,/'

_ 50 -

E

E

Jr-

s^°-

f

30 -

/

< 45 cm
> 90 cm

20

40

60 80 100

Oyster age (weeks)

120

140

Figure 6. Piankatank River reef oysters that set in August 1993 and
were sampled for the disease studies. Sizes are presented as a function
of oyster ages and depth of residence below mean low water (<45 cm
and >90 cm).

areas into containers in an endemic area (Ray 1953, 1954. Mackin
1962, Paynter and Burreson 1991). Placement in containers pro-
vides a greater degree of experimental control, but artificial den-
sities of oysters can lead to results different from those in naturally
set populations, where distances vary from oyster to oyster.

The prevalence and intensities of infections of P. marinus in
reef oysters generally followed the patterns dictated by tempera-
ture and salinity trends observed in earlier studies (e.g.. Soniat
1985, Andrews 1988. Burreson 1991. Burreson and Ragone-Calvo
1996, Ray 1996). P. marinus infections in oysters rose in the
spring, peaking in October and November, and declined in the
winter months into the spring. Intensities of infections were more
nearly reflective of previous reports in that the peaks for the two
depths were in October and November and the minima in May of
the following year (Fig. 2b). Therefore, assuming that temperature
and salinity values approximate those of the study period and
given the infection intensities and levels, oyster mortalities from P.
marinus can be expected to begin 13 months after setting, rising
most significantly 22 months after setting.

Considering the fact that the salinity values recorded during the
first year after setting did not go below 10 ppt, and most of the time
were > 16 ppt, it is reasonable to assume that infections from these
two diseases did not occur before May 5. 1994, when sampling
began. This assumption is based on other studies that observed
when a population becomes infected, the infections do not disap-
pear (or decline) unless the salinity decreases below 10 ppt for an
extended period (Andrews and Hewatt 1957, Ragone and Burreson
1993. Burreson and Ragone Calvo 1996).

Previous studies have noted that oysters are refractory to ac-
quiring infections in the first year of life (Ray 1953, 1954) and
become increasingly more susceptible into the second year, with
significant prevalence, incidence, and mortality being observed
then (Andrews and Hewitt 1957). That pattern was observed in the
present study (Figs. 2a,b and 4a,b). As mentioned above, the com-
plicating factor was the low level of infection pressure from H.
nelsoni in the first year of life at the reef. P. marinus infections in
oysters are dose-dependent (Mackin 1962. Chu and Volety 1997).
and small oysters probably filter less volumes of water to acquire
enough infective cells of the parasite to initiate an infection (Bur-
reson 1991, Burreson and Ragone Calvo 1996). Results from the
current study indicate that in the first 1 1 months of life, only a very
small number of oysters on the oyster reef become infected with

346

VOLETY ET AL.

the two species of pathogens. The question that cannot be an-
swered is whether the primary factor in encouraging or permitting
a rapid increase in prevalence was temperature, oyster age, or
salinity. The best answer probably is that all three played interac-
tive roles.

Data for adult oysters imported from the upper James River
seed beds, which represent a disease-susceptible population, con-
firmed that the two pathogens were present in the study area and
in the neighboring area of the lower York River (Figs. 3a. b and
5a,b). They were used primarily to indicate presence or absence of
H. nelsimi. because it is known to fluctuate greatly, some years
being nearly absent from the lower York River region. (Andrews
1988, Haskin and Andrews 1988).

It is interesting to note that P. marinus infection prevalence was
the same in oysters held in the York River and at the Piankatank
River reef during 1994. but in 1995 was expressed earlier in the
Piankatank River and prevalence reached 100%, 15 weeks before
those held in the York River. One would have expected that the
oysters held in the York River would have shown a higher preva-
lence, because salinities were about 5 ppt above those of the Pi-
ankatank River and thus would have presented more favorable
salinities for expression of P. marinus. On the other hand, in 1 994.
H. nelsoni infections were nearly nonexistent in the Piankatank
River reef oysters; whereas, in the York River stock, infections
were above a prevalence of 60% during the summer and fall of
1994 (Fig. 5a). These observations reaffirm the necessity of having
a stock of susceptible, adult oysters present in a study where ju-
venile and young oysters are being observed.

The reef oysters s2 years old can reasonably be compared to
the imported oysters in terms of response to the diseases. Although
P. marinus prevalences and intensities of infections were similar in
the two groups of oysters at the reef, H. nelsoni data suggest that
the reef-set oysters were more resistant to those infections. Im-
ported oysters at the reef reached a peak of 68% infection (Fig. 5a):
whereas, the reef oysters peaked at 36 and 45%, depending upon
the depth of residence (Fig. 4a). Likewise, the intensities of H.
nelsoni infections had a mean level of 2 in imported oysters as
opposed to 1 .3 in the reef oysters. Therefore, some advantage
seems to have been obtained for the reef-set oysters, if we can
neglect minor age differences.

The data for H. nelsoni was somewhat surprising, because only
one oyster was found to be infected in the first year of life (Fig.

4a). and the population did not otherwise begin to show infections
until the oysters were over 1.5 years old. It is possible that this lag
can be attributed to: ( I ) the oysters being young and. thus, less
susceptible, as has been reported from other studies; and (2) the
fact that even the susceptible, imported adult oysters did not ac-
quire very many infections at the Piankatank River reef (Fig. 5a)
in the first year of life. It was clear that H. nelsoni was present in
strength in the nearby York River (Fig. 5a) but not in the reef area,
thus illustrating the patchiness in distribution of the reservoir of
infective cells, at least in that part of the Chesapeake Bay. The
decline in prevalence and intensity of H. nelsoni in reef oysters
(Fig. 5a,b) was more precipitous that has been previously reported
using imported susceptible adult oysters (Haskin and Andrews
1988). In 1995, a different picture of//, nelsoni infection distri-
butions in the imported, adult oysters was observed (Figs. 5a.b).
The prevalences and intensities were quite similar at the two sta-
tions, with the infections appearing earlier at the York River sta-
tion and lasting longer in the population. Nine more weeks of data
were obtained from the York River stock, because the stock at the
reef was depleted by mortalities earlier, probably caused by P.
marinus infections (Fig. 5a,b).

Given the decreased susceptibility of oysters to P. marinus and
//. nelsoni at residence depth of s45 cm compared with oysters at
290 cm, it seems that piling of shells and constructing artificial
reefs is a better strategy for rejuvenating oyster stocks than spread-
ing thin layers of oyster shells on the bottom in estuarine and
coastal areas. Future studies should investigate the survival differ-
ences in oysters at different depths and the factors responsible for
differences in growth rates of oysters at different depths.

ACKNOWLEDGMENTS

This project was funded, in part, by the Virginia Council on the
Environment's Coastal Resources Management Program through
Grant NA370Z0360-01 (task 16) of the National Oceanic and At-
mospheric Administration, Office of Ocean and Coastal Resource
Management Act of 1972 as amended. We thank Kenneth Walker
and Ian Bartol for assistance in field operations, and Ms Juanita
Walker for her technical assistance. Insightful comments on the
previous drafts of this manuscript by Dr. William S. Fisher are
greatly appreciated. Contribution number 1062 of the U.S. Envi-
ronmental Protection Agency. Gulf Ecology Division.

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York.
Andrews. J. D. 1988. Epi/.ooliology of the disea.se caused by the oyster

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II. The fungus disease cau.sed by Dermncy.sliJiiiiii miiriniiin in oysters

of Chesapeake Bay. Ecol. Moiiof>i: 27:1-26.
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CriisMislreu iiiyiniiii on a constriiclcd intcrtidal reef Hull. Mar. Sci.

61:8X1-897
Burrell. V. G. Jr.. M. Y. Bodo & J. J. Manzi. 19X4. A comparison of

seasonal incidence and intensity of Perkinsus marinus between subliilal

and intertidal oyster populations in South Carolina. / World Maricui

Soc. 15:301-309.
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resistant stocks. / Shellfish Res. 10: 417-423.

Burreson, E. M. & L. M. Ragone Calvo. 1996. Epizootiology of ft'rt;;i.v».«
marinus in Chesapeake Bay, with emphasis on data since 1985. J.
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Burreson. E. M., E. Robinson & A. Villaba. 1988. A comparison of par-
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Res. 7:19-23.

Chu. F. L. E. & A. K. Volely. 1997. Disease processes of the parasite
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Dahlback. B. & L. A. H. Gunnarsson. 1981. Sedimentation and sulfate
reduction under a mussel culture. Mar. Biol. 63:269-275.

Hargis. W. J. & D. S. Haven. 1999. Chesapeake oyster reefs, then- Inipor-

Dermo and MSX in Oysters on Artificial Reefs

347

tance and destruction and guidelines for restoring them. pp. 27-28. In:
M. W. Luckenbach. R. Mann and J. A. Wesson (eds.). Oyster Reef
Habitat Restoration: A Synopsis and Synthesis of Approaches. Virginia
Institute of Marine Science Press. Gloucester Point. Virginia,

Haskin. H. H. & J. D. Andrews. 1988. Uncertainties and speculations about
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Mackin, J. G. 1962. Oyster disease caused by Dennocystulium mariimm
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Quick. J. A,. Jr, & J, G, Mackin. 1971, Oyster parasitism by Labyrinth-
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Ragone. L. M, & E, M, Burreson, 1993, Effect of salinity on infection
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Gloucester Point. Virginia,

Jounuil of Shellfish Research. Vol. 19, No. 1, 349-352. 2000.

PREVALENCE OF PERKINSUS SPP. IN CHESAPEAKE BAY SOFT-SHELL CLAMS, MYA
ARENARIA LINNAEUS, 1758 DURING 1990-1998

SHAWN M. MCLAUGHLIN' AND MOHAMED FAISAL^

'Nalioiuil Ocean Senice. NOAA

Center for Coastal Environmental Health and Biomolecular Research
Cooperative Oxford Laboratory
904 S. Morris St.
0.xford, Maryland 21654-9724
'Virginia Institute of Marine Science
School of Marine Science
The College of William and Mary
Gloucester Point. Virginia 23062

ABSTRACT Prevalence and intensity of Perkinsus spp. infections were determined in soft-shell clams Mya arenaria during 1990 to
1998 based upon incubation of rectal tissues in Ray's fluid thioglycoUate medium. During the study, soft-shell clams were collected
from 18 sites in the upper Chesapeake Bay in Maryland. Enlarged hypnospores were found in -7% (1 14/1.705) of the soft-shell clams.
Peak prevalences occurred in the fall of 1992 with -53% (16/30) at Piney Point and 50% (15/30) at Ea.stern Neck, and in August 1995
with -64% (18/28) and -37% ( 1 1/30) at Cedar Point and Piney Point, respectively. This investigation provides evidence that Perkinsus
spp. infections in soft-shell clams are more common than previously thought.

KEY WORDS: soft-shell clam. Perkinsus spp.. Chesapeake Bay. Maryland, infection, intensity
INTRODUCTION

Protozoa of the genus Perkinsus have been associated with
significant losses of feral and cultured species of bivalve mollusks
worldwide. Previous reports of Perkinsus sp. infections in soft-
shell clams Mya arenaria of the Chesapeake Bay have been un-
common (Andrews 1954), and its geographic distribution remains
to be determined. Recently. McLaughlin and Faisal (1998a) re-
ported the presence of Perkinsus spp. in Maryland soft-shell clams
and described the associated histopathological alterations. Most of
the infections observed were light in intensity, limited to the gills
and palps, and evoked cellular host responses including encapsu-
lation of invading parasites. As infection intensity increased, the
parasite was found in nearly all tissues of the soft-shell clam,
sometimes causing adverse host effects (McLaughlin and Faisal
1998a, 1999). Interestingly, two species of Perkinsus were recently
isolated from hemolymph and gills of soft-shell clams collected
from the Chesapeake Bay and propagated in vitro (McLaughlin
and Faisal 1998b). Morphology, life cycle, and molecular charac-
terization studies showed similarities between the soft-shell clam
hemolymph isolate and P. marinus. and provided evidence that the
gill isolate was an undescribed Perkinsus sp. (McLaughlin and
Faisal I998a,b, Kotob et al. 1999a.b).

In the assay routinely used for the detection and quantitation of
Perkinsus spp. cells in bivalves, host tissues are incubated in Ray's
fluid thioglycoUate medium (RFTM) and enlarged hypnospores
then stained with Lugofs iodine (Ray 1952), In soft-shell clams,
the use of rectal tissue in thioglycoUate assays was found to be
effective for diagnosing advanced Perkinsus spp. infections
(McLaughlin and Faisal 1999). A positive result implies that the
infection has progressed from the early encapsulation stage within
gill tissues to a more systemic infection spreading into various
tissues of the infected clam. In this paper, we report infection
prevalences and intensities of Perkinsus spp. in Mya arenaria
collected from 18 sites in the Chesapeake Bay during 1990 to

CHESAPEAKE BA

Figure. 1. Sampling sites in the upper Chesapeake Bay (1 = Swan
Point; 2 = Eastern Neck; 3 = Love Point; 4 = Piney Point; 5 = Eastern
Neck Island; 6= Cedar Point; 7 = Cabin Creek; 8 = Wye River; 9 =
Howell's Point; 10 = Gibson Island) with PcrA/HSHs-positive soft-shell
clams Mya arenaria based upon incubation of rectal tissues in Ray's
fluid thioglycoUate medium (Ray 1952). Scale: 1 cm = -6.9 miles
(-11.04 km)

349

350

McLaughlin and Faisal

TABLE 1.

Prevalence (percent infected) and intensity (weighted prevalence) iMackin 1962) of Perkinsiis spp. in softshell clams Mya arenaria from sites

in the upper Chesapeake Bay (" = 30).

Temperature

Salinity

Percent

Weighted

Location

Date

(°C)

(ppt)

Infected

Prevalence

Swan Point

December 1990

9.0

7.5

7

0.07

Swan Point

March 1991

8.0

5.0

0

0.00

Swan Point

July 1991

27.5

10.5

0

0.00

Howell's Point

August 1991

20.0

9.2

10

0.27

Wye River

August 1991

25.4

12.5

3

0.03

Swan Point

October 1991

20.0

12.5

23

0.33

Swan Point

January 1992

5.0

14.5

3

0.03

Little Choptank

March 1992

—

—

0

0.00

Love Point

April 1992

7.7

11.5

3

0.03

Swan Point

April 1992

5.0

10.0

0

0.00

Piney Point

April 1992

9.9

9.3

0

0.00

Bishop Head Point

April 1992

—

—

0

0.00

Sandy Point

April 1992

12.8

3.8

0

0.00

Gibson Island'

April 1992

8.0

12.0

0

0.00

Cabin Creek

June 1992

24.9

13.1

7

0.06

Swan Point

July 1992

3.6

11.0

0

0.00

Love Point

July 1992

25.8

10.8

0

0.00

Sandy Point

July 1992

23.8

10.0

0

0.00

Wye River

July 1992

23.8

13.0

0

0.00

Swan Point

September 1992

0

12.0

7

0.10

Piney Point

September 1992

22.6

5.0

53

1.17

Gibson Island

October 1992

15.9

16.3

26

0.40

Eastern Neck

October 1992

13.6

14.0

50

1.50

Swan Point

February 1993

3.0

7.0

0

0.00

Nichols Point

June 1993

20.4

4.0

0

0.00

Piney Point

June 1993

19.1

4.2

0

0.00

Cedar Point-

June 1993

17.0

4.2

0

0.00

Swan Point

August 1993

25.1

10.0

17

0.02

Piney Point

August 1993

25.0

9.8

10

0.30

Eastern Neck

August 1993

25.1

10.0

10

0.20

Love Point

August 1993

25.3

11.0

0

0.00

Gibson Island

August 1993

26.0

8.2

0

0.00

Rock Point

August 1993

—

—

0

0.00

Swan Point

October 1993

13.7

14.0

3

0.03

Swan Point

March 1994

4.4

5.0

0

0.00

Swan Point

June 1994

26.6

3.5

0

0.00

Love Point

June 1994

22.0

8.0

0

0.00

Sandy Point

June 1994

25.0

4.9

0

0.00

Eastern Neck Island

June 1994

25.0

5.5

0

0.00

Pier 1

June 1994

26.2

5.0

0

0.00

Love Point

July 1994

26.6

5.0

0

0.00

Swan Point

July 1994

26.6

3.5

0

0.00

Eastern Neck

August 1994

25.2

6.5

0

0.00

Swan Point

August 1994

25.6

7.1

0

0.00

Huntingt'ield

September 1994

22.2

10.0

0

0.00

Swan Point

September 1994

22.2

9.0

0

0.00

Swan Creek

September 1994

22.2

8.0

0

0.00

Swan Point

February 1995

2.2

6.0

10

0.10

Piney Point

February 1995

3.1

6.0

3

0.03

Eastern Neck Island

February 1995

3.1

6.0

3

0.03

Swan Point

July 1995

24.2

6.0

0

0.00

Eastern Neck Island

July 1995

25.0

1 n.o

0

0.00

Cedar Point'

August 1995

23.5

14.0

64

X.f^l

Piney Point

August 1995

23.6

1 3.5

37

1.33

Swan Point

August 1995

23.0

12.5

13

0.20

Swan Point

October 199(1

15.6

T ■)

13

0.20

Swan Point

July 1998

25.6

4.0

7

0,10

' n = 28.
^i = 29.

Prevalence and intensity were based upon incubation ol rectal tissues in Ray's lluid thioglycollate medium (Ray 1952).

Pekkinsus Prevalence in Mya arenakia

351

1998. Prevalences and intensities were determined using rectal
tissues in thioglycollate assays.

MATERIALS AND METHODS

Clam Collections

More than 1,700 soft-shell clams were collected in 57 samples
(-30 clams/sample) from 18 sites in the Chesapeake Bay (Fig. 1 )
from 1990 to 1998 by hydraulic escalator dredge. Clams from each
sampling site were held on ice and transported immediately to the
wet lab facility at the Cooperative Oxford Laboratory (COL), Ox-
ford. Maryland. Clams were held in 76-L glass aquaria supplied
with Tred A\on River water or artificial seawater at temperatures
between 8 and 20 °C until processed (1-2 days). Salinities were
adjusted to approximate those of the collection sites (5-16 ppt).

Ray 's Fluid Thioglycollate Assays

Pieces of rectum were excised from each clam and incubated in
RFTM (Ray 1952) for 5-7 days. Incubated tissues were subse-
quently macerated on glass slides, stained with Lugol's iodine, and
examined for spherical blue-black bodies characteristic of Perkin-
sus spp. (Ray 1952). Infection intensity was estimated using a
semiquantitative scale from 0 (negative) to 7 (extremely heavy
infection), modified from Ray (1954) and Mackin (1962).
Weighted prevalences were determined by adding the individual
assigned intensities and dividing by the number of clams sampled
(Mackin 1962).

RESULTS

Perkinsus spp. infections were found in -7% (114/1.705) of
soft-shell clams examined. Infected clams were found at 10 of the
18 sites surveyed (Fig. 1). Temperatures and salinities at sites
positive for Perkinsus spp. ranged from 2.2 to 25.4 ° C and from
2.2 to 16.3 ppt (Table 1 ). Peak prevalences of soft-shell clam
Perkinsus spp. usually occurred in the late summer and fall when
salinities and temperatures were highest. A peak in prevalence
occurred in the fall of 1992 with -53% (16/30) at Piney Point and
50% (15/30) at Eastern Neck (Table 1 ). Prevalence also peaked in
August 1995 with -64% (18/28) and -37% ( 1 1/30) at Cedar Point
and Piney Point, respectively. As shown in Table 2, intensities of
infections in the rectal tissues ranged from extremely light (stage
1) to heavy (stage 5). Extreme cases (stages 6 and 7) were ob-
served in less than 1%- (9/1705) of the soft-shell clams examined.
The maximum weighted prevalences observed were 1.17 at Piney
Point in September 1992 and 1.57 at Cedar Point in August 1995.

DISCUSSION

Previous reports oi Perkinsus spp. infections in soft-shell clams
are rare. In Virginia, Perkinsus sp. was reported to occur infre-
quently in soft-shell clams and the low infection intensities ob-
served were not associated with histopathological lesions or mor-
talities (Andrews 1954). Similarly, histological examination of
over 3.500 soft-shell clams collected from 20 sites in Maryland
during 1969 to 1989 revealed only occasional occurrences of Per-
kinsus spp. (Sara V. Otto, Maryland Department of Natural Re-

TABLE 2.

Infection intensities of Perkinsus spp. in softshell clams, Mya arenaria, from sites in the upper Chesapeake Bay based upon incubation of

rectal tis.sues in Ray's fluid thioglycollate medium (Ray 1952).

Intensity of Infection (Stages 1-7)

Site

(» = 30)

Date
(month & year)

1

# (-%)

#(-%).

# (-%)

#(-

j)

5
#(-%)

6

1
#(-%)

Total

#(-%)

Swan Point
Howell's Point
Wye River
Swan Point
Swan Point
Love Point
Cabin Creek
Swan Point
Piney Point
Gibson Island
Eastern Neck
Eastern Neck
Swan Point
Piney Point
Swan Point
Swan Point
Piney Point
Eastern Neck Island
Cedar Point'
Piney Point
Swan Point
Swan Point
Swan Point

December 1990
August 1 99 1
August 1 99 1
October 1991
January 1992
April 1992
June 1992
September 1992
September 1992
October 1992
October 1992
August 1993
August 1993
August 1993
October 1993
February 1995
February 1995
February 1995
August 1995
August 1995
August 1995
October 1996
July 1998

2(7)

1(3)

1(3)

4(13)

1(3)

1(3)

2(7)

1 (3)

8(27)

6(20)

6(20)

4(13)

1(3)

1 (3)

3(10)

I (3)

1 (3)

5(18)

6(20)

2(7)

1 (3)

2(7)

1(3)
1(3)

1 (3)

3(10)

1(3)

2(71

3(10)

1(3)

1(3)

5(18)
3(10)
2(7)

3(10)
1(3)

3(11)
1(3)

1(3)
1(3)

1(3)
1(3)

2(7)

1(3)

1 (3)

2 (7)

2(7)

3(11)

1(3)

1(3)

1(3)
1 (3)

1(3)

2(7)

2(7)
3(10)
1 (3)
7 (23)
1(3)
1(3)
2(7)
2(7)

16(53)
8(27)

15 (50)
3(10)
5(171
3(10)
1(3)
3(10)
1 (3)
1 (3)

18(64)

11(37)
4(13)
4(13)
2(7)

Stage 1 = extremely light. 2 = very light, 3 = light, 4 = moderate, 5 = heavy. 6 = very heavy. 7 = extremely heavy.

352

McLaughlin and Faisal

sources. Oxford. Maryland, pers. comm.). McLaughlin and Faisal
(1999) demonstrated that positive thioglycollate assays using rec-
tal tissue denote advanced, generalized infections of Perkinsiis
spp. in soft-shell clams. This observation was verified in histologic
preparations. Hence, the findings from our present study suggest
that Perkinsus spp. infections may be increasing in soft-shell clams
of the Chesapeake Bay. Indeed, soft-shell clams have been shown
to host more than one species of Perkinsus, one closely related to
P. marinus and the other an undescribed Perkinsus sp. (McLaugh-
lin and Faisal 1998a.b; Kotob et al. 1999a.b). The lack of an
effective tool for distinguishing between species of Perkinsus
within a host limits our ability to attribute the increased prevalence
to one or both .soft-shell clam Perkinsus species.

This apparent elevation in Perkinsus spp. infections in soft-
shell clams of the Chesapeake Bay parallels increased prevalences
and range extensions of P. marinus infections in the eastern oyster.
Increased occurrences of the oyster parasite were attributed to high
salinities from successive droughts during the 1980s, concurrent
mild winter temperatures, and movement of infected oysters (Bur-
reson and Calvo 1996). Range extensions of P. marinus parasites
were further hypothesized to be associated with genetic changes in
the host or parasite and/or environmental parameters (Ford 1996).
Coincidental increases in soft-shell clam Perkinsus spp. in the
Chesapeake Bay may be attributable to similar factors. For ex-
ample, the extension of oyster P. marinus into all productive oyster
grounds in the Chesapeake Bay in the late 1980s and early 1990s
(Burreson and Calvo 1996) coincided with the increased occur-

rence of soft-shell clam Perkinsus spp. observed in this study.
Indeed. P. marinus was first reported in oysters in Swan Point,
Chester River, in 1987 (Burreson and Calvo 1996) less than 3
years before Perkinsus spp. infections were observed in soft-shell
clams from the same site. Similarly, reduced infection levels of P.
marinus in Chesapeake Bay oysters during 1994 (Burreson and
Calvo 1996) were concurrent with reduced soft-shell clam Perk-
insus spp. prevalences in the same year.

The high prevalences of soft-shell clam Perkinsus spp. at some
sites in Maryland during 1992 coincided with the lowest recorded
catch of soft-shell clams since 1962 (Connie Lewis. Maryland
Department of Natural Resources, Annapolis, Maryland, pers,
comm.). The continued low harvests of soft-shell clams in the
Chesapeake Bay suggest further investigations on impacts of the
parasite to M. arenaria fisheries are warranted.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the Maryland Department
of Natural Resources and the Maryland Department of the Envi-
ronment for collection of clams. We also thank the histology staff
at the Cooperative Oxford Laboratory (COL) for processing of
clams. The research was supported by a grant from the National
Oceanic and Atmospheric Administration (NOAA). Virginia Sea
Grant College Program and the U.S. Spain Joint Commission on
Scientific and Technological Cooperation, Madrid. Spain. Virginia
Institute of Marine Science contribution # 2290.

LITERATURE CITED

Andrews, J. D. 1954. Notes on fungus parasites of bivalve mollusks in
Chesapeake Bay. Proc. Natl. Shellfish. Assoc. 45:157-16.1.

Burreson. E. M. & L. M. Ragone Calvo. 1996. Epizootiology o( Perkinsus
inctrinus disease of oysters in Chesapeake Bay. with emphasis on data
since 1985. .1. Shellfisli Res. I5:17--14.

Ford. S. E. 1996. Range extension by the oyster parasite Perkinsus marinus
into the northeastern United States: Response Ui climate change? J.
Sliellfish Res. 15:45-56.

Kotob, S. I., S. M. McLaughlin, P. Van Berkum & M. Faisal. 1999a. Char-
acterization of two Perkinsus spp. from the softshell clam Mya
arenaria using the small subunit ribosomal RNA genes. J. Euk. Mi-
crnhiol. 46:4.19-444.

Kolob. S. I.. S. M. McLaughlin. P. Van Berkum & M. Faisal. 1999b.
Discriminalion between iwo Perkinsus spp. isolated from the soft-shell
clam Mya aremuia by sequence analysis of two internal transcribed
spacer regions and 5.8S ribosomal RNA genes. Parasitology 1 19:.16.1-
368.

Mackin. J. G. 1962. Oyster disease caused by Dermocystidium mariiuim
and other microorganisms in Louisiana. Publ. Inst. Mar. Sci. Univ. Tex.
7:1.12-229.

McLaughlin. S. M. & M. Faisal. 1998a. Histopathological alterations as-
sociated with Perkinsus spp. infection in the soft-shell clam Mya
arenaria. Parasite 5:26.1-271.

McLaughlin. S. M. & M. Faisal. I998h. /;; vitro propagation of Iwo Per-
kinsus species from the soft-shell clam Mya arenaria. Parasite 5:341-
348.

McLaughlin, S. M. & M. Faisal. 1999. A comparison of diagnostic assays
for detection of Perkinsus spp. in the soft-shell clam Mya arenaria.
Aquaeulture 172:197-204.

Ray. S. M. 1952. A culture technique for the diagnosis of infections with
DernuHvstiilium inarinuni. Mackin, Owen and Collier, in oysters. Sci-
ence 1 16:360- .16 1.

Ray. S. M. 19.54. Biological studies of Dennocystiiiium marinmn. Rice
Inst. Pamphlet, Spec, Issue. The Rice Institute. Houston, Texas,

Journal of Shellfhh Research, Vol. 19. No. I, 353-359, 2000.

SUMMER MORTALITY OF PACIFIC OYSTERS, CRASSOSTREA GIGAS (THUNBERG):
INITIAL FINDINGS ON MULTIPLE ENVIRONMENTAL STRESSORS IN PUGET SOUND,

WASHINGTON, 1998

DANIEL P. CHENEY, BRIAN F. MACDONALD. AND
RALPH A. ELSTON

Pacific Shellfish Institute
Olympia. Washington 98501

ABSTRACT A study was begun in late 1997 in Puget Sound. Washington, and Tomalas Bay. California, to characterize more
precisely the summer mortality ot the Pacific oyster ( Cra,v.so5/rra gigas) in a variety of culture conditions and locations and to describe
definitively the relationship of summer mortality to infectious diseases. Water quality and seasonal factors also were identified. A field
component investigated the oysters' thermal stress response and assessed induced thermal tolerance as a means to reduce mortalities.
In addition, management practices for commercial cultivation were evaluated as measures to reduce the frequency and extent of oyster
losses. Our evaluation of the 1998 data from the summer mortality project supports earlier reports on the rate and timing of mortality
events. There were differences in the mortality rates among the varieties of oysters tested, with triploid oysters having consistently
higher mortality rates than diploid oysters planted in comparable plots. Trends in mortalities were toward higher rates at or immediately
after neap tides when dissolved o.vygen was lowest and during periods of elevated air and water temperatures. Relative densities of the
phytoplankton Gymnodinium splendens. Ceralium spp., and Psuedo-nitzschia spp. were higher during the late summer; dissolved
oxygen concentrations were correspondingly low, and oyster mortalities were high during this same period. It is likely that Pacific
oysters at the study sites experienced varying degrees of chronic stress attributable to multiple environmental factors. Evaluations of
effects of those stressors and development of oyster health management strategies are continuing.

KEY WORDS: Pacific oyster, Crassoslrea gigas. disease, mortality, env ironment

INTRODUCTION

Pacific oyster Crassostrea gigas (Thunberg) production on the
U.S. West Coast has not expeiienced the catastrophic losses from
disease plaguing the East Coast; however, mass mortalities occur
peiiodically and continue to threaten commercial production. The
syndrome known as summer mortality has been known on the
Pacific Coast for at least 40 years. Sharp increases in mortality
from June to September are the classic example of summer mor-
talitv . The magnitude of the losses has been estimated as up to 50%
of the harvestable crop in a given summer, but losses are highly
variable by specific location and year. Reduction of these high
losses is an integral part of a larger Pacific oyster health manage-
ment program, being carried out by West Coast research organi-
zations and shellfish farmers, to increase production and sales of
seed and edible Pacific oysters.

Background

As early as the 1940s, serious losses of Pacific oysters were
reported from Japanese culture locations (Koganezawa 1974).
Mortalities occurred during the summer months, and at times ex-
ceeded 60%. Although various age groups were affected during
an episode, the more severe losses occurred in the older and larger
oysters. On the west coast of North America, oyster mortalities
have been reported since the late 1950s from Washington. Cali-
fornia, and British Columbia (Glude 1975). Again, older oysters
seemed to be more susceptible, but the timing and degree of loss
was variable. The most severe episodes occurred in shallow, nu-
trient-rich embayments in late summer when seasonal tempera-
tures were highest.

Japanese researchers advanced a theory of metabolic imbalance
related to accelerated reproductive maturation (Imai et al. 1965,
Tamate et al. 1965). They concluded that oyster mortalities were
related to reproductive maturation and environmental conditions
present in growing areas before the end of July. The thought was

that mass mortalities in Matsushima Bay associated with high
temperatures and nutrient-rich waters led to accelerated reproduc-
tive development.

In Washington state, a similar metabolic imbalance was pro-
posed, although regional water temperatures are generally lower
than those in the Japanese growing areas (Perdue 1983, Perdue et
al. 1981). Scholz et al. (1973) believed mortality was associated
with gonad resorption. These studies also noted a high variability
in mortality rates between growing areas in close proximity.

Studies to produce resistant oysters and genetically altered
broodstocks were conducted by the University of Washington in
the 1970s and 1980s. Selective breeding experiments showed that
survival could be increased significantly, but it was accompanied
by a severe decline in production yields (Beattie 1984). Surviving
oysters were smaller, slower growing, and thinner than susceptible
oysters and had little commercial use. The subsequent develop-
ment of triploid Pacific oysters, which have poorly developed
gonads, offered a promising option to reduction of summer kills
(Allen et al. 1989).

The role of infectious agents in summer mortalities of Pacific
oysters and other Crassoslrea species has been studied extensively
in Asia, the United States, and Europe (Sindermann 1990, Elston
1993). Early studies by Japanese investigators discounted the role
of a bacterial disease now known as nocardiosis, because a corre-
lation with infection and mortality could not be established (Nu-
machi and Oizumi 1965). However, nocardiosis has recently been
associated with some episodes of summer kill (Elston et al. 1987.
Friedman 1990. Friedman et al. 1991). A herpes-like virus infec-
tion has been seen in larvae and seed oysters of Pacific oyster from
hatcheries on the Atlantic coast of France (Nicolas et al. 1992.
Renault et al. 1994, Renault IFREMER. pers. comm. 1997). These
authors reported summer mortalities of 80-90% in infected seed.
Similar herpes-like infections have not been observed in U.S. West
Coast Pacific oysters.

353

354

Cheney et al.

PURPOSE AND OBJECTIVES

In late 1997, the Sea Grant Oyster Disease Research Program
funded a muhiyear study to further investigate summer mortahty
in Pacific oysters. This project was designed to test hypotheses
relating to mortalities of Pacific oysters on the U.S. West Coast
and to recommend measures to reduce those mortalities. The spe-
cific objectives were the following.

1. Perform studies that will provide health and disease infor-
mation for Pacific oysters in areas experiencing high fre-
quencies of summer mortality.

2. Carry out a comprehensive survey of environmental condi-
tions occurring in the affected areas.

3. Evaluate in field trials the influences of environmental fac-
tors on stress proteins, morbidity, and mortality of diploid
and triploid Pacific oysters, under a variety of culture con-
ditions.

4. Identify and test short-term mortality reduction options for
shellfish farmers and resource managers; and make recom-
mendations for longer term study.

5. Compile information on Pacific oyster mortality from shell-
fish farmers and researchers on the U.S. and Canadian West
Coast.

This report describes the first-year activities and findings of
that study. Project activities reviewed in this report include:

1 . experimental design and setup of study sites and treatment
groups in Puget Sound. Washington;

2. monitoring of mortality, condition indices, growth, and as-
sociated fauna and flora; and

3. monitoring of water quality parameters and phytoplankton.
Related activities during the same period, not reported here

included:

1. compiling information on summer mortality events from
Tomales Bay, California, and other locations; and

2. sampling for stress protein, conducting histopathology
analyses, and assessing pathology associated with mortality.

METHODS AND MATERIALS

Site Selection

The original project design called for field sampling and
screening stations in two locations in south Puget Sound (Mud Bay
and Totten Inlet) and one in north Puget Sound (Sequim Bay). The
Mud Bay and Sequim Bay sites were believed to have a high
incidence of summer mortality; whereas, the Totten Inlet site was
selected as a low mortality control. Each site was dedicated for the
duration of the project on private oyster grounds owned by Taylor
ShclUish Farms and the Jamestown S'Klallam Tribe.

Preliminary siting meetings were held with shellfish farmers
and researchers in February 1998. As a result of these meetings,
the decision was made that the project would benefit from two
additional Puget Sound sites. One site was to be near the commu-
nity of Allyn, in North Bay, south Puget Sound. This site was
being intensively monitored for toxic phytoplankton and other ma-
rine algae, had experienced consistent annual mortalities and. in
1997, had very high levels of both Vibrio jxiriiluwnuilylicii.K and
paralytic shellfish toxicity (PSP). The second site was in Eld Inlet
and located to continue observations on Pacific oysters imported in
1994 from Tasmania. During a 1997 to 1998 study, these oysters
were found to have both lower mortality and higher growth rates
than a comparable populalion of nali\e Pacific oysters (Kitlcl

1998). Finally, late in the summer of 1998. a sixth site was added
in Tomales Bay, California. Sampling of this site was directed at
an histopathological analysis of seed mortality during the Septem-
ber to October period. No environmental or water quality data
were gathered. The locations of each of the five Puget Sound sites
are shown in Figure 1.

Site Design and Setup

The experimental design called for a combination of shellfish
health, environmental, and general biological monitoring at each
study site. In conjunction with pathogen screening, we monitored
water quality, stress proteins, oyster size, condition, mortalities,
associated fauna and flora, and other relevant features. Experi-
ments were designed to challenge, under commercial conditions,
seed, yearling, and market-ready oysters held on the bottom and in
culture bags placed on the bottom and on racks. All plots were
located in or immediately adjacent to commercial grounds.

All experimental sites in Puget Sound were established be-
tween mid-April and late May 1998. Approximate bottom eleva-
tions mean lower low water (MLLW) were: Sequim Bay 0.0 m (0
ft), Totten Inlet 0.9 m (3 ft). Mud Bay 0.3 m ( 1 ft), Allyn 0.3 m ( 1
ft), and Eld Inlet 0.0 m (0 ft). Nearly 26.000 diploid and triploid
oysters of varying sizes were transplanted from existing farm beds
and nursery facilities. An additional 2,000 oysters were stockpiled
to provide replacement animals and specimens for histopathology
and stress protein sampling. The make-up and density of oysters in
the culture treatments at each site are shown in Table 1.

Instrumentation was also deployed for continuous or repeated

Puget
Sound

eattle

Mud Bay

Fijiiirf. 1. Study sites I'aiitlc oyster summer mortality project IWH-
IW), I'ujjel Sound. Washington. Lines and lilled circles In the inset
maps point to the 1998 summer mortality sampling; sites.

Pacific Oyster Summer Mortality

355

TABLE 1.

Oyster type, treatment, culture type, sample unit, and number of oysters used at experimental oyster summer mortality study sites in Puget

Sound, summer-fall 1998.

Culture

No.

Number

Oysters

Oyster

Sequim

Totten

Type

Treatment

Type'

Density

Unit

Units

Allyn

Mud Bay

Bay

Inlet

Diploid

Seed

Singles

50

/bag

3-6

300

0

300

150

125

/bag

3-6

750

0

730

375

200

/bag

3-6

1.200

0

1,200

600

Totals

2,250

0

2,250

1,125

Seed

Cultched

100

/sq m

3.2

0

320

320

0

300

/sq m

3.2

0

960

960

0

650

/sq m

3.2

0

2,080

2.080

0

Totals

0

3,360

3,360

0

Yearling

Cultched

80

/sq m

1.6-3.2

256

256

0

128

300

/sq m

1.6-3.2

960

960

0

480

Totals

1,216

1,216

0

608

Market-ready

Cultched

80

/sq m

1.6-3.2

256

256

256

128

300

/sq m

1.6-3.2

960

960

960

480

Totals

1,216

1,216

1,216

608

Market-ready

Singles

25

/bag

3-6

150

0

150

75

75

/bag

3-6

450

0

450

225

50

/bag

5

0

250

0

0

40

/bag

15

0

600

0

0

Totals

600

850

600

300

Tasmanian

Market-Ready

Singles

50

/bag

5

0

250

0

0

50

/bag

15

0

750

0

0

Totals

0

1,000

0

0

Total Diploid

5,282

7,642

7,426

2,641

Triploid

Yearling

Singles

80

/sq ni

1.6

0

0

0

128

300

/sq m

1.6

0

0

0

480

Totals

0

0

0

608

Yearling

Singles

150

/bag

2

0

0

0

300

Market-ready

Singles

80

/sq m

1.6-3.2

256

256

0

128

300

/sq m

1.6-3.2

960

960

0

480

Totals

1,216

1,216

0

608

Total Triploid

1,216

1,216

0

1,516

Total Diploid and Triploid

6,498

8,858

7,426

4,157

' Cultched seed: 2-15 mm diameter seed attached to shell. 4-8 seed per shell; single seed: 15-30-mm-diameter singles; yearling: 1-2 years old and
80-100 mm-long singles; market ready: 2-3 years old 100-1 50-mm long singles or in clusters. These oysters were spread evenly on the bottom or placed
in 1.0 X 0.5 = m lO-mm plastic-mesh growout bags.

monitoring of key water quality parameters. Onset® miniature data
loggers were placed in waterproof cases and installed on the bot-
tom at each study site to sample ambient water and air tempera-
tures. We also assembled and deployed similar Onset* data log-
gers fitted with dissolved oxygen (DO) probes and operational
amplifiers for long-term, continuous monitoring of DO.

Site Surveys and Monitoring

Field Sampling and Mortality Monitoring

Size data and condition indices were collected for each group
of animals during the setup phase of the project. Since setup, up to
1 1 sampling sessions have been performed at each of the five
Puget Sound sites. Monitoring of the test sites began before the
anticipated onset of the summer mortality events in late June and
continued during each low tide cycle, approximately every 7 to 14
days. Moribund oysters and outright mortalities within each of the
test plots were recorded, flagged, and documented photographi-
cally during each survey. Mortalities were counted when there was

obvious shell gape, and the animals lacked any closure response.
Oysters considered moribund were gaping but were able to pro-
duce a closure response.

Moribund animals were removed and preserved for histological
inspection. Live specimens were collected routinely for use in the
histopathology and stress protein portions of this study. The origi-
nal densities of the test plots were maintained by replacing animals
removed from the plots with others stockpiled in nearby plots,
planted at similar densities.

Environmental Monitoring

Intertidal water and air temperatures were recorded continu-
ously, a reading was taken every 24 to 30 min at each site begin-
ning in mid-May 1998. Continuous DO data (readings every 15 to
30 min) were also obtained at the Mud Bay site in Eld Inlet,
beginning July 1 1. 1998. YSI® oxygen-temperature-salinity and
pH meters were used to measure these water quality parameters
during selected flood and ebb tides. A water sampler bottle was

356

Cheney et al.

used to collect water samples at depth. Meteorological data were
collected from available weather instrumentation. Tide data were
computed with tide prediction software.

Phytoplankton samples were collected by fme-mesh (10-(i.m
mesh). 0.25-m diameter ring nets at the Mud Bay and Allyn sites
during each sample period. Living or fomialin-fi.xed net samples
were examined using light microscopy to generate a species list
and determine species relative abundance (dominant, many. few).

An oyster condition index (CI) was derived from 11 to 31
randomly selected market-size oysters in the May to June 1998
period at the Sequim. Allyn, and Mud Bay sites. A second series
of samples were taken in October to November 1998 at the above
sites and in Totten Inlet. The CI was used to determine the quality
or "fatness" of oysters. All CI calculations employed the gravi-
metric method discussed in Schumacker et al. (1998).

RESULTS

Shellfish Mortalities

Substantial mortalities of Pacific oysters occurred at the Totten
Inlet, Mud Bay, Allyn Bay. and Tomalas Bay sites during the
middle to late summer. Mortalities at the Sequim Bay and Eld Inlet
sites were negligible.

Cumulative and average daily mortalities at four of the Puget
Sound sites are shown in Table 2 for market-ready diploid and
triploid oysters. Mortalities for the reported time periods ranged
from 40 to 56% for triploid oysters in Totten Inlet, Mud Bay, and
Allyn Bay; and 31 to 45% for diploid oysters in Totten Inlet and
Mud Bay. Considerable variation was seen in seed oysters, with
Totten Inlet experiencing higher die-offs than either Allyn or Se-
quim bay. We have limited sampling information from the Tas-
manian stocks at the Eld Inlet site. Mortalities at this site over a
83-day period were 3.6% for Tasmanian Pacific oysters. 9.6% for
a control stock of native Pacific oysters, and 1.4% for Pacific
oysters transplanted from Mud Bay. The trend that Kittel (1998)
reported seemed to be continuing.

The progression of Pacific oyster mortalities throughout the
summer was followed by means of weekly to monthly field ob-
servations. Mortalities were calculated as a percentage average
daily mortality between each sample period for Sequim Bay. Tot-
ten Inlet, Mud Bay. and Allyn datasets (Fig. 2). Sequim Bay did
not exceed 0.04% per day at any time, and 7 out of 10 samples
were less than or equal to 0.01%. The other sites experienced
elevated mortalities beginning in early July in Totten Inlet and
early August in Mud Bay and Allyn Bay. Totten Inlet was un-
usual— very high mortality occurred within the first 4 weeks of
onset, mortality declined to a low level after that. Allyn and Mud

TABLE 2.

Cumulative percentage mortalities of I'acirie oysters for sinjjie seed,

diploid and triploid. and low- and hinh-denslty treatment groups at

oyster summer mortality study sites, summer-fall IVMS.

3.5%

3.0%

f £. 2.5%

2 '-i 2.0%

g o 1.5%

i ^ 1.0%
a.

0.5%

0.0%

li |-^ Diploid -a- Triploid

|Mu<.

-r^

1 1

, 'iT

III \\vk

Tl

Jlk

ll

ui,™>

lyifcii

(] iwnUtr^Lf'w

r

Iri

IfiP

Hfio

5

m

Vf^n

" V

\ \\w

,/iM/^

^*-

"

HI" i|

M ,— J^^ ^

t

- — 1=^ t=i

30

25 U
20 i

5/15 5/29 6/12 6/26 7/10 7/24 6/7 8/21 9/4 9/18 10/2 10/16 10/30
Date (month/day)

Figure. 2. Percentage daily mortality (left-hand axis) and ambient wa-
ter and air temperatures (right-hand column) at Puget Sound summer
mortality study sites, summer-fall 1998.

bays had increasing but variable mortalities beginning later in the
summer, mortalities continued at moderate levels through the end
of September.

Triploid mortalities began eariier in the summer and spiked
more rapidly and at a higher rate than the diploid treatments. Daily
diploid mortalities did not exceed 0.6%; whereas, the average rate
of triploid treatments approached 2.5%. Triploid mortalities in
Mud Bay and Allyn Bay were also high; however, they tended to
track the diploid die-off closely. Triploid mortalities were most
pronounced in Allyn (Fig. 2). The high and sudden mortality in
Allyn was unfortunate, because the commercial beds were largely
planted as triploids. All triploid oysters in those commercial beds
exhibited a rate and timing of mortality onset that was similar to
the treatment plots.

Ell viroiimental Monitoring

All sites for the summer mortality sampling are in protected
estuarine embayments. The Sequim Bay, Allyn Bay, and Mud Bay
sites are in shallow heads of inlets strongly intluenced by tidal
exchange and freshwater input from the surrounding uplands. Wa-
ter column data for Sequim Bay. Totten Inlet. Mud Bay, and Allyn
Bay for the summer to fall 1998 period are shown in Table 3. High
pH levels were recorded during the early summer, probably re-
tlecting high primary productivity during this period. Salinities
were depressed only for brief periods after heavy rainfall events in
Mud Bay and Allyn Bay.

Sequim Totten Mud Bay Allyn Temperature

Days observed 197 138 184 I.S5

Single seed 13% 65% 16%

Diploid low density 3.5% 45% 31% 12%

Diploid high density 2.2% 42% 42% 28%

Triploid low density 56% 40%

Triploid high density 50% 52% 55%

Ambient temperatures were monitored al all survey sites during
the 1998 sampling period (Fig. 2). Tidal period played a large role
in the duration and range of recorded temperatures. Low daytime
tides coupled with intense insolation resulted in high ambient air
temperatures and elevated water temperatures on the incoming
tide. Peak temperatures neared 53 C (or 127 "F) and frequently

Pacific Oyster Summer Mortality

357

TABLE 3.
Water column conditions for all samples taken at oyster mortality study sites, summer-fall 1998.

Secchi

Temp

D.O.

D.O.

Sal.

Depth

(C)

% sat.

(mg/L)

pH

(ppti

(m)

Sequim — Surface (;i = 3|

Average

9.6

Maximum

15.3

Minimum

6.0

Totten— Surface (n = 17)

Average

18.6

Maximum

26.5

Minimum

12.1

Mud Bay— Surface (n = 18)

Average

19.2

Maximum

27.4

Minimum

10.5

Mud Bay— Bottom to -3.75 m

(H = 15)

Average

17.5

Maximum

25.0

Minimum

10.7

Allyn — Surface (h = 13)

Average

17.9

Maximum

22.8

Minimum

9.3

Allyn— Bottom to -3.5 m (ii =

13)

Average

16.8

Maximum

20.3

Minimum

10.2

53.0
66.0
35.0

98.1

141.4

67.2

71.2

106.0

39.4

74.7

100.9

39.4

85.8
115.1
56.5

79.4
119.8

59.5

5.3

7.7

25.7

6.6

7.9

28.3

2.9

7.5

21.3

7.7

8.0

28.1

10.6

8.5

29.5

5.4

7.5

26.0

5.8

7.7

24.9

8.6

8.6

29.1

3.3

7.2

19.9

6.0

7.7

28.0

1.2

8.4

8.2

29.6

2.2

3.2

7.3

25.9

0.6

7.1

8.0

26.5

18.0

9.2

9.3

29.0

27.2

4.5

7.4

15.9

6.6

6.4

7.9

27.5

20.0

9.3

8.3

29.1

27.2

4.8

7.5

23.1

15.3

exceeded 40 °C during low-tide exposure in Totten Inlet (Fig. 2).
Air temperatures exceeded 25 °C during low tide at all stations
between late June and early September.

Dissolved Oxygen

Dissolved oxygen (DO) concentrations recorded above the
sediment surface in Mud Bay fluctuated from less than 1 mg/L
(ppm) to nearly 12 mg/L. Summertime DO was closely linked with
the tidal cycles. A long period of neap tides with low and slack
water during the evening resulted in a daily and successive reduc-
tion in DO. For example, from September 20 through October 2,
the DO was between 0.5 and 2 mg/L for about 9 days (Fig. 3).
Similar, but shorter duration, declines in DO were also recorded in
middle and latter parts of August. A closer look at the late Sep-
tember extreme low DO event revealed the following: ( 1 ) the DO
was lowest at slack water; (2) if the meter was exposed (tide below
about 0.3 m MLLW) the DO spiked, indicating a measurement in
air; and (3) water temperature was very constant and declined only
slightly in the early morning. From the second week of October
onward, records from Mud Bay showed a gradual increase in DO.
with numerous strong peaks (DO above 8 mg/L) corresponding to
low-tide exposure to the air and circulation during strong spring
tides.

Phytoplankton

Phytoplankton taxa observed in samples taken from Puget
Sound summer mortality sites are listed in Table 4. A total of 32
samples were screened from June 23 to December 2, 1998.
Samples taken through mid-July were composed primarily of cen-
tric and pennate diatoins and Ceraliitin spp. Beginning in mid-July

and continuing through early October. Gyiiinodiniiiin splendens
and Ceratium spp. were typically dominant taxa, followed in rela-
tive abundance by Chaetocreros spp., Coscinodiscits spp., and
Psuedo-nitzschia spp. Dinotlagellates declined after mid-October,
and Dityhtm brightwellii. Coscinodiscus spp., and Psuedo-
nitzschia spp. were the dominant taxa.

Condition Index

Average condition indices of market-size oysters from summer
mortality study sites ranged from 8.78 to 11.08 in spring 1998
(Table 5). The same size group of animals sampled in fall 1998 had

9/20 9/22 9/24 9/26 9/28

Date (Month/Date)

9/30

10/2

Figure. 3. Tide levels (lines) and dissolved oxygen (dots) 6 cm above
the sediment surface at the Mud Bay, Puget Sound, summer mortality
study site, September 20 through October 2, 1998.

358

Cheney et al.

TABLE 4.

Dominant phytoplankton taxa from samples taken at oyster
mortality stud} sites, summer-fall 1998.

Diatoms

Centric

Chaetocreros spp.
Cn.sc'modiscus spp.
Ditylum hrightwellii
Eiicampiu zodiuctis
Giiinanlia spp.
Rhizosolenia setigeni
Skeleloiwma coskUiim
Thallassiosini spp.
Pennate

Pseudo-nitzschia spp.
Dinoflagellates

Proceuuiim spp.
Gymnodiniwn sangKineiini
NiKtilmu scintillans
Ceratium spp.
Alexandrium spp.
Protoperidiniiim spp.

average indices of 4.78 to 8.78. Triploid and diploid oysters had
similar indices in the fall sampling. Allyn had the lowest average
inde.x of three sites in the spring, and over-all lowest indices in fall
sampling. Many of the Allyn oysters were visibly thin and trans-
parent.

DISCUSSION

The published literature and anecdotal reports suggest many of
the mortalities occurring in Pacific oysters are the result of mul-
tiple factors or stressors, including pathogens, elevated tempera-
tures, low DO, xenobiotic stress, and the physiological stress as-
sociated with reproduction. It is likely that multiple chronic stress-
ors may combine to bring about mortalities and that an oyster's
ability to deal with a particular seasonal stress, such as tempera-
ture, may be the deciding factor as to whether that organism will
survive. Our evaluation of the first year's data from the summer
mortality project supports observations reported by Glude (197.5)
on the rate and timing of mortality events. In addition, there
seemed to be significant differences in the mortality rates among
the varieties of oysters tested. Triploid oysters al the Mud Bay,
Allyn Bay, and Totten Inlet test sites experienced a cumulative
mortality rate 8 to 28 percentage points higher than the mortality

rates of diploid oysters planted in comparable plots (Table 2).
Mortality events trended toward higher rates at or immediately
after neap tides when DO was lowest. Extreme air temperature
spikes and increasing ambient water temperatures were also oc-
curring at the same time. However, high temperature alone may
not be lethal. Many oysters at the Totten Inlet site survived re-
peated exposure to temperatures over 40 °C (Fig. 2). We have not
yet ineasured the internal temperatures of Pacific oysters in par-
allel with our temperature readings recorded by our data loggers.
Larger oysters with thick shells, positioned vertically and partially
embedded in sediment would probably have lower internal tem-
peratures than the external (environmental) temperatures recorded
by the data loggers.

A preliminary assessment of phytoplankton occurring in the
study sites suggests a possible link between the densities of Gym-
nodiniiim sangiiineiim. Ceratium spp., Psiiedo-nilzschia spp.. and
other dominant taxa with the on.set of summer mortality. G. soii-
ifiiineiim was abundant in Mud Bay and Allyn Bay, and at times
the water in Mud Bay had a pronounced orange-red tint. Dissolved
oxygen concentrations were low and mortalities were high during
this same period. These observations parallel early accounts of the
interactions between oyster mortality and phytoplankton. Nightin-
gale (1936) reported numerous occurrences of "red-tides" associ-
ated with G. sangiiineiim (densities ranged from 37 to 15,800
cells/mL). He attributed losses of Olympia oysters Ostrea liirida
directly or indirectly to those red-tide blooms. Nightingale (1936)
also assessed the effects of G. sangiiineiim in aquaria containing
Olympia oysters. He used cell densities typically seen in Puget
Sound blooms and observed responses ranging from excessive
mucus production to shell closure and a cessation of feeding. More
recent literature has associated G. sangiiineiim with fish kills and
toxicity (Steidinger and Tangen 1996), and Carolyn Friedman
(Bodega Marine Laboratory, pers. comm. 1998) reported seed
mortalities in Tomales Bay during C. sangiiineiim blooms.

It is likely that oysters at the Puget Sound summer mortality
study sites experience varying degrees of chronic stress because of
the water quality and biological changes we observed at those
locations. Therefore, a critical factor for survival may be the oys-
ter's ability to elicit a satisfactory response to a suite of summer
stressors. The Pacific oyster summer mortality project will con-
tinue through 2001 to describe the primary stressors affecting oys-
ter survival, assess the effects of specific phytoplankton taxa.
evaluate the role of infectious disease, and identify practical meth-
ods and tools to increase the predictability and management of
mortality events. Future project reports will discuss hislopatholog-

TABLE 5.
Condition indices of Pacific oysters at summer oyster mortality study sites.

Spring 98
Diploid

Fall 1998

Diploid

Triploid

Low

High

Low

High

Scquini Bay
Totten Inlel
Mud Bay
Allyn

II.OX ± I.W

1 1.07 ± 1.81
8.78 ± 2.8.^

7.92 ± 1 .70

10.78 ± 1.46

8.25 + 2. 1. S

539 ± I .^.S

8.94 ± 2.35
9.89 + 2.12
7.88 ± 2.95
4.45 ± 1.27

8.78 ± .1.2.3
4.78 ± 0.90

8.07 ±2.17

5.08 ± 1.32

Figures are the sample mean index values ±one standard deviation (/i = 15 to 30) for diploid oysters in the spring of 1998. and diploid and triploid oysters
at low and high licalnicnt ilcnsiilcs sampled in the fall cil 1998.

Pacific Oyster Summer Mortality'

359

ical events leading up to and resulting in oyster mortality, and
physiological and biochemical responses of the animals to elevated
temperatures and other stressors.

ACKNOWLEDGMENTS

This project is a cooperative industry-research-agency effort.
Shellfish industry partners provided culture sites, shellfish product
(single seed, cultched seed, and mature oysters), material and sup-
plies, laboratory support, and field staff for site setup. They in-

cluded Taylor Shellfish, Inc.: Jamestown S'Klallam Tribe; Chelsea
Sea Farms; and Hog Island Shellfish Farm. Scientific contributors
included: Gary Cherr and Amro Hamdoun at the University of
California at Davis. Bodega Marine Laboratory: Rita Horner, Uni-
versity of Washington Department of Oceanography; and Sher-
wood Hall, U.S. Food and Drug Administration, Office of Sea-
food. This paper is funded by a grant from the National Oceanic
and Atmospheric Administration. The views expressed herein are
those of the authors and do not necessarily reflect the views of
NOAA or any of its sub-agencies.

LITERATURE CITED

Allen. S. K.. S. L. Downing & K. K. Chew. 1989. Hatchery manual for
producing triploid oysters. Washington Sea Grant Pub). WSG 89-3. 27
pp.

Beattie. J. H. 1984. Effects of growth and mortality differentials on pro-
duction among selected stocks of the Pacific oyster Crassosirea gigas
Thunberg. / Slwllfish Res. 5:49.

Elston. R. A. 1993. Infectious diseases of the Pacific oyster. Cnissostrea
gigas. Aniui. Rev. Fish Dis. 259-276.

Elston. R. A.. J. H. Beattie. C. Friedman, R. P. Hedrick & M. L. Kent.
1987. Pathology and significance of fatal infiammatory bacteraemia in
the Pacific oyster, Crassosirea gigas. J. Fish Dis. 10:121-132.

Friedman, Carolyn S. 1990. Nocardiosis of the Pacific oysters, Crassosirea
gigas Thunberg (Oy.ster). Ph.D. dissertation. University of California.
Davis, California. 135 pp.

Friedman, C. S.. J. H. Beattie. R. A. Elston & R. P. Hedrick. 1991. Inves-
tigation of the relationship between the presence of a Gram-positive
bacterial infection and summer mortality of the Pacific oyster. Cras-
sosirea gigas Thunberg. .^quactillure 94:1-15.

Glude. J. B. 1975. A summary report of the Pacific Coast oyster mortality
investigations 1965-1972. Proceedings of the Third U.S.-Japan Meet-
ing on Aquaculture at Tokyo, Japan. October 15-16, 1974. p. 28.

Imai, TK. Numachi, J. Oizumi & S. Sato. 1965. Studies on the mass
mortality of the oyster in Matsushima Bay. II. Search for the cause of
mass mortality and the possibility to prevent it by transplantation ex-
periment, (in Japane.se, English summary). Bull. Tohokii Regional Fish.
Res. Uib. 25:27-38.

Kittel, M. T. 1998. Comparative analysis of Tasmanian Pacific oysters
Crassosirea gigas after grow-out in Washington State. World Aqua-
culture Society, Aquaculture '98, Las Vegas, Nevada, p. 297.

Koganezawa, A. 1974. Present status of studies on the mass mortality of
cultured oysters in Japan and its prevention, pp. 29-34. In: Proceedings
of the Third U.S.-Japan Meeting on Aquaculture. Tokyo, Japan, Oc-
tober 15-16, 1974.

Nicolas, J. L., M. Comps & N. Cochennec. 1992. Herpes-like virus infec-

tion of Pacific oyster larvae, Cras.soslrea gigas. Bull. Eur. .\ssoc. Fish
Palhol 12:11-13.

Nightingale, H. W. 1936. Red water organisms, their occurrence and in-
fluence upon marine aquatic animals with .special reference to shellfish
in waters of the Pacific Coa.st, Argus Press. Seattle, Washington. 24 pp.

Numachi, K. & J. Oizumi. 1965. The pathological changes of the oyster
caused by Gram-positive bacteria and the frequency of their infection.
Bull. Tohoku Reg. Fish. Res. Uib. 25:39-47.

Perdue. J. A. 1983. The relationship between the gametogenic cycle of the
Pacific oyster, Crassosirea gigas and the summer mortality phenom-
enon in strains of selectively bred oysters. Ph.D. dissertation. Univer-
sity of Washington. Seattle. Washington. 205 pp.

Perdue, J. A.. J. H. Beattie & K. K. Chew. 1981. Some relationships be-
tween gametogenic cycle and summer mortality phenomenon in the
Pacific oyster (C. gigas) in Washington state. / Shellfish Res. 1:9-16.

Renault, T., N. Cochennec, R. M. Le Deuff & B. Chollet. 1994. Herpes-
like virus infection of Japanese oyster [Crassosirea gigas) spat. Bull.
Eur. Assoc. Fish Pathol. 14:64-66.

Scholz, A. J., R. E. Westley & M. A. Tarr. 1973. Pacific oyster mass mor-
tality studies: seasonal summary report no. 4. Washington Department
of Fisheries. 30 pp.

Schumacker, E. J, B. R. Dumbauld & B. E. Kauffman. 1998. Investigations
using oyster condition index to monitor the aquafic environment of
Willapa Bay Washington. World Aquaculture Society, Aquaculture
'98, Las Vegas. Nevada, p. 478.

Sindermann, C. J. 1990. Principal diseases of marine fish and shellfish, vol.
2. Academic Press. San Diego, California. 521 pp.

Steidinger. K. A. & K. Tangen. 1996. Dinoflagellates. pp. 387-584. In:
C. R. Tomas (ed.). Identifying Marine Diatoms and Dinoflagellates,
Academic Press, San Diego, California.

Tamate, H. K., K. Numachi, K. Mori, O. Itikawa & T. Imai. 1965. Studies
on the mass mortality of the oyster in Matsushima Bay: pathological
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Journal ofShettfish Rcsi-ciirh. Vol. ly. No. 1, 361-364, 2000.

THE EXPERIMENTAL ANALYSIS OF TIDAL CREEKS DOMINATED BY OYSTER REEFS:

THE PREMANIPULATION YEAR

RICHARD DAME,' DAVID BUSHEK,^ DENNIS ALLEN,^
DON EDWARDS,' LEAH GREGORY,' ALAN LEWITUS,^
SARAH CRAWFORD,' ERIC KOEPFLER,' CHRIS CORBETT,"*
BJORN KJERFVE,^ AND THEO PRINS' "^

^Depcirtincnt of Marine Science

Coastal Carolina University

Conway. South Carolina 29528
'Belle W. Baruch Institute for Marine Biology & Coastal Research

University of South Carolina

Georgetown. South Carolina 29442

Department of Statistics

University of South Carolina

Columbia. South Carolina 29208
"^Department of Marine Science

University of South Carolina

Columbia. South Carolina 29208
'Rijkes Institute for Coastal Research

4330 EA Middelburg

The Netherlands

ABSTRACT We report here the e.xperiniental design and observations from the premanipulation year of an ecosystem-level study
investigating the hypothesis that oyster reefs control the structure and function of intertidal creeks. A group of eight tidal creeks in
North Inlet. South Carolina, USA, dominated by oysters, Crassostrea virginica (Gmelin), were studied using a replicated BACI
(Before-After Control-Incident) design in which all creeks are sampled simultaneously. Before the start of the premanipulation year,
oyster biomass in the creeks was adjusted to 8 g db/m'. Detailed geomorphological observations were made on each creek as the study
began. Nutnents and chlorophyll a were measured weekly in each creek and exhibited seasonal and interannual influences. Intensive
planktonic-inicrobial loop samplings were conducted seasonally and suggested a diatom-dominated winter community controlled by
nutrient availability and a microtlagellate-dominated summer community controlled by grazing. Nekton biomass exceeded oyster
biomass in most creeks during the summer. As expected, oyster growth decreased from summer to winter, and survival was higher in
winter. In the study's second, or manipulation year, the role of oysters will be tested by removing them from four creeks.

KEY WORDS: ecosystems, estuarine, oysters, creeks, plankton, nekton, field experiment, microbial loop

INTRODUCTION Despite numerous speculations and scaled-up estimates of

the influences of filter-feeding reefs on estuaries and creeks (Clo-
em 1982, Officer et al, 1982. Dame et al. 1986. Newell 1988,

Within southeastern Atlantic Coast marsh-esiuarine ecosys-
tems, tidal creeks are channels that allow the movement of organ-

isms and materials between the marshes and mud-tlats and the Alpine and Cloem 1992, Ulanowicz and Tuttle 1992), no compre-

deeper portions of the estuary. Intertidal oyster reefs are promi- hensive in situ ecosystem-scale expenments have been performed

nent, intensely heterotrophic components of these creeks. These '« observe these impacts directly. We report here the experimental

reefs can make an impact on biodiversity and productivity in design and premanipulation year observations of a field study that

h ne sv terns hv '^'''^ '^^ hypothesis that oyster reefs control the structure and tunc-

""T 7rovkiing three-dimensional (3-D) structures that increase 'io" o*' "dal creeks in which they are a dominant feature. When

habitat heterogeneity and supply space to support diverse '-omplete, this study (known as CREEK) will have compared eco-

assemblages of benthic and nektonic organisms (Wells system-scale differences among eight tidal creeks before and alter

1961, Dame 1979. Tsuchiya and Nishihira 1986, Zimmer- 'he removal ot oysters from tour ot the creeks,
man et al. 1989. Breitburg et al. 1995, Wenner et al. 1996.
Breitburg 1999),

2. modifying tidal creek morphology and hydrodynamics by Environmental Setting
structurally altering creeks, changing tidal flow patterns, and

increasing water residence times (Keck et al. 197.3. Prey and The observations used in this investigation are from a group of

Basan 1978. Bahr and Lanier 1981, Lenihan et al. 1996), tidal creeks located in the near pristine North Inlet marsh-estuarine

3. filtering large amounts of particulate material from the water ecosystem. North Inlet (33' 20' N, 79" 10' W) is located near the
column and releasing large quantities of inorganic and or- city of Georgetown on the northeastern coast of South Carolina,
ganic nutrients into creek waters (Haven and Morales- USA. The approximately 3.400 ha system is composed of salt
Alamo 1970, Newell 1988, Dame et al. 1989. Dame 1993, marshes dominated by Spartina atterniflora (2,500 ha) and tidal
19%). creeks with intertidal oyster reefs (850 ha). A coastal maritime

361

MATERIALS AND METHODS

362

Dame et al.

Replicated BACI Design

Before

After

^C1

X,,

Control
Sites

Impact
Sites

t

time

Intervention point

Figure 1. A graphical representation of the Before-After Control-
Incident (BACI) statistical design.

forest borders the estuary and generates intermittent fresliwater
streamflow from approximately 1.000 ha. The climate of the area
is subtropical, with average water temperatures ranging from 8 °C
in January to 30 °C in July and August. The system is subject to
semidiurnal tides, with an average range of 1 .5 m. North Inlet has
no salinity stratification and usually has very little freshwater in-
put. Owing to shallow water depths and vigorous tidal exchange,
creeks in North Inlet are well mixed and nearly always vertically
homogeneous with respect to dissolved substances.

Statistical Considerations

We use a replicated BACI {Before-After Control-Impact) de-
sign (Fig. 1 ). with eight similar tidal creeks as replicates. Creeks
were assigned to one of four blocks based on their physical loca-
tions within the estuary and suspected or known spatial differences
at this scale. Blocking was deemed important because Clambank
Creek creeks drain an upland area; whereas. Town Creek creeks do
not border any uplands, and because there is a salinity gradient
from north to south with those creeks further south more likely to
experience low salinity spillover from Winyah Bay during fresh-
ets. The Before manipulation year began in March 1997 and ended
in February 1998. The After manipulation year began in March
1998 following the removal of oysters from four randomly se-
lected creeks, two each in Clambank Creek and Town Creek. Thus,
the CREEK study satisfies a number of concerns raised by Hurl-
bert (1984): (1) there are control creeks: (2) the creeks are repli-
cated: and (3) the creeks are sampled repeatedly, both before and
after the intervention. In addition, the design heeds the recommen-
dation of Stewart-Oaten et al. (1986) by sampling all creeks si-
multaneously. The statistical analysis after the intervention year is
an adaptation of Stewart-Oaten et al.'s ( 1986) proposed analysis.
This paper describes only the before or premanipulation year to
highlight system variability and to identify potential sources of that
variability.

Creek Geomorphology

The eight tidal creeks used in this study are located on two
larger order creeks, Town Creek and Clambank Creek, and are
within I km of each other (Fig. 2). The observed creeks are typi-
cally ephemeral (i.e.. dry at low tide). A detailed topographic-
bathymetric survey of each creek and its basin was conducted

Ma4 '

^^^^^^^^^I^H

CREEK 4

CREEK 2 ^K

CREEKS

CREEK 3

Plin||

CREEK 6

TOWN

j^^^^^^^mRg' CREEK 1

CREEK

CREEK

CREEK 7

CREEK 8

■^^^3

1

Figure 2. .\n arcal depiction of the study area.

Tidal Creek Experiment

363

TABLE 1.
Structural dimensions of the eight experimental tidal creeks.

Creek

Dimension

1

T

3

4

5

6

7

8

Length (m)

177

1(11

164

229

138

232

174

423

Length u/tributaries (m)

359

IM

284

254

153

306

309

517

Width at mouth (m)

7.6

5.8

6.7

4.6

4.9

3.1

6.7

9.5

Cross-sectional area at moi

th

(in")

5.4

3.2

4.7

2.5

2.5

2.6

4.8

5.7

Water volume (m')

667

321

527

520

446

391

623

1 .423

Utilizing a Topcon total station. All elevations were referenced to
a common datum that, in turn, was referenced to eight U.S. Geo-
logical Survey permanent benchmarks. The data were used to es-
timate creek length, width, cross-sectional area at mouth, suiface
area, and water volume.

Physical and Chemical Variables

Beginning in early March 1997 and continuing until late Feb-
ruary 1998. water samples were taken once a week from each
study creek for chemical analysis. The samples were taken ap-
proximately midway between the daytime high and low tide
stages. Water samples were taken from the center of each creek
mouth at a depth of 1 m below the surface but not closer than about
0.3 m from the bottom. Triplicate samples were collected from
each creek, and all creeks were sampled within 45 niin. The
samples were immediately placed in ice and rushed to the labora-
tory for analysis. Temperature was measured at each site as
samples were collected. Salinity and concentrations of ammonium,
nitrate -i- nitrite, orthophosphate. and chlorophyll a were deter-
mined using standard techniques. All water chemistry parameters

were logarithmically transformed to ensure equal variability, but
temperature and salinity observations were not transformed. The
Grubbs test (Grubbs and Beck 1972) was used to determine out-
liers; only two observations were deleted.

Plankton and Microbial Loop Observations

The planktonic food web in the experimental system was ex-
amined using a series of bioassay experiments. These studies were
conducted at a morning tnid-ebb tide on five dates (March 20. June
13. July 27, September 13. and December 9) in 1997. Replicate
samples were collected at each of the eight experimental creeks
and dispensed into 1-L acid-cleaned polycarbonate bottles.
Samples were incubated under various treatments designed to ex-
amine the effect of substrate enrichment or reduced grazing pres-
sure on phytoplankton community biomass (chlorophyll a). The
treatments included 4 (j.m NHj addition. 20 ixm glycine addition,
and a 20: 1 dilution treatment used to reduce grazing pressure on
phytoplankton by decreasing encounter rates between microzoop-
lankton and phytoplankton prey (Landry and Hassett 1982). Le-
witus et al. (1998) have found, from experiments involving serial

Creek 3

1 sq meter mesh, 2x vertical exaggeration

Figure 3. An example of a 3-D surface plot for Creek 3 showing geomorphological relief.

364
ppt

Dame et al.

CREEK SALINITY
1997-1998

38

18--

14 ■■

10

34+ ♦

♦ ♦

♦ ♦ ♦ ♦ ♦

♦ ♦ ♦
30 + ♦ ♦ ♦ ♦

♦ ♦

♦ ♦ ♦ ♦

26 + ♦ ♦ ♦

♦ ♦

♦ ♦

22+ ♦

♦
♦

♦ ♦

♦ ♦

♦ ♦ ♦

♦ ♦ ♦ ♦

♦ ♦♦♦♦♦ ♦ ♦

♦ ♦ ♦ ♦

♦ ♦ ♦ ♦ ♦ ♦

♦ ♦ ♦

♦ ♦ ♦

♦ ♦

♦ ♦

♦ ♦

♦ ♦ ♦ ♦

♦
♦

♦ ♦ ♦ ♦

♦ ♦♦♦ ♦ ♦♦♦♦
♦ ♦ ♦ ♦

♦ ♦

♦

♦ ♦

♦ ♦

♦

♦
♦
♦

♦ ♦ ♦

♦ ♦

♦

♦ ♦

♦ ♦

♦
♦

+

+■

+■

+

Mar-97

May-97

Jul-97

Nov-97

Jan-98

Sep-97
DATE

Figure 4. Time plot of salinity data (ppt) from the eight experimental tidal creeks during the premanipulation year of 1997 to 1998.

dilution of North Inlet water, that a 20:1 dilution fell within the
range where grazer reduction over 72 hours was saturated. Bottle.s
were incubated in raceways containing flowing estuarine water to
simulate tidal creek temperatures. Overhead fluorescent cool white
bulbs provided uniform irradiance adjusted to a light-dark cycle
simulating natural conditions. Water samples were mechanically
stirred (gently) at uniform rates between bottles. Chlorophyll a was
measured daily at midday over the 72-hour time course.

Nekton Abundance and Biomass

Nekton abundance and biomass were determined for each creek
in March. June. August, and November of 1997. Simultaneous
collections of nekton were made with block nets set at early morn-
ing slack high tide at all eight creek mouths. Catches were re-
moved from the block nets, and pools within each creek bed were
seined at low tide to provide a complete assessment of fish and
motile macroinvcrtebralc use of the creeks. All samples were fro-
zen and subsequently sorted to the species level. Total abundance
and biomass were deterinined for each species. Up to 100 indi-
viduals of each species were measured to the nearest 2.0 mm. Total
wet weight biomass per cubic meter of water volume was the
primary unit used to compare catches from creeks with different
volumes. A factor of 0.25 was used to convert wcl weights to dry
weights (Caspers 19.57).

Oyster Biomass, Growth, and Siirviral

Before the premanipulation year observations began, oyster
biomass in each creek was estimated from 10 (.|uatlrats (0.2.5 nrl

distributed at different elevations along the length of the creek.
Oyster biomass in each creek was adjusted to an average of 8 g dry
body weight/m'' of water. The grams dry body per m^ relationship
was used, because it more realistically describes the benthic-
pelagic coupling of the oysters to the water column (Dame 1993).
During the premanipulation year, oyster growth and survivorship
were observed by placing plastic-mesh bags containing 25 marked
and measured oysters in each of the eight e.xperimental tidal
creeks. Because the creeks are ephemeral, and tidal exposure is a
critical factor in bivalve physiology, bags were placed at four
locations approximately equidistant along the mainstem of each
creek at approximately the same measured elevation. Summer ob-
servations were made between July and October and fall— winter
observations were from October to February. Growth was mea-
sured as change in length, measured to the nearest 0.1 mm. Bio-
mass to length relationships determined by Dame (1972) were
used to calculated dry body biomass.

RESULTS

Creek Geomorphology

The detailed topographic-bathymetric survey of each of the
experimental creeks was used to generate longitudinal profiles,
hypsometric curves, storage curves, and 3-D surface maps. The
results of these surveys are summarized in Table 1 . and an example
ot a 3-D surface map is given in Pig. 3. The creeks range in length,
includini; branches, from 1 53 to 5 1 7 m. and hankfull w ater volume

TiDAi. Creek Experiment

365

ranges from 23 1 to 1 .423 m' (Table 1 ). The surface-t- volume ratio
by creek ranged from 1.3 to 2.6. There was less than an order of
magnitude difference for any measure between creeks.

Physical Environment

Temperature ranged from 8 to 35 'C and displayed a well-
defined seasonal trend. Analysis of variance (ANOVA) of all tem-
perature observations shows that date (time) explains 99.2% of the
variation in these data (P < 0.01). Salinity data ranged from 12 to
36 ppt (Fig. 4). There was a distinct period of lower and more
variable values in all creeks during the December 1997 to March
1998 period. While 91.1% of the variability in the salinity obser-
\ ations were attributed to date, a significant (P > 0.01 ) amount of
variability (1.1%) was attributable to creeks.

Water Chemistry

Chlorophyll a concentrations range from near zero in the winter
to about 42 (j-g/L in the summer, and depict an annual curve when
plotted (Fig. 5). Results of the ANOVA of the premanipulation
year data show that 91.6% of the total variability was attributable
to time of year and 3.8% was related to creek differences. There
were significant differences among dates and blocks of creeks, but
not among creeks within blocks. Ammonium concentrations are

shown in Figure 6. Values are near zero in autumn and winter and
are near 7 |xm/L in spring and summer. ANOVA revealed that
about 84% of the variability was attributable to date and 4.4% was
attributable to creek. Blocks of creeks were not significantly dif-
ferent; however, there were cases where creeks within blocks were
different. Phosphorus concentrations were low (near zero to about
1.5 (j.m/L). with maximum values in summer and minimum in
winter. Concentrations of nitrate + nitrite were also low (near zero
to 2.7 |jLm/L), with maximum values and variability in winter and
spring. ANOVA showed that variability of nitrate-i-nitrite and
phosphate was similar, although less of the variability in
nitrate-l-nitrite was explained.

Plankton

The seasonal patterns exhibited by chlorophyll a in response to
NH"* addition, glycine addition, or dilution were generally similar
among creeks (Figs. 5 and 6). For water collected from March to
July, the addition of NHj occasionally stimulated chlorophyll a
concentrations in the water from Clambank tributaries (creeks 1—1)
but not Town Creek tributaries (creeks 5-8), which is consistent
with the lower ambient NHj concentrations in the former group
(Fig. 7). During the summer months, the Town Creek tributaries
averaged higher concentrations of NHj than those of the Clambank

MQ/I

CREEK CHLOROPHYLL a
1997-1998

50

40-.

30--

20--

10--

+
+

+
t +

Ittn^*^

+ ;:;■ + [:*:: + *

+

+

Mar-97

May-97

Jul-97

Nov-97

Jan-98

Sep-97
DATE

Figure 5. Time plot of chlorophyll a concentrations (pg/L) from the eight experimental tidal creek.s for the premanipulation year of 1997 to 1998.
(triplicates shown).

366

Dame et al.

|im/l
12

CREEK AMMONIUM
1997-1998

10--

8--

6-.

4..

+

+

^ + u
+ + ■■ *

+ + ±

i i 1*1 p

tj±

* T' i}^:^

JIP + T

+

Mar-97

May-97

Jul-97

Nov-97

Jan-98

Sep-97
DATE

Figure 6. Time plot of ammonium concentrations (nm/L) from the eight experimental tidal creeks for the premanipulation year of 1997 to 1998.
(triplicates shown).

tributaries. Dissolved organic carbon (DOC) concentrations were
lower in the Town Creek group during this same period.

In almost all cases, the dilution treatment led to greater stimu-
lation of chlorophyll production than either of the substrate addi-
tions. The dilution effect was greatest in the summer when nano-
phytoplankton dominated the community and minimal during the
winter and early spring when a microplanktonic diatom-dominated
community regulated by nutrient availability was prevalent (Fig. 8).

Oysters

Oyster growth was higher in summer than in winter and was
not significantly different between creeks (Fig. 9). Oyster survi-
vorship was higher in winter than summer, and no significant
differences between creeks were e\'idcnt (Fig. 10).

Nekton

Seasonal variations in total nekton biomass were consistent
among creeks. Biomass was highest in all creeks during the sum-
mer (Fig. I 1). Lowest biomasses were obser\ed in March, when
water temperatures and salinities were lowest. During the summer
months, nekton biomass is generally equal to or as much as five
times greater than oyster biomass; whereas, the reverse is true in
winter. More than 60 species of fishes, shrimps, and crabs (mostly
young of the year) were identified. Seasonal shifts in species domi-

nance were strong and consistent among creeks. Each season, the
same creeks supported the highest or lowest nekton densities.
These differences seem to be related to geomorphological differ-
ences among the creeks.

DISCUSSION

The choice of the appropriate statistical methods for detecting
trends and perturbations in ecosystems has been a lively area of
debate in the ecological literature (Hirsch el al. 1991). Green
(1979) proposed a multifactor ANOVA approach for detecting
changes in a single stream for which there are observations up-
stream and downstream of the perturbation. Green's design was
criticized by Hurlbert (1984) for lacking spatial and temporal rep-
lication. Hurlhcrl introduced the lerm pscudoreplication to de-
scribe multiple measurements of a single study site versus what he
considered true replication: measurements of multiple study sites
at multiple time points. Stewart-Oaten et al. (1986) refined Green's
( 19791 approach by proposing that the difference between control
and impact or manipulated sites measured at multiple lime points
before and after the nianipulation was the appropriate response
variable for analysis. They called this approach BACI.

Although salinity observations seem to provide evidence of the
strong inlerannual FNSO climatic event of 1997 to 1998, the tem-
perature data only reflect a normal seasonal pattern. ENSO events

Tidal Creek Experiment

367

Creeks 5 and 6

Mar

Jun

Sept Dec

-NH4
-Glycine
- Dilution

«» c

C <l

'" *^ -C

ffl C -^
u o (>'

if"
111

CD S O

c S o
.2 i

C

-1

Creeks 3 and 4

Mar

B

Sept Dec

Creeks 7 and 8

D

2

^

^

1

Q

/ J

-4

s

1

Mar Jun Jul

Sept

Dec

-1

Figure 7. Results of addition and dilution incubation experiments on tlie planlttonic community in the eight experimental creel^s during the
premanipulation year 1997 to 1998. A value of 0.(1 indicates no difference in chlorophyll a between experiment and control after 72 hours of
incubation, and a value of 1.0 indicates a 100% increase in chlorophyll a in the experiment.

Summer

v:^^^^^^^*

Light-limited
Hig/i NH4, DON

Flagellates
Picoplankton

Regenerated NH4 +DON

Winter/Spring

Light t-sa tura ted
Low NH4, DON

Figure 8. A graphical representation of the two different states of the planktonic web within the premanipulated creeks. Solid arrows indicate
stronger relationships.

368

Oyster Growth

Dame

ET AL.

g db/m^

50

□ Summer
■ Winter

45
40
35

30

-

25
20

i

T

15

1

1

10

1

1

5

1

1

0

MACROFAUNAL BIOMASS - 1997

12 3 4 5 6 7 8
Creek

Figure 9. Oyster growth in tlie experimental creeks during winter and
summer seasons in the premanipulation year 1997 to 1998.

are thought to influence the southeastern Atlantic coast by increas-
ing winter precipitation and decreasing air temperatures
(Ropelewski and Halpert 1986. Philander 1990. Hanson and Maul
1991 ). Although higher than normal freshwater discharge to south-
eastern estuaries with a higher groundwater table should be ex-
pected during these events, these features were only statistically
observed from the northeastern coast of South Carolina to Florida
(Kuhnel et al. 1990). During the 1997 to 1998 El Nifio. the coastal
zone of the Carolinas received over 200% of normal precipitation
for this period. In North Inlet, a normally euhaline estuary, the
increased rainfall depressed salinities below 15 ppt for one 3-week
period during the December 1997 to March 1998 period. It is
probably this precipitation that depressed salinities and increased
the variability of our salinity observations during the winter period.
Chlorophyll a concentrations are a measure of the major phy-
toplanktonic food source of the dense oyster populations within
North Inlet. The data in Figure 5 are similar to those reported
previously for North Inlet (see Lewitus et al. 1998). There seems
to be a seasonal transition in the microbial food web structure and

Oyster Survival

□ Summer
■ Winter

1

8

2 3 4 5 6 7
Creek

Figure Id. .Summer and winter oyster survival in the cxpcrimentiil
creeks during the premanipulation year 1997 (o 1998.

NEKTON
biomass

■ March

■ June

□ August
Q November

12 3 4 5 6 7 8

CREEK

Figure 11. Seasonal variations in nekton biomass (g db/m^) in the
eight experimental creeks during the premanipulation year 1997 to
1998.

regulation, from a microplanktonic diatom-dominated community
regulated by nutrient supply during the winter to a nanoflagellate-
prevalent phytoplankton bloom regulated by microzooplankton
grazing in the summer (Fig. 7). The generally consistent lack of a
nitrogen response by the phytoplankton strengthens the possibility
that oyster removal may lead to nitrogen limitation.

Ammonium is the major inorganic nitrogen source in North
Inlet, because the estuary is a bar-built, high-salinity salt marsh
with little freshwater input. This form of inorganic nitrogen is also
a major excretory product of oysters and. thus, a major component
in the regeneration of nitrogen in this system (Dame 1993). Ni-
trogen is typically limiting in many coastal and estuarine systems
( i.e., there is an inverse relationship between nitrogen and chlo-
rophyll during the bloom period); however, this is not the case in
North Inlet. In historical data from North Inlet (Lewitus et al.
1 998), and in the data presented here, there is a positive correlation
between chlorophyll a (Fig. 5) and ammonium (Fig. 6) concentra-
tions. This relationship suggests that ammonium is not limiting
during the summer bloom and that other factors, such as grazing,
are important (Lewitus et al. 1998). Concentrations of nitrate +
nitrite are elevated during the same winter period as reduced sa-
linities, suggesting terrestrial runoff as a source and nutrient limi-
tation as a control on the winter phytoplankton.

Although nekton use of the intertidal creeks was not uniform in
space or time, the occurrence of spatially stable patterns suggests
that temporally stable features of the physical habitat were impor-
tant determinants of use. Geomorphological and associated hydro-
graphical features of intertidal creeks may be key attributes of
habitat quality as it relates to the nursery function of salt marsh
channels. High nekton biomass may, in turn, exert significant in-
tluenccs on water quality criteria that affect the creek system (i.e..
NH"* concentrations). The cquixaleni magnitudes of nektonic and
oyster biomass within these tidal creeks implies thai there may be
a much more complicated and hitherto untorescen control of nu-
iricnt cycling within these syslenis.

The premanipulation year studies reported here show that: ( I )
annual and interannual environmental cycles are evident; (2) no
l\v 11 creeks are the same, but they are all similar in that S.^^r of the
observed variability was explained by date; (3) the plankton com-
nnmilv exists in Iwo stales, which are regulated differently; (4)

Tidal Creek Experiment

369

oyster growth and survivorship are nomiah and (5) nekton are
much more important than previously thought and may match
o\ sters in their influence on tidal creek systems. These observa-
tions will certainly be elucidated by the results of the postmanipu-
lation (oyster removal) year.

ACKNOWLEDGMENTS

The authors express their gratitude and heartfelt thanks to all of
the students and volunteers who have participated in this project.
Their efforts, energy, and stimulus were invaluable. The project
was supported by an award (number DEB-95-0957) from the Na-
tional Science Foundation. This is publication No. 1209 of the
Belle W. Baruch Institute for Marine Biology & Coastal Research.

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Joiinwl of Shellfish Research. Vol. 19. No. 1. 371-377. 2000.

OYSTER REEF RESTORATION: CONVERGENCE OF HARVEST AND
CONSERVATION STRATEGIES

DENISE L. BREITBURG,' LOREN D. COEN,"

MARK W. LUCKENBACH,^ ROGER MANN/ MARTIN POSEY,^

AND JAMES A. WESSON*

The Academy of Natural Sciences

Estiiahne Research Center

10545 Mackall Road

St. Leonard. Mainland 20685
'South Carolina Department of Natural Resources

Marine Resources Research Institute

P.O. Box 12559

Charleston, South Carolina 29422-2559
^Eastern Shore Laboratory

Virginia Institute of Marine Science

College of William and Maiy

Wachapreague, Virginia 23480

School of Marine Science

Virginia Institute of Marine Science

College of William and Maiy

Gloucester Point. Virginia 23062

Department of Biological Sciences

University of North Carolina at Wilmington

Wilmington. North Carolina 28403
^Virginia Marine Resource Commission

P.O. Box 756

Newport News. Virginia 23607

ABSTRACT Oyster reef restoration, protection, and construction are important to meeting harvest, water quality, and fish habitat
goals. However, the strategies needed to achieve harvest and conservation goals have often been considered to be at odds. We argue
that these goals are. in fact, compatible and that the same strategies will promote a sustainable harvest of the resource, increased
filtration of estuarine waters, and increased provision of structured habitat for finfish. crabs, and other organisms that utilize oyster reefs
or receive benefit indirectly from them. Creation or designations of unharvested sites (refuge sites) are key components of these
strategies. Unharvested reefs have the potential to provide vertical relief, which is typically destroyed by harvest practices, to act as
a source of larvae, which potentially increases the supply of harvestable oysters, and to protect those individuals most likely to have
some resistance to disease. Furthermore, proper monitoring and design of refuge and restoration efforts are critical to providing
information needed to improve the success of future restoration efforts, and will simultaneously enhance the basic information needed
to understand the ecology of oysters and their role in estuarine and coastal systems.

KEY WORDS: oyster reef, restoration, water quality, harvest, fish habitat. Cnissoslrea virginica. sanctuaries

INTRODUCTION growth habitat for subsequent recmits to the oyster poptilation. In

addition, most harvesting practices are destructive to the reef ma-
Oyster reef restoration is a recognized need by resource agen- tri.x. reducing the vertical relief and damaging structural integrity
cies in most states along the Atlantic and Gulf of Mexico coasts of in excess of that caused by removal of the individual oysters ac-
the United States. In general, the initial impetus for these programs tually marketed (Hargis and Haven 1999. Lenihan and Micheli
has been declining harvests and standing stocks of oysters that are 1999). Shell repletion programs attempt to mitigate this habitat
at an all time low (MacKenzie et al. 1997a, MacKenzie et al. removal and destruction by adding shell as substrate for settlement
1997b. Luckenbach et al. 1999. Coen and Luckenbach 2000 and of oyster larvae. A consequence of these repletion efforts has been
references therein). Although numerous factors have been impli- a shift toward put-and-take fisheries (Coen and Luckenbach 2000).
cated in these declines, a consistent factor has been the destruction Recognition of oyster reefs as valuable estuarine habitats that
of reef habitat during the harvesting process (Hargis and Haven provide a range of ecosystem services is increasing (Coen and
1999. Lenihan and Micheli 2000). To date, most oyster restoration Luckenbach 2000, Coen et al. 1999b). The original goal of restor-
programs have focused on improving oyster habitat as a means of ing and enhancing fishery stocks has been augmented, and in a few
enhancing the commercial fishery (Luckenbach et al. 1999, Coen instances, superceded, by two additional goals: ( 1 ) improving wa-
and Luckenbach 2000). Harvest of oysters involves removal of the ter quality (by removing a portion of the phyloplankton standing
reef sub.slrate and. therefore, a decrease in available settlement and stock) and (2) providing a structured habitat thai may increase

371

372

Breitburg et al.

secondary production, including production of finfish and decapod
crustaceans, such as crabs (Fig. 1 ) (Wenner et al. 1996, Coen et al.
1999a. Coen 1999b). Extrapolations from laboratory filtration
rates (Newell 1988, Powell et al. 1992), direct field measurements
(Dame 1996 and references therein), and ecosystem-level model-
ing (Ulanowicz and Tuttle 1992) have clearly demonstrated that
oyster reefs can have significant impacts on material processing
and energy flow in estuarine systems. The recognition of the im-
portance of oysters' ability to reduce phytoplankton biomass as a
result of their filtering capabilities coincides with an increased
concern over eutrophication in coastal waters. Increased anthro-
pogenic loadings of nutrients make the ecosystem-level role of
suspension feeders (such as oysters) all the more critical at the
same time that overharvest and disease have reduced populations
through much of their range.

Furthermore, descriptive and experimental studies have pointed
to the importance of oyster reefs as habitat for commercially and
ecologically important fmfish and decapod crustaceans (see Wells
1961, Bahr and Lanier 1981. Stanley and Sellers 1986, Breitburg
1992, Breitburg 1999. Wenner et al. 1996. Coen et al. 1999a. Coen
1999b, Harding and Mann 1999). Although few specifics are
known about the relationships among oyster reef structure, oyster
population structure, and the provision of these ecosystem ser-
vices, it is likely they are related to the vertical relief of reefs, the

size and numbers of reefs, the overall estuarine habitat landscape,
habitat health, and the population density and age structure of
oyster populations. Seemingly, this sets up a conflict between the
goals of fisheries exploitation and those of ecological restoration
and conservation. With recent revisions to the Magnuson-Stevens
Fishery Conservation and Management Act (1996) this conflict
might be expected to intensify (Coen et al. 1999b).

In this paper, we address the challenge of simultaneously
achieving all three goals of oyster reef restoration (fisheries, water
quality, and habitat), highlight ecological processes that may make
the feasibility of meeting all three goals more or less difficult, and
discuss the potential benefits of melding research and restoration
activities. We emphasize our belief that these goals are generally
compatible and the importance of keeping all three goals in mind
to achieve sound habitat and resource management and restoration.
Many of the ideas in this paper stem from discussions at the special
session and workshop on oyster reef restoration organized by L.
Coen and M. Luckenbach at the 2nd International Conference on
Shellfish Restoration held in Hilton Head, South Carolina, in No-
vember 1998. Our intent is to summarize some of the major themes
and explore the constraints associated with sustaining the goals of
fisheries exploitation and habitat conservation, not to provide a
comprehensive review of the workshop and presentations or to
address all of the issues related to oyster restoration rai.sed therein.

Primary
production

Appropriate

physical

habitat

Sustainable
harvest

Improved
water
quality

Habitat for

fmfish, decapods

& benthic invertebrates

Protection of
other habitats

Requirements

Benefits of
Reef Restoration

Figure I. Resdinilion of oyster reefs has three primary goals: increasing sustainable harvests of oysters, improving water (|ualily through the
removal of phytoplankton hiomuss. and increasing structured hahllal utili/.ed by finfish, crahs. henlhic invertebrates, and (especially for
intertidal reefsl birds. In addition, studies by Meyer and colleagues indicate the possibility that oyster reefs can play a .significant role in reducing
shoreline erosion and protecting salt marsh habitat (see Meyer et al. 19%, Meyer et al. 1997).

Oyster Restoration for Harvest and Conservation

373

Although many areas of uncertainty remain, we believe a pattern
of con\ergence is emerging (see recent reviews by Lenihan and
Peterson 1998. Coen et al. 1999b. Luckenbach et al. 1999. Coen
and Luckenbach 2000).

COMPATIBILITY OF HARVEST AND ECOLOGICAL GOALS
OF OYSTER REEF RESTORATION

Are sustainable harvest and ecological goals of oyster reef res-
toration compatible? The relationships between production and
biomass. as well as between the fishery and ecological benefits of
unharvested refuge areas, contribute to our belief that the answer
is yes. Figure 2 illustrates the possible relationships between pro-
duction and biomass. Maximum production of a resource is
achieved at a biomass lower than the maximum potential biomass
because of proces.ses ranging from self-shading in phytoplankton.
to age-dependent growth declines, to prey depletion that occurs at
high population densities of consumers. In part, the degree to
which harvest and ecological values of reefs coincide will depend
on which of the family of curves depicted in Figure 2 best de-
scribes estuarine oyster populations. Maximum sustainable yield
strategies in fisheries generally focus on keeping a population near
its maximum rate of production but on the descending portion of
the curve (i.e.. biomass greater than that at maximum production),
where overharvesting of the resource is less likely to occur than
along the ascending portion of the curve (see Applegate et al.l998.

Restrepo et al. 1998 for a comprehensive discussion of these
curves in a fishery management context). Because maximum fil-
tration rates and maximum production are both related positively
to per capita growth rates (Powell et al. 1992, Hoffman et al.
1995), population densities producing high levels of sustainable
harvests should also be those that lead to a high (possibly maxi-
mal) ecological benefit of water filtration by oysters. Finally, al-
though less well understood, we argue that "more is better" in
terms of the habitat oysters provide for fish, crabs, and other
benthic organisms, but, as with the other goals, there is a decreas-
ing benefit portion of the curve. Something short of complete
coverage by oysters is needed to produce a diversity of benthic
habitats that includes soft bottom, submerged aquatic vegetation.
salt marsh, oyster reefs, and clam beds, where these have naturally
or historically co-occurred. As important, many fish and decapods
orient toward the edges of reefs and do not simply utilize the large
interior areas (Powell 1994. Breitburg 1999). It is critical to keep
in mind that even if the optimal biomass for harvest and ecological
goals do not coincide precisely, movement toward all three goals
requires increasing oyster biomass in most estuarine systems.

The more the production versus biomass curves are skewed to
the right (e.g., curve C rather than curve A in Fig. 2), the higher
will be the optimum oyster standing stock for a sustainable fishery
and the greater will be the coincidence between biomass levels
optimizing the filtration capacity of the oyster population and the
provision of habitat for other biota. Several features of oyster
biology, as well as ecological interactions among oysters, the

o
o

o

Oh

B II

max

Biomass

Figure 2. Relationship between production and biomass. Theoretical considerations suggest that maximum production will often occur at one
half the maximum biomass (.Applegate et al. 1998). However, interference competition and resource depletion can skew the curve to the left (.\),
and increased efficiency or reproductive success at high densities can skew the curve to the right (Cl. We suggest that under most conditions,
oyster populations will be described by curves B or C, making harvest, water quality, and habitat restoration goals compatible.

374

Breitburg et al.

physical environment, and other biota suggest a high-biomass-
high-productivity relationship, with greatest success for all three
goals occurring with well-developed or •'mature" high-relief reefs.
High density within oyster beds is likely the optimal condition for
the oysters themselves, because the preferred settlement substrate
for oyster larvae is oyster shell (e.g., Hidu 1969. Luckenbach et al.
1997, Bartol and Mann 1999), the fertilization success of sessile
animals is increased at high densities (Levitan 1991. Levitan et al.
1992). and the subtidal reefs will maintain greater vertical relief,
reducing sedimentation effects and enhancing local flow rates
(Lenihan and Peterson 1998). High aerial coverage by oysters
should provide insurance against the strong spatiotemporal vari-
ability in physical and biotic factors that can influence both spat set
and the health of adults (Lenihan and Peterson 1998). For systems
with limited water exchange and/or small tidal creeks with rela-
tively large tidal ranges (> 1-2 m). minimum reef area may be
essential for maintenance of local populations. In more open sys-
tems, increased cover may provide a buffer against local distur-
bances and recruitment variability.

IMPORTANCE OF HARVEST REFUGES

Unharvested (refuge) areas are critical to achieving both har-
vest and ecological roles of oyster reefs. Refuge areas protect
brood stock and. as a result, can enhance oyster populations in
surrounding harvested areas that are many times the size of the
refuge itself (Wesson 1998). Moreover, in areas affected by oyster
diseases, refuges provide protection for individuals that may have
some resistance to disease. In harvested areas, the largest oysters.
which are the individuals that have survived in the presence of
disease pressure and have the highest fecundity, are the ones culled
from the population (Rothschild et al. 1994. Coen et al. 1999b).
Protecting some reefs from harvest should, therefore, serve to en-
hance the vigor of stocks.

In addition, harvest-free sanctuaries allow reefs to develop and
retain vertical relief and structural complexity that are important to
both oysters and associated fauna. Vertical relief can provide oys-
ters with the means to avoid near-bottom oxygen depletion and
high sedimentation rates, and to take advantage of increased flow
velocity and enhanced growth rates (Lenihan et al. 1996, Lenihan
and Peterson 1998, Lenihan et al. 1999). In addition to reef eleva-
tion, vertical complexity of the reef itself (i.e., the presence of high
culms interspersed with low areas) enhances fish and decapod
utilization (e.g., Breitburg et al. 1995, Breitburg 1999, Coen et al.
1999b. Harding and Mann 1999, Posey et al. 1999. Coen and
Luckenbach 2()()()) and may protect oyster spat from predation
(Wesson 1998. unpubl. data, Giotta and Coen 1999). Because har-
vesting reduces vertical complexity, these habitat functions may
benct'it from creation of unharvested (refuge) areas (Coen et al.
1999b. Lenihan and Micheli 1999). However, there is also a view
that some thinning may enhance intertidal oyster populations
(Lenihan and Micheli 1999, W. Anderson, South Carolina Depart-
ment of Natural Resources, pers. comm.).

Refuges also provide a tool at the landscape level that allows
reefs to be placed in areas that are protected or closed to harvest
and that will maximize desired functions (reviewed in Lenihan and
Peterson 1998, Coen et al. 1999b, Luckenbach et al. 1999. Coen
and Luckenbach 2()()()). For example, low-salinity refuge areas in
the Maryland portion ol the Chesapeake Bay are designated to
protect oyster brood stock in areas generally unaffected by either
Perkinsus (Dermo) or Haplosporidiiim (MSX) (Bushek and Allen
1996a.b. Paynler 1999, Coen and Luckenbach :()()()). .Similarly.

designated areas closed to direct harvesting for health reasons may
act as refugia as an indirect result of their value as habitat and
brood stock reserves (Coen and Luckenbach 1999).

SPATIAL CONSIDERATIONS

There is still much to be learned about the importance of the
location of restored oyster reefs within an estuarine landscape
(Posey et al. 1998, Coen et al. 1999b). Whitlach and Osman (1999)
have developed a metapopulation demographic model of oyster
populations that illustrates the importance of dispersal between
spatially distinct subpopulations to the persistence of oyster reefs.
The foregoing discussion about brood stock sanctuaries and the
dispersal of larvae from them to nearby reefs clearly illustrates the
importance of reef position within a landscape to the development
of reefs and potential fisheries production. Further, the location of
reefs will affect the ecosystem services that they provide (see
Lenihan and Peterson 1998, Coen et al. 1999b, Coen and Luck-
enbach 2000). For instance, restoring or constructing reefs in lo-
cations key to intercepting waters with high nutrient loadings and
the associated high phytoplankton biomass should be possible.
Similarly, the proximity to other structured habitat may be impor-
tant to the function of oyster reefs (Micheli 1997. Coen et al.
1 999b). Reefs could be sited in areas with little or no other struc-
tured habitat so that they could function as important "stepping
stones" or migration corridors along the landscape. Alternatively,
if data indicate the advisability of doing so. reefs could be sited in
close proximity to other structured habitat to maximize interac-
tions and connections between, for example, submerged aquatic
vegetation or salt marsh grass and oyster reef assemblages.

A particularly intriguing ecosystem service provided by con-
structed oyster reefs adjacent to salt marshes has been discussed by
Meyer et al. (1996. Meyer et al. 1997). In addition to providing
structured habitat for fauna, these reefs stabilize the creek banks
and reduce erosion of adjacent marshes (Meyer et al. 1996. Mayer
et al. 1997. Meyer and Townsend 2000). As more information is
gathered, the role of oyster reefs in erosion control may be deter-
mined to be as important as their other ecological services. Reefs
with substantial vertical relief that reach the surface of the water
may dissipate much of the energy generated where fetch on open
bodies of water allows substantial energy to accumulate.

Regardless of other spatial considerations for oyster reef res-
toration and creation, several aspects of the placement of reefs
within the landscape will intluence their success both in terms of
reef longevity and their measurable, short-term impact on the sur-
rounding habitat. Successful siting of reefs generally depends upon
locating substrate capable of supporting the added shell (without
rapid burial), and therefore, generally favors their construction on
tootprints of historical oysters reefs. In addition, placement of
brood stock sanctuaries should consider local circulation to maxi-
mize retention and recruitment of resultant larvae. This philosophy
has dictated the placement of constructed reef sanctuaries in the
Virginia portion of the Chesapeake predominantly in small sub-
csluaries with limited watersheds, small tidal excursions, and basin
topographies that encourage gyre-like circulation near the river
mouths (Haven et al. 1981. Southworth and Mann 2000. Wesson
unpubl. data).

STRATEGIES FOR RESTORATION

The harvest and eciilogical goals of oyster restoration are most
likely to be compatible where management efforts focus on the
ultimate goals, and the harvest is managed as a sustainable rather

Oyster Restoration for Harvest and Conservation

375

than a "put-and-take" fishery. For example, targets lor the amount
of acreage lor oyster restoration and protection could be set by
determining the \ olume of water to be filtered within a given time
or by determining the ratio of unharvested to harvested area re-
quired to sustain a target harvest quantity. We argue that such
goal-oriented target setting is more likely to achieve the desired
result than setting targets based upon historical oyster populations.
Moreover, it is important to consider that restoration efforts pro-
ceed one step (i.e.. one or a few reefs) at a time and that metrics
to gauge the success of these efforts need to reflect both the value
of the individual projects and their contribution toward the ulti-
mate goal. For instance, the harvest potential of an individual reef
expressed in terms of the biomass that may be harvested sustain-
ably per unit area (rather than as the number of bushels of market-
sized oysters in the standing stock) embodies both the productivity
of the reef and the total area necessary to achieve the desired
harvest levels. Similarly, the fishery value of a protected (unhar-
vested) refuge area based on its potential contribution to harvest in
other areas after allowing for a number of years of reef develop-
ment is a more reasonable assessment of the value of a refuge than
would be a simple calculation of the number of acres taken out of
the active fishery. Likewise, measures of the ability of a unit area
of reef to filter a specified volume of water or to support a speci-
fied biomass of tlnfish. decapods, shorebirds. or other target spe-
cies will be more useful metrics than attempts to define the con-
tribution of a single reef to the percent of the entire water mass
filtered each day or to the biomass of a particular fish within an
entire estuary.

LEARNING FROM RESTORATION EFFORTS

Restoration efforts, when properly designed and monitored,
present an unparalleled opportunity to improve our understanding

of both the optimal design for future restoration efforts and the
ecological role of oyster reefs in coastal systems (Table 1 ). There
are two key elements required to maximize the information from
restoration efforts. The first is careful planning in the design and
siting of reefs to match the restoration efforts with the information
desired. For example, in areas such as the northern portion of the
Chesapeake Bay and Delaware Bay where subtidal reefs were
likely the historical norm, there may be concern that reefs not
visible from but near the surface of the water may present navi-
gation hazards. However, constructing reefs in deep water (thus,
creating no navigation hazard) can expose oysters and associated
biota to low dissolved oxygen concentrations during summer. By
constructing and monitoring replicated reefs similar in size and
relief (and thus cost) at shallow and deep sites, the optimal depth
for reef placement in future restoration efforts could be deter-
mined. Simultaneously, important basic information could be gath-
ered on the similarities and differences in the oyster populations
and the ecological functioning of deep and shallow oyster reefs.
More generally, by designing restoration efforts to allow compari-
sons between reasonable alternatives, it becomes possible to an-
swer many important restoration questions. These include such
questions as: (1) Does the benefit (i.e., growth, recruitment, or
survival of oysters) derived from the construction of high vertical
relief beds outweigh the costs of constructing such reefs? (2) Do
oyster reefs placed near other structured habitats (such as SAV
beds or tidal marsh areas) have higher or lower habitat value for
finfish? (3) Is the extended "footprint" (i.e., area of increased
oyster recruitment surrounding restored reefs) greater near har-
vested or unharvested restoration sites? (4) Does the addition of
juvenile or adult brood stock oysters (either wild or hatchery-
reared) increase long-term productivity of a reef sufficiently to
justify the costs? (5) Does the benefit of oysters" water filtration

TABLE 1.
Examples of restoration efforts.

Restoration Action

Improvement in Restoration Practices

Improvement in Understanding
of Oyster Reef Function

1 . Reefs constructed at different depths

2. Reef construction using different base
materials

3. Reef construction with \'ar>'ing spatial
dispersion patterns

4. Position constructed reefs in varying
proximity to other landscape elements

5. Reefs constructed in areas with different
tidal ranges and water quality and
harvesting status

6. Reefs constructed with varying shapes
and vertical structure

Importance of reef depth to successful
restoration

Evaluation of alternative materials for
successful restoration

Aid in the placement and spatial
arrangement of restored reefs

Aid in the placement and spatial
arrangement of restored reefs

Aid in the successful restoration and
protection of habitats that might
otherwise not be protected or restored
successfully

Aid in the placement and construction of
restored reefs

Relationship between depth and
recruitment, growth and survival of
oysters and reef associated biota

Relationship between construction material
and development of oyster populations
and reef associated biota

Evaluation of the role of reef spacing
patterns in maximizing oyster
recruitment and providing habitat for
mobile species

Evaluation of the importance of reef
placement within a landscape for
achieving restoration goals

Enhance appreciation of EFH or critical
habitat roles; provide better
understanding of biogeographic
differences among sites differing in
physical regimes

Evaluation of reef morphology
relationships for habitat goals

Restoration efforts can be designed in ways that will provide information critical for improving future restoration work. In addition, they provide the
opportunity for large-scale ecosystem manipulations that may greatly improve the understanding of the structure and functioning of coastal systems. The
examples of these opportunities in the table are intended to be illustrative, not exhaustive.

376

Breitburg et al.

capabilities vary with location, depth, habitat type, shape, etc.? (6)
How do the shape and vertical complexity of reefs affect habitat
function? (7) How do the answers to these and other related ques-
tions differ among sites and systems (e.g., intertidal versus subtidal
oyster reef habitats, areas with significantly different tidal ranges,
etc.)?

The second element required to maximize information from
restoration efforts is the necessity for adequate monitoring to
evaluate their success (see discussions in Coen and Luckenbach
2000). The specific type and intensity of monitoring will be de-
termined by the goals of any particular restoration effort, the com-
parisons being made (as above), the target levels being set for
improved harvest and ecological benefits, and ultimately the avail-
able funding. In addition, evaluation of both the biological impact
of reef restoration (both harvest and ecological benefit goals) and
the economic considerations may often be important. Experiences
from the past several decades with restoration of other marine and
coastal habitats consistently point to the need for well-designed
monitoring studies to evaluate the success of restoration efforts
(see Thayer 1992). As pointed out by Zedler ( 1992), monitoring to
assess success and research that can help clarify how to meet
restoration goals, are often not supported adequately by the entities
that fund the actual restoration projects (discussed also in Coen
and Luckenbach 2000). A significant challenge for oyster reef
restoration efforts will be developing potential funding sources to
support both large-scale habitat manipulations and long-term
monitoring and assessment activities.

By combining carefully planned and targeted restoration efforts
with adequate monitoring of the results, it will be possible to
obtain information on topics about which little is known. Some of
these topics (see also Table 1 ) are: ( 1 ) the characteristics of oyster
reefs that are important for transient finfish and crab populations;
( 2 ) the area beyond the boundaries of the actual restoration effort
in which both oysters and associated biota are affected under a
range of hydrographic conditions: (3) the importance of the spatial
arrangement of reefs within an estuarine landscape: and (4) the
potential for oyster reefs to play a role in reducing shoreline ero-
sion. These are not simply topics of academic interest but relate to
the core goal of restoring oyster harvests as a sustainable fishery
and minimizing anthropogenic effects to our coastal systems. In
addition, among the most critical issues for future restoration ef-
forts may be the questions: Where can sufficient reef substrate be
obtained? and What oyster strains should be used to restore areas
where oysters have long been in decline? Alternative substrates
take on an increasingly significant role, as does the potential prob-
lem of introducing nonindigenous species or new disease strains
with the importation of oyster shell from other regions (Bushek
and Allen 1996a, Busheck and Allen I99(ib. Bushek 1997. Coen et
al. unpublished. G. Rui/ pers. comm.).

FlITURK .STKPS

Despite uncertainties surrounding many aspects of reef resto-
ration. It is important to move forward with restoration efforts: it
is clear thai reef restoration has the potential to provide strong
benefits to both the harvest and ecological functions of oyster reefs
in coastal systems. Most important, restoration efforts should tar-
get all three functions of natural reefs: harvest, the provision of
structured habitat, and the potential for improved water quality.
Rather than an ad\'ersarial relationship between fisheries and con-
servation interests in this regard, wc suggest there are enough
similarities of interests and approaches — especially the desire to
optimize the amount and location of settlement substrate — that

compatible strategies may be achieved. A critical feature of
achieving this compatibility will be clearly expressing the benefits
of reef restoration (depicted in Fig. 1 ). and relating each benefit in
a quantifiable way to reef and oyster production.

ACKNOWLEDGMENTS

We thank all participants for the free exchange of ideas that
contributed to the interesting and productive symposium and
workshop on oyster reef restoration at the 2nd International Con-
ference on Shellfish Restoration. In particular, we thank R. Dame,
D. Bushek, and H. Lenihan for comments and input into the or-
ganization of the session and associated workshop. Participation
by D. Breitburg, M. Luckenbach, and R. Mann was funded in part
by the US-EPA Chesapeake Bay Program. South Carolina Sea
Grant Consortium provided major funding (#NA46RG0484) for L.
Coen. North Carolina Sea Grant provided funding for M. Posey.
This is Contribution # 436 from the Marine Resources Research
Insfitute, SCDNR.

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Joiinial of Shellfi!'!' Reseiirch. Vol. 19, No. 1, 379-386. 2000.

USE OF OYSTER SHELL TO CREATE HABITAT FOR JUVENILE DUNGENESS CRAB IN
WASHINGTON COASTAL ESTUARIES: STATUS AND PROSPECTS

BRETT R. DUMBAULD,' EILEEN P. VISSER/
DAVID A. ARMSTRONG,^ LAUREN COLE- WARNER,'
KRISTINE L. FELDMAN,- AND BRUCE E. KAUFFMAN'

' Washington Department of Fish and Wildlife

Willapa Bay Field Station

P. O. Box 190

Ocean Park. Washington 98640
-School of Fisheries Box 357980

University of Washington

Seattle. Washington 98195
^Seattle District U.S. Army Corps of Engineers

P. O. Box 3755

Seattle, Washington 98124

ABSTRACT The deployment of oyster shell in estuarine intertidal areas to create habitat tor juvenile Dungeness crab {Cancer
magisier) is now used routinely as mitigation for '•unavoidable losses" of crab during dredging operations in Grays Harbor and Willapa
Bay along the southwest coast of Washington State. Feasibility studies were conducted in 1986 to 1987 for a U.S. Army Corps of
Engineers project to widen and deepen the navigation channel in Grays Harbor. Since that time, several studies have elucidated the
ecology of crab and other organisms that recruit to the created shell reefs. Studies have also refined the procedures used to calculate
crab losses caused by dredging and crab production in the shell habitat. The shell does serve as crab habitat: however, initial
assumptions about the longevity of the shell have proved to be overly optimistic, because the shell can sink or be covered with silt
before the end of the first summer after deployment. In addition, competition with the shore crab. Hemigrapsus oregonensis. has
displaced juvenile Dungeness crab. We summarize results of these studies and present initial results from an ongoing mitigation effort
that seeks to produce a more persistent living oyster reef in Willapa Bay.

KEY WORDS: Dungeness crab, dredging, mitigation, oyster shell, Washington State

INTRODUCTION

A large project that widened and deepened the navigation chan-
nel of Grays Harbor estuary in Washington State. USA (Grays
Harbor Navigation Improvement Project. GHNIP) continues to be
the impetus for numerous studies on the potential effects of dredg-
ing on the environment (USACOE 1982. USACOE 1989). Early
on. Dungeness crab {Cancer magister. Dana. 1852) was identified
as one of the most important species impacted by dredging opera-
tions in Grays Harbor (Tegelberg and Arthur 1977, Stevens and
Armstrong 1984). and numerous studies were conducted to define
those impacts (Armstrong et al. 1987, McGraw et al. 1988, Wain-
wright et al. 1992),

State and federal agencies directed the U.S. Army Corps of
Engineers (USACOE) to minimize the loss of Dungeness
crab caused by dredging or to mitigate for unavoidable losses
(USACOE 1989). Based on observations that shell deposits found
in the intertidal areas of Grays Harbor serve as habitat for newly
settled Dungeness crab (Armstrong and Gunderson 1985, Gunder-
son et al. 1990. Jamieson and Armstrong 1991. Eggleston and
Armstrong 1995). an extensive pilot study was conducted to dem-
onstrate that shell deposits could be artificially created by placing
oyster shell on the mudflats (Dumbauld et al. 1993). The habitat
created was expected to enhance intertidal recruitment and sur-
vival of juvenile Dungeness crab and provide an economical
means of mitigating for the loss of older, subtidal crab attributable
to dredging. The GHNIP was approved and dredging completed in
1990 by removing 10 million m'' of sediment from the navigation
channel in Grays Harbor. To the extent possible within the con-

fines of an economical dredging program (McGraw et al. 1988),
the USACOE scheduled dredging operations to coincide with low
crab abundance. Nonetheless, impacts occurred, and the USACOE
utilized a model to estimate the losses at 100,000 adult equivalents
(Wainwright et al. 1992). This represented less than 1% of the
estimated crab population, but this loss was significant enough that
USACOE was obligated to mitigate for the loss by creating inter-
tidal shell habitat. A series of large shell plots were created on the
inteilidal mudflats of Grays Harbor from 1992 to 1998 to mitigate
for the loss of crab caused by the initial construction of the chan-
nel, annual incremental maintenance, and other subsequent dredg-
ing projects.

In this paper, we review a pilot study and associated research
that led to the current policy of mitigating for the loss of Dunge-
ness crab by creating intertidal shell habitat in Washington State
coastal estuaries. Problems are described that continue to be ex-
perienced in the implernentation of the policy by constructing full-
scale shell reefs, and potential solutions that have been developed
based on continued research are summarized. We also introduce a
more recent project that seeks to mitigate for the loss of crab
attributable to a test dredging project in Willapa Bay, Washington.
A significant difference in the mitigation strategy for the latter
project is the attempt to take advantage of natural oyster recruit-
ment occurring in this estuary. This is designed to create a living
oyster reef much like the reefs being restored on the East Coast of
North America (discussed elsewhere in this symposium proceed-
ings). Our intent in this paper is to describe a novel approach to
creating habitat for a decapod crustacean on the West Coast of
North America, and also to highlight the perils and practical les-

379

380

DUMBAULD ET AL.

sons learned from implementing such large-scale restoration ef-
forts.

PILOT STUDY

Early observations indicated juvenile Dungeness crab recruit to
and survive in shell deposits (typically death assemblages from the
eastern softshell clam Mya arenaria L., 1758) and in aquaculture
areas where Pacific oysters (Crassostrea gigas. Thunberg. 1793)
are raised for human consumption (Armstrong and Gunderson
1985, Gunderson et al. 1990. Jamieson and Armstrong 1991 ). We
conducted a pilot study to test the hypothesis that additional shell
placed in the intertidal area of Grays Harbor. Washington, would
.serve as a refuge for and increase survival of juvenile crab, thereby
mitigating for substantial losses attributable to subtidal dredging
during construction of the GHNIP (Dumbauld et al. 1993). Oyster
shell was placed intertidally at three locations in Grays Harbor
(Fig. 1. small plots). At each location, three 15 x 15 m plots were
constructed, and treatments were; ( 1 ) heavy shell cover (2-3 shell
layers thick; (2) light shell cover (one shell layer thick); and (3)
shell piles (approximately 1 m in diameter and 0.5 m high). Crab
recruited to the shell plots from May to July and by late August
grew to fourth-sixth instars (J4-J6, 17-31 mm carapace width.

Grays Harbor

u
u

u

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0.

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Willapa Bay

South Bend

124'08'W

46* 24'N

CW). then presumably emigrated to the subtidal. Results indicated
that location within the estuary influenced crab abundance and that
the three-dimensional configuration of the shell was also important
(Fig. 2). Although there was statistical interaction between the
location and treatment terms, when low numbers and variable
results from the South Bay were removed from the analysis, it was
determined that crab density was significantly higher as three-
dimensional habitat increased in the sequence: shell piles > heavy
shell cover > light shell cover. Subsequent research has shown that
the shell habitat provides small crabs refuge from predation and
that the thick shell treatment also provides better habitat than ee-
Igrass (Fernandez et al. 1993).

The integrity of the shell habitat was measured 1 and 2 years
after shell placement. The heavy shell treatment seemed to survive
winter storms and sedimentation better than the piles treatment and
crab continued to use the plots in subsequent years, albeit in lower
densities. Because the heavy treatment was also the most practical
to implement on a large scale. USACOE chose this configuration
as part of the preferred alternative in the supplemental Environ-
mental Impact Statement (EIS) (USACOE 1989).

LARGE-SCALE IMPLEMENTATION: PROBLEMS
AND LESSONS

Shell Retention

The GHNIP received approval from state and federal agencies
and dredging removed 10 million m' of sediment from the navi-
gation channel of Grays Harbor in 1990. Results of the pilot study
were used to plan for shell mitigation on a large scale. Recognizing
the importance of site selection, the USACOE conducted studies in
1990 to 1991 (Armstrong et al. 1992). Three of four 0.4-ha plots

^ Light

South Channel South Bay Neds Rock

I'lHurt' I- Map "f (irays Harbor and Willapa lta>. Washinnlon. sIioh- KImirf 2. Time averai-ed (May-Soplemhcr) 0+ crab density (± 1 SD)

inj; Uicalions of larm'-sealc slull milination plots at I'acnian lI'Cl and in linhl. Iu'a\\, and shell pile habitat conlljiuralions created al three

South Channel (SC'I in (Jrays Harbor (•) and three smaller plots in locations in Crajs Harbor estuar> durin): a 1986 pilot stud) (adapted

Willapa Bay (A I. from Dumbauld et al. 1W3).

Use of Oyster Shell for Dungeness Crab Habitat

381

constructed in 1990 disappeared because of shell sinking into the
substrate or sedimentation, so. in a second experiment conducted
in 1991. test plots (30 m x 30 m) were placed at eight intertidal
locations distributed throughout the Grays Harbor estuary. Shell
sank below the surface at the majority of these sites within the first
2 weeks after construction. Tests, using a very sensitive surveyor's
level to detemiine relative elevations and produce contour maps,
showed that the process most responsible was subsidence attrib-
utable to bioturbation by resident thalassinid shrimp (Fig. 3). Sedi-
mentation occurred as well (i.e.. shell remained above grade level
but was covered by additional sediment), but this process seemed
to be correlated with shrimp density.

Full-scale mitigation began in 1992 in Grays Harbor with con-
struction of two large shell plots 6.7 ha and 2.2 ha at locations
know n as South Channel and Pacman respectively. (Fig. 1 ). Shell
retention by August of that year was only 329f at Pacman because
of relatively high shrimp density (60 burrows/m"). and because the
shrimp present were ghost shrimp (Neotrypaea califoniiensis.
Dana. 1854). which cause more sedimentation than mud shrimp
(Upogehia pugeWensis. Dana. 1852) (Dumbauld et al. 1996.
Dumbauld et al. 1997). Shell retention was about 70% at South
Channel, where a mix of mud shrimp and ghost shrimp were
present but at much lower density (20 burrows/m"). Although
remaining shell at both sites produced juvenile crab, all subsequent
mitigation projects in Grays Harbor have been at the South Chan-
nel location because of better shell retention. Based on pilot study
results, the USACOE predicted that shell retention would level off
at 50-80% after the first 2 years (USACOE 1989): this was not
realized in the full-scale mitigation project (Dinnel 1996. Fig. 4).
An exponential curve fits the data, but the slope of the retention
curve is site dependent. Similar studies in Willapa Bay have since
linked shell subsidence directly to abundance of shrimp and the
species of shrimp present (Feldnian et al. in press. Fig. 4). Finally.
USACOE hoped that harrowing the shell as practiced by the com-
mercial oyster industry (Sayce and Larson 1966). might return
shell to the surface and provide for additional shell life and. there-

CD
>

O

o

c

(U

o

100 ^

80
60
40

\1 20-

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0

• Predicted

■ GH Mix
WB Mud Shrimp
WB Ghost Shrimp
WB Treated

Years Post Construction

Figure 4. Shell retention over time as predicted bj USACOE (•) and
realized at the plots constructed in the South Channel area of Grays
Harbor in 1992 (■). Both ghost and mud shrimp were present at this
site. Also shown are similar retention curves for Willapa Bay at a site
with ghost shrimp present (D), a site where only mud shrimp were
present ( ^), and a site that had ghost shrimp present, but was pre-
treated with the pesticide carbaryl to control the shrimp (A).

fore, crab recruitment. This was to be carried out in year 4 and was
projected to return surface cover to 90% of the original. Initial
tests, although never quantified, indicated that harrowing would
not succeed.

100 H

>

80

o

o

—

60

0)

sz

(/)

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40

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May 1991
May 1 990
June 1991

-\ — ' — ' — ' — ' — r

100 200 300

400

500

600

Burrow Counts (number nrf )

Figure 3. Relationship between shrimp burrow counts and percentage shell cover 1-2 months after deployment at 14 sites in Grays Harbor
estuary. Note the significant negative correlation (r = -0.68, P< 0.1)5) and approximate threshold near a count of 100 burrows/m" above which
shell retention is less than 10% (after Armstrong et al. 1992).

382

DUMBAULD ET AL.

Faunal Composition

As part of a monitoring plan, composition, density, and/or bio-
mass of several taxa were measured in the created shell habitat
and, in some cases, compared with fauna in adjacent open mud-
flats. In addition to recruitment and density of juvenile Dungeness
crab, two issues of interest were: (1) amount and composition of
other fauna that may serve as food for Dungeness crab; and (2) the
recruitment and spatial distribution of both species of thalassinid
burrowing shrimp.

Certain fishes (e.g., the saddleback gunnel. Pholis ornala. Gi-
rard, 1854) and small crustaceans (mostly amphipods such as
Eogammanis coiifervicohis, Bousfield. 1979, and Corophiiim spp.)
in addition to Dungeness crab had much higher biomass (and
density) in shell as compared to open mud (Williams 1994, Fig. 5).
We found a relationship between the depth of shell and relative
density and biomass of gammarid amphipods (up to 6.000 indi-
viduals or 4g/m~ ash free dry weight, AFDW), but no relationship
with juvenile Dungeness crab density (Fig. 6). Several important

prey taxa, including juvenile bivalves and polychaetes, occurred at
comparable biomass (0.7 and 0.5 g AFDW/m", respectively) in
shell and mud (Fig. 5). The quantities of small prey suggested the
resident juvenile Dungeness crab population would have sufficient
food to grow and develop to an instar size (J4-J5), at which size,
they emigrate from the intertidal to subtidal environment (Visser
1997). Further research indicated that predation on juvenile clams
{Macoina haltliica. L.. 1758) increased in the shell habitat because
of enhanced Dungeness crab abundance, holding the standing
stock of clams to a similar level as that found in the open mud
(Iribame et al. 1994, Iribame et al. 1995).

Recruitment and distribution of 0+ (young-of-the-year)
thalassinid shrimp differed across the two dominant habitats in
accord with species. Related (o the use of shell as habitat for
juvenile crab was whether shell would also reduce burrowing
shrimp density and have potential as an alternative to using car-
baryl to control these species that have a negative impact on oyster
culture operations (Dumbauld et al. 1996. Dumbauld et al. 1997).
Very high densities of O-i- ghost shrimp were found in adjacent

39
B

Q

<

c
a

Infaunal Taxa

Figure 5. Mean total biomass (ash-free dry weight, AFDW)* I.O/nr l±l SKl of major inraunal taxa from shell and open mud control plots in
Grays Harbor ponied across locaticms and months. C'nih biomass estimates are based on values for ,I2-.|.^ instars (after Williams 1994).

Use of Oyster Shell for Dungeness Crab Habitat

383

(0

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o

a.

0 SOUT>1 CHANNEL

■ GRASS CREEK SOOTH

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a a «

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0 D a D a

• 0 0

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0*0* a .

Shrimp Recruitment

SHELL DEPTH (cm)

Figure 6. Amphipod and crab densities in shell samples from August
1990. A significant relationship between amphipod (Eogammariis con-
fervicolus) density and shell depth is evident (A) whereas, there is no
relationship between Dungeness crab density and shell depth (B). Also
given is a scale for biomass (AFDW, g) for amphipods (after Arm-
strong et al. 1992).

open mudflat habitat and in mud overlying shell that had sunk
below the surface (Feldman et al. 1997). Conversely, high densi-
ties of 0+ mud shrimp were found in naturally occurring Mya shell
deposits and in oyster shell mitigation plots (Feldman et al. unpubl.
data. Fig. 7). Feldman et al. (in press) concluded that shell gener-
ally reduces population density of juvenile ghost shrimp because
of its function as a physical barrier and settlement deterrent and/or
as a refuge for high densities of O-i- Dungeness crab, a predator.
Mud shrimp seem less affected by surface cover of heavy shell,
and, although their burrowing activity is less than that of ghost
shrimp, they could pose a greater long-term threat to shell integ-
rity.

Competition

During 1992, the initial year of shell plot construction, the
abundance of Dungeness crabs that recruited to the plots was high,
but few other crabs were seen. However, in subsequent years, the
abundance of yellow shore crabs [Hemignipsiis oregonensis.
Dana, 1851) found in the shell plots increased dramatically, and a
negative correlation was noted between the density of these two
species (Fig. 8). Further detailed investigations indicated shore
crabs recruited as juveniles to the plots via larval settlement and
also as adults by moving from adjacent areas (Visser 1997). Visser
found the mechanism responsible for the inverse relationship be-
tween the two species of crab to be direct competition for space
and not predalion. .Shore crabs are predominantly herbivores and

200

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v

>

<n

i

+
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a.

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Q.

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1

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Subsurface
shell

Epibenthic
shell

Mya shell

50

85

40

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30

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10

Mucj Subsurface Epibenthic Mya shell

shell

shell

Figure 7. Number of young-of-the-year (YOY) thalassinid shrimp re-
cruiting to open mud and various categories of shell cover, including
shell that had already been buried below the surface (subsurface shell),
a thick layer of oyster shell (epibenthic shell), and deposits of eastern
softshell clam shells. Ghost shrimp (top) were most abundant in open
mud and sand, and mud shrimp (bottom) were most abundant in shell
deposits, reflecting both settlement behavior as well as timing relative
to predation pressure by resident YOY crab.

scavengers, although they do consume some Dungeness megalo-
pae. Shore crabs are very active competitors for refuge space and
won an average of 787^ of interspecific encounters when they were
of comparable size to the Dungeness crab (Visser 1997). Because
Dungeness crabs recruit to the shell on an annual basis and emi-
grate later at larger size to the subtidal, they are at a size disad-
vantage when they settle in areas where shore crabs have had time
to colonize, because the latter are year-round residents. Visser
(1997) also found that Dungeness crab megalopae tend to avoid
settling in areas where shore crabs are present. This result was
entirely unexpected and forced USACOE to reconsider the strat-
egy of obtaining multiyear crab recruitment from shell plots that
were more than 1 year old. Instead, they had to rely on new habitat
construction every year.

Interannual Variability in Settlement

Dungeness crab display cyclic abundance, in part because of
their pelagic larval life history, which results in variable recruit-
ment to the benthos (McConnaughey et al. 1992, McConnaughey
1994) found a 40-fold variation in interannual settlement along the

384

DUMBAULD ET AL.

a

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• — '350

-

^'

«300

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s-

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0)

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03

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3

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0

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D oo

50

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0

^ — '-n 'd

o °t o m 'o o '

Grays Harbor Mitigation Plots

100

200

300

400,

Hemigrapsus Density (rri )

500

Figure 8. Density of 0+ Oungeness crab in shell mitigation plots at the
South Channel location in Grays Harbor as a function of total density
of shore crab {Hemigrapsus nregonensis) collected in the same samples
from 1992 through 1994 (after Armstrong et al. 1992).

Washington coast that was inversely related to the strength of
alongshore and cross-shelf transport during the previous 4-5
month larval period. Crab settlement is also variable from location
to location within the estuary because of wind-driven surface cur-
rents. However, Eggleston and Armstrong ( 1995) found that post-
settlement mortality was more influential than larval supply in
controlling the density of Jl instars at two locations in Grays
Harbor. Density at settlement has varied substantially in the
GHNIP constructed shell mitigation plot and to various other pro-
jects that have occurred since 1992 in the South Channel area of
Grays Harbor; however, the average crab density in newly created
shell in August was not significantly different between years (Fig.
9). Based on the results of the pilot study, USACOE chose a
conservative average of 10 crab/m" (August) to estimate crab pro-
duction for shell mitigation. This average has been met in all years,
including 1992, the first year of habitat construction.

Crab Production and Dredge Entrainment Models

An adult equivalent loss model was developed to determine the
number of crabs killed by dredging in the GHNIP (Wainwright et
al. 1992). The model is based on a dredge entrainment function
that relates the number of crabs entrained (estimated directly based
on counts taken from a modified dredge) and the number of crabs
present (estimated from trawls conducted simultaneously). Natural
mortality estimates are used to con\ ert different age classes of crab
into adult equivalents. The model also incorporated seasonal and
spatial information on crab density and size composition from the
Grays Harbor estuary and USACOE dredging scenarios of gear
type and amount of sediment dredged to project the niMiiber of
crabs entrained and killed. The model produced an estimate of
162, ()()() age 2-h crab (near maturity) killed during GHNIP con-
struction, which equates to about 73.000 3+ crab (age of recruit-
ment to the fishery). The same mortality estimates were used lo
calculate the number of O-k crabs necessary to mitigate for the loss,
which came to about 9.5()().()0() crabs.

The original approach that USACOE took for estimating the
number of O-h crab produced by shell mitigation was based on the

E

3

CO

c

(U

Q
n

CO

O

240

200

160

120

80

40

0

June
August

90

92

94
Year

Figure 9. Average density of 0+ Dungeness crab (± 1 SD) collected in
shell mitigation plots at the South Channel location in Grays Harbor
from 1990 through 1998. Data were collected in 1990 to 1995 by the
University of Washington and in 1996 lo 1998 by Grays Harbor Com-
munity College.

simplistic view that when crabs emigrate from the plots in July
they are considered ■"produced." The standing slock density in
August was multiplied by an estimate of the amount of shell re-
maining (total area multiplied by percentage shell cover) to give a
production figure. This approach was later modified, and a pro-
duction model developed that takes into account time, molt inter-
val, growth, mortality, and multiple cohorts of settling crab (Arm-
strong et al. 1995). The model uses J2 density as a starting point
and estimates mortality and growth from intertidal shell plot data.
Results suggested either a J3 or J4 (last instar that inhabits the shell
before emigrating) production unit could be valid, and J4 was
selected by agency and USACOE biologists. USACOE recently
refined all previously calculated mitigation estimates using this
production model, and, in most cases, a higher estimate was ob-
tained than that of the August standing stock method. This is
thought to be primarily because of emigration, which begins before
the August sampling date. The production model was updated and
improved again based on 1998 field data showing initial settlement
densities were extremely high, but survival rates were rather low.
Additions to the production value were also made for crabs that
were larger than the J2 starting point at the initial May sampling
date. This accounts for settlement that can occur (to a minor ex-
tent) as early as March to April in Grays Harbor. Thus, the pro-
duction model takes mortality rates for J2-J5 crab for each shell
plot sampled and applies them to measured field density of each
instar over the average time that instar spends at size before molt-
ing. This \alue is multiplied by the total habitat available for each
moiuh and summed to give the total number of 0-H crabs produced
per plot.

SOLUTIONS AND PROSPECTS

Since 1995. an interagency work group has met to evaluate and
update the L'SACOE crab miligalion program, and most recently,
the agencies signed a strategy agreement (USACOE et al. 1998).
The revised crab mitigation strategy is intended to update the
Dungeness crab avoidance and mitigation measures outlined in the

Use of Oyster Shell for Dungeness Crab Habitat

385

original Environmental Impact Statement written for the GHNIP
(USACOE 1982) in order to keep the program relevant to
USACOE. the project sponsor, and agency concerns. It addresses
a number of the factors listed abo\ e. including:

1. a renewed focus on and credits for avoidance of impacts,
including reduced over-all dredging, a re-examination of
clamshell dredging, and timing of downstream dredging to
avoid peak months of crab abundance;

2. a commitment to shell mitigation for impacts remaining
from past dredging and for any future impacts:

3. use of the production model (Armstrong et al. 1995) to
calculate the number of young-of-the-year crab "produced""
by the shell mitigation plots: and

4. continued efforts to promote impact avoidance to re-
evaluate crab mitigation efforts, including semiannual meet-
ings of the crab mitigation workgroup.

Finally, we introduce a project that attempts to mitigate for the
loss of crab resulting from a test dredging project in Willapa Bay,
Washington (Dumbauld and Kauffman 1999). This project repre-
sents a new twist in the mitigation strategy, because it attempts to
create a living oyster reef as habitat for juvenile crabs. USACOE
conducted test channel dredging on the sand bar at the mouth of
Willapa Bay in August 1997 to determine the effectiveness of a
new tool (agitation dredging with sidecasting) in this environment.
A fairly small amount of material was removed (61.164 m"*). Bot-
tom trawls for crabs were conducted, and USACOE agreed to
mitigate for the loss of 1.931 crabs (age 2-I-). Given the potential
for future mitigation projects in Willapa Bay, the crab workgroup
used this mitigation project to determine whether techniques ap-
plied in Grays Harbor would work equally well in Willapa Bay. A
second and perhaps more relevant goal for this work group was to
investigate whether living oyster reefs could be created, because
oysters spawn and set naturally in this estuary. The shell mitigation
project was conducted in the Washington State oyster reserves in
Willapa Bay. The reserves offered se\'eral advantages, including:
( 1 ) the Washington Department of Fish and Wildlife manages the
reserves to protect and preserve oyster resources and, therefore,
they offered an optimal site for examining creation of living oyster
reefs; (2) the reserves are located in areas that typically receive
larval oyster set; and (3) because oyster production is an existing
activity on the reserves, permitting was simplified.

Approximately 2.567 bushels (90 m^) of oyster shell were
placed at each of three separate intertidal locations on the reserves
to examine both crab and oyster recruitment in 1998 (Fig. 1 ). Shell
placement sites were carefully selected to avoid areas with high
thalassinid shrimp density; shell retention was approximately 659^
in the first year. Crabs recruited to all three shell plots, but num-
bers were higher on the two plots located closer to the mouth of the
estuary. Using the production model (Armstrong et al 1995). com-
bined recruitment to all three plots was calculated to be about
147,000 J4 crab, which satisfied the mitigation requirement. Oys-
ters recruited to the shell plots in late summer 1998 and began to
form visible live oyster reefs in 1999. particularly at the station
closest to the estuary mouth, where they were more abundant and
grew faster. Crab recruitment was also highest at this location in
1999, but was markedly lower than the previous year. We hope to
continue to monitor these plots to assess the value of the habitat
provided by living oyster reefs as the oysters mature.

This is a brief overview of what has been and continues to be
an interesting, but sometimes arduous process to mitigate for lost
resources. One additional factor, not mentioned so far, is cost.

Dinnel (1996) calculated that the cost of placing shell has in-
creased since the inception of the project to the point where each
crab, worth approximately $3 to the fisher, now costs USACOE at
least $70 to replace through shell mitigation. With .some of the
refinements noted above, he notes this might be reduced by 50-
60%; however, it is clear that avoiding impacts in the first place is
still the most cost-effective tool. To the extent possible. USACOE
and the other agencies involved have acknowledged this in the
latest mitigation strategy (USACOE et al. 1998) by embracing
creative steps to avoid crab loss as well as refining models to
estimate crab production within shell. The cost-benefit ratio may
be enhanced even further should it be possible to create living shell
reefs that function as crab habitat beyond the initial year of con-
struction as well as providing other ecosystem services.

ACKNOWLEDGMENTS

Funding for the studies reviewed here was provided by numer-
ous sources, including the Seattle District, U.S. Army Corps of
Engineers (contract to David Armstrong DACW67-85-0033 and to
Brett Dumbauld at WDFW. DACW67-98-M-0255). Washington
Sea Grant (contract to David Armstrong NA36RG0071-01), the
Willapa Bay/ Grays Harbor Oyster Growers Association, and the
Western Regional Aquaculture Center. Thanks to Loren Coen and
Mark Luckenbach for the invitation to attend and present this work
at ICSR and the Seattle District. U.S. Army Corps of Engineers for
providing funds to do so. Finally, thanks to myriad colleagues,
students, agency employees, and others, too many to list, who have
been and continue to be integral components of these projects.

LITERATURE CITED

Armstrong. D. A.. M. Fernandez & E. Visser. 1995. Estimated production
of 0+ Dungeness crab (Cancer mai>is!er) in intertidal shell mitigation
habitat. Grays Harbor estuary. 1990 to 1994. Final Report to the Seattle
District. U.S. Army Corps of Engineers, Seattle, Washington. 17 pp.

Armstrong. D. A. & D. R. Gunderson. 1985. The role of estuaries in
Dungeness crab early life history: A case study in Grays Harbor. Wash-
ington, pp. 145-170. In: B. R. Melteff (ed.). Proceedings of the sym-
posium on Dungeness crab biology and management. Alaska Sea Grant
Rept. 85-3. University of Alaska. Fairbanks. Alaska. 424 pp.

Armstrong. D. A.. O. Iribarne. P.A. Dinnel. K.A. McGraw. J. A.'Schaffer.
R. Palacios. M. Fernandez. K. Feldman & G. Williams. 1992. Mitiga-
tion of Dungeness crab. Cancer mugi.ster. losses due to dredging in
Grays Harbor by development of mtertidal shell habitat: Pilot studies
during 1991. Final report for the Seattle District. U. S. Army Corps of
Engineers, Seattle, Washington. 36 pp.

.Armstrong. D. A., T. C. Wainwright. J. Orensanz. P. A. Dinnel & B. R.
Dumbauld. 1987. Model of dredging impact on Dungeness crab in
Grays Harbor. Washington. Final Report for Battelle Laboratories and
Seattle District. U. S. Army Corps of Engineers. School of Fisheries.
University of Washington. Seattle. Washington. FRI-UW-8702. 167
pp.

Dinnel. P. 1996. Historical pe^^pective of the Grays Harbor crab mitigation
project. Final report to David Evans and Associates and the Seattle
District. U.S. Army Corps of Engineers. SeatUe, Washington. 50 pp.

Dumbauld. B. R.. D. A. Armstrong & K. L. Feldman. 1996. Life history
characteristics of two sympatric thalassinidian shrimps, Neolrypaea
californiensis and Vpogehia piigetlensis. with implications for oyster
culture. J. Crust. Biol. 16:689-708.

Dumbauld. B. R.. D. A. Armstrong & T. L. McDonald. 1993. Use of oyster
shell to enhance intertidal habitat and mitigate loss of Dungeness crab
(Cancer magister) caused by dredging. Can. J. Fish. Ac/iun. Sci. 50:
381-390.

Dumbauld, B. R.. D. A. Armstrong & J. R. Skalski. 1997. Efficacy of the
pesticide carbaryl for thalassinid shrimp control in Washington state

386

DUMBAULD ET AL.

oyster iCrassosrrea gii^as. Tliunherg. 1793) aquaoulture. J. Shellfish
Res. 16:503-518.

Dumbauld. B. R. & B. E. Kauffman. 1999. Mitigation for juvenile Dunge-
ness crab loss due to a test dredging project in Willapa Bay. Washing-
ton. Final report to the Seattle District U.S. Army Corps of Engineers.
Seattle. Washington. Washington Department of Fish and Wildlife.
Tech. Rept. FPT 99-04. 22 pp.

Eggleston, D. B. & D. A. Armstrong. 1995. Pre- and postsettlement de-
terminants of estuarine Dungeness crab recruitment. Ecol. Monogr.
65:193-216.

Feldman. K. L.. D. A. Armstrong. D. B. Eggleston & B. R. Dumbauld.
1997. Effects of substrate selection and postsettlement mortality on
recruitment success of the thalassinidean shrimp Neotnpaea califomi-
ensis to intertidal shell and mud habitats. Mar. Ecol. Prog. Ser. 150:
121-136.

Feldman. K. L.. B. R. Dumbauld, D. A. Armstrong. T. H. DeWitt & D. C.
Doty., in press. Oysters, crabs, and burrowing shrimp: An environmen-
tal conflict over aquatic resources and pesticide use in Washington
state's (USA) coastal estuaries. Estuaries

Fernandez. M., O. Iribarne & D. Armstrong. 1993. Habitat selection by
young-of-the-year Dungeness crab. Cancer magister. and predation
risk in intertidal habitats. Mar Ecol. Prog. Ser. 92:171-177.

Gunderson. D. R.. D. A. Armstrong. Y. Shi & R. A. McConnaughey. 1990.
Patterns of estuarine use by juvenile English sole {Parophrys vetulus)
and Dungeness crab [Cancer magister). Estuaries 13:59-71.

Iribarne. O.. D. Armstrong & M. Fernandez. 1995. Environmental impact
of intertidal juvenile Dungeness crab habitat enhancement: Effects on
bivalves and crab foraging rate. / E.xp. Mar. Biol. Ecol. 192:173-194.

Iribarne. O.. M. Fernandez & D. Armstrong. 1994. Does space competition
regulate density of juvenile Dungeness crab. Cancer magister Dana, in
sheltered habitats? J. Exp. Mar. Biol. Ecol. 183:259-271.

Jamieson, G. & D. A. Armstrong. 1991. Spatial and temporal recruitment
patterns of Dungeness crab in the northeastern Pacific. Mem. Queensl.
Mus. 31:365-381.

McConnaughey. R. A.. D. A. Armstrong, B. M. Hickey & D. R. Gunder-
son. 1992. Juvenile Dungeness crab (Cancer nuigister) recruitment
variability and oceanic transport during the pelagic larval phase. Can.
J. Fish. Aquai. Sci. 49:2028-2044.

McConnaughey, R. A., D. A, Armstrong, B. M. Hickey & D. R. Gunder-
son. 1994. Interannual variability in coastal Washington Dungeness
crab (Cancer magister) populations: Larval advection and the coastal
landing strip. Fish. Oceanogr. 3:22-38.

McGraw, K.. L. L. Conquest. J. O. Waller. P. A. Dinnel & D. A. Arm-
strong. 1988. Entrainment of Dungeness crabs. Cancer magister Dana,
by hopper dredge in Grays Harbor. Washington. J. Shellfish Res. 1:
219-231.

Sayce, C. & C. Larson. 1966. Willapa oyster studies — Use of the pasture
harrow for the cultivation of oysters. Comm. Fish. Rev. 28:21-26.

Stevens, B. G. & D. A. Armstrong 1984. Distribution, abundance , and
growth of juvenile Dungeness crabs. Cancer magister. in Grays Harbor
estuary, Washington. Fish. Bull. 82:469-483.

Tegelberg. H. & R. Arthur. 1977. Distribution of Dungeness crabs (Cancer
magister) in Grays Harbor, and some effects of channel maintenance
dredging. Final report to the Seattle District. U.S. Army Corps of
Engineers by the Washington Department of Fisheries. 94 pp.

U. S. Army Corps of Engineers (USACOE). 1982. Interim feasibility re-
port and Final Environmental Impact Statement. Grays Harbor, Che-
halis, and Hoquiam Rivers, Washington, channel improvements for
navigation. Seattle District, U.S. Army Corps of Engineers, Seattle,
Washington. 570 pp.

U. S. Army Corps of Engineers (USACOE). 1989. Final Environmental
Impact Statement Supplement No. 1 , Grays Harbor. Washington, Navi-
gation Improvement Project. Seattle District, U.S. Army Corps of En-
gineers, Seattle, Washington. 115 pp.

U. S. Army Corps of Engineers. U.S. Fish and Wildlife Service. Wash-
ington Department of Ecology. Washington Department of Fish and
Wildlife. Quinault Indian Nation. National Marine Fisheries Service,
and U,S. Environmental Protection Agency. 1998. Revised Crab Miti-
gation Strategy Agreement, Environmental Assessment, and Evalua-
tion Report. Seattle District, U.S. Army Corps of Engineers, Seattle,
Washington, 31 pp.

Visser. E. P. 1997. Competition, cannibalism, and prey limitation: Factors
compromising the effectiveness of shell habitat mitigation for early
benthic phase Cancer magister in Grays Harbor, WA. Ph.D. disserta-
tion. University of Washington, Seattle, Washington. 1 16 pp.

Wainwright. T. C. D. A. Armstrong, P. A. Dinnel. J. M. Orensanz & K. A.
McGraw. 1992. Predicting effects of dredging on a crab population: An
adult equivalent loss approach. Fish. Bull. 90:171-182

Williams, G, D. 1994. Effects of habitat modification on distribution and
diets of intertidal fishes in Grays Harbor estuary. Washington. Ma.sters
thesis. School of Fisheries, University of Washington. Seattle. Wash-
ington, 53 pp.

Jourmil of Slu'Uthh Research. Vol. 19, No. 1, 387-395. 2000.

TOWARD DESIGN CRITERIA IN CONSTRUCTED OYSTER REEFS: OYSTER RECRUITMENT
AS A FUNCTION OF SUBSTRATE TYPE AND TIDAL HEIGHT

FRANCIS X. O'BEIRN,' MARK W. LUCKENBACH,'
JANET A. NESTLERODE,- AND GREGORY M. COATES'

^Eastern Shore iMboratory

Virginia Institute of Marine Science

College of William and Mary

Wachapreagiie. Virginia 23480
"Virginia Institute of Marine Science

College of William and Mary

Gloucester Point, Virginia 23062

ABSTRACT Restoration of degraded oyster reef habitat generally begins with the addition of substrate that serves as a reef base and
site for oyster spat attachment. Remarkably, little is known about how substrate type and reef morphology affect the development of
oyster populations on restored reefs. Three-dimensional, intertidal reefs were constructed near Fisherman's Island, Virginia; two reefs
in 1995 using surfclam (Spisiila solidissiina) shell and six reefs in 1996 using surfclam shell, oyster shell, and stabilized coal ash.
We have monitored oyster recruitment and growth quarterly at three tidal heights (intertidal, mean low water, and subtidal) on each
reef type since their construction. Oyster recruitment in 1995 exceeded that observed in the two subsequent years. High initial densities
on the 1995 reefs decreased and stabilized at a mean of 418 oyster/nr. Oyster settlement occurred on all reef types and tidal heights
in 1996; however, postsettlement mortality on the surfclam shell and coal ash reefs exceeded that on the oyster shell reefs, which
remained relatively constant throughout the year (mean = 935 oysters/m"). Field observations suggest that predation accounts for most
of the observed mortality and that the clam shell and coal ash reefs, which have little interstitial space, suffer greater predation. Oyster
abundance was consistently greatest higher in the intertidal zone on all reefs in each year studied. The patterns observed here lead to
the preliminary conclusion that the provision of spatial refugia (both intertidal and interstitial) from predation is an essential feature
of successful oyster reef restoration in this region. In addition, high levels of recruitment can provide a numerical refuge, whereby the
oysters themselves will provide structure and increase the probability of an oyster population establishing successfully on the reef,

KEY WORDS: oyster, Crassostrea virginicci. habitat restoration, recruitment substrate, intertidal, Virginia

INTRODUCTION

The marked decline in oyster resources in the mid-Atlantic
region throughout much of this century have been attributed pri-
marily to increased harvest pressure, a direct consequence of in-
effective resource management (Haven et al. 1978, Rothschild et
al. 1994, Frankenberg 1995). Furthermore, the increased preva-
lence of the protistan parasites Perkinsus marinus ("Dermo") and
Hciplosporidiiim nelsoni ("MSX") (Burreson and Ragone 1996)
and over-all environinental degradation have accelerated declines
in oyster numbers over the last three decades. There is a general
consensus that oyster reefs were once a dominant feature of much
of the lower Chesapeake Bay. contributing considerable biological
and geological structure to the system. Historically, oysters in this
system likely affected systemwide trophic structure and water
quality (Newell 1988, Ulanowiz and Tuttle 1992), while providing
considerable physical structure, which, in turn, facilitated the de-
velopment of diverse benthic communities. The need to restore
oyster resources and oyster reefs, not only for their direct harvest
but also for the ecological services they provide, has been recog-
nized recently (Lenihan 1996, Coen and Luckenbach in press,
Coen et al. 1999).

To date, efforts to restore the resource have been focused in
areas where the oysters were abundant and extensive but have been
reduced to subtidal "footprints" of foimer reefs. Restoration at-
tempts carried out in areas previously devoid of oysters (as de-
scribed herein) have been few. Typically, restoration of a degraded
oyster reef has involved the addition of substrate to serve as a reef
base and site for spat attachment and subsequent oyster growth.
Oyster shell resources and/or the funds to purchase them are often

in limited supply; therefore, the interest in evaluating both how to
use oyster shell most effectively and the efficacy of using alter-
native substrates as reef bases is considerable. Attention has re-
cently been given to the importance of vertical relief of reefs on
oyster growth, survival, and disease dynamics (Bartol and Mann in
press; Lenihan et al. 1996, Lenihan and Peterson 1998); however,
there remains a paucity of infonnation on the degree of relief
necessary to maximize oyster settlement, recruitment, and subse-
quent survival. Furthermore, numerous studies have investigated
the use of alternative substrates to oyster shell (Soniat et al. 1991,
Haywood and Soniat 1992, Haywood et al. in press). These studies
have generally been laboratory or small-scale field experiments
and have not clarified how these substrates might be used to maxi-
mize oyster recniitment, growth, and survival in the context of
large-scale reef restoration. These issues have increasing relevance
as restoration efforts proceed throughout the extensive range of the
eastern oyster. This report focuses on a large-scale field experi-
ment in the lower Chesapeake Bay, Virginia, which related oyster
recruitment, growth, and survival to reef substrate types and tidal
height. The results have relevance for the choice and placement of
materials and the development of design criteria for oyster reef
restoration.

SITE DESCRIPTION

The study site is located near Fisherman's Island, Virginia,
U.S.A., in the vicinity of the mouth of Chesapeake Bay (Fig. 1).
This is a polyhaline site with a tidal amplitude of approximately
1.25 m. Marsh islands, intertidal Hats, and subtidal bottom within
the area are all owned by the Commonwealth of Virginia and the

.^87

388

O'Beirn et al.

m COAL ASH

^ CLAM SHELL ^

^ OYSTER SHELL

Figure 1. Location of study area near Fisherman's Island, Virginia. Reefs with an alphanumeric label were monitored continually throughout
the period of the study. Reefs are not drawn to scale.

federal government and are managed by the U.S. Fish and Wildlife
Service as part of the Eastern Shore of Virginia National Wildlife
Refuge. In April 1995. two intertidal reefs, approximately 8,000
m" (2 acres) each, were constructed at the site as part of a reme-
diation project funded by the Chesapeake Bay Bridge Tunnel Dis-
trict. The reefs were created by placing approximately 40,000 Vir-
ginia bushels (~ 1 .973 m"*) of surfclam (Spisiila solidissima) shells
on two intertidal mudflats (see A and B in Fig. 1). The reefs
extended from - 0..^ m below to 0.5 ni above MLW. The reef
designated A in Figure 1 had greater surface area at higher tidal
elevation than reef B. Irregular patterns of mounds, ridges, and
furrows existed across the reef surface as a result of the planting
technique (deployment from barges by water cannon). Hereafter,
the clam shell reefs, constructed in 1995. are designated as 95
Clam reefs.

Eleven additional reefs (Fig. I ) were constructed in 1996 with
funding from the Aquatic Reef Habitat Program. Virginia Power
Company, and the Virginia Oyster Repletion program. Five of
these reefs were constructed with surfclam shells, two with oyster
{Crassostrea vir^inica) shells, and four with slabili/ed coal com-
bustion by-products (fly ash). The latter material, constructed us-
ing 88% fly ash stabilized with 12% (w:w) Portland cement, is
described in greater detail in Andrews et al. ( 1997) and has been
shown to pro\ ide an environmentally suitable substrate for oyster
settlement and iirowth (Allien et al. 1496). Limited a\ailabilitv of

oyster shells resulted in the smaller number of reefs (/; = 2)
constructed with that material. A total of 39.920 bushels (1,965
m') of surfclam shells, 7.000 bushels (325 m'') of oyster shell, and
20,150 bushels (994 m') of coal-ash pellets were used to construct
the reefs. Two reefs of each substrate type, ranging in size from
162 to 364 m". were selected for monitoring (reefs 1-6 in Fig. 1 ).
The reefs were oriented in a north-south direction, with seven
reefs in one row and four reefs in another row to the west. A
channel ranging in width from 10 to 40 m separates the two rows.
Hereafter, the reefs constructed in 1996 are designated as Oyster,
96 Clam, and Ash.

MATERIALS AND METHODS

Quadrate Sampling

Sampling of the reefs for determination of oyster abundance
and size was initiated in October 1995. On each of the reefs se-
lected for monitoring (two of each substrate type: A. B. and 1-6 in
Fig. 1). quadrate samples (;i = 3) were collected from each of
three tidal heights. The tidal heights were 0.25 m below mean low
water (hereafter called Subtidal). at ttiean low water (hereafter
called MLW). and 0.25 m above MLW (hereafter called Inter-
tidal). Replicate quadrates (0.0625 m"; /; = 3) were placed hap-
ha/ardh within each lidal height stratum (Subtidal. MLW, and
Intertidal) on replicate reefs (« = 2) of each reef substrate type

Oyster Habitat Restoration Substrate Suitability

389

95 Clam

Time

Figure. 2. Oyster abundance (number per m", mean ± SD) from three
tidal iieights throughout the stud) on the 95 Clam shell reefs. * No
replicate quadrates were taken during this sampling period.

(Oyster. 95 Clam, 96 Clam, and Ash) to give a maximum of 72
samples per sampling period. Within each quadrate sample, all reef
substrate was retained to a depth of 15 cm but did not include
underlying sediments if encountered. Samples were transported to
the laboratory on ice (if necessary) and were processed immedi-
ately. Processing involved the enumeration of all live oysters in
each sample. In addition. 50 oysters from each tidal height, on each
reef sampled, were measured to the nearest 0.1 mm. Sampling took
place on a quarterly basis in an attempt to detect seasonal changes
in oyster abundance.

Interstitial Space Estimates

The volume of interstitial space for each of the sub.strates used
to construct reefs in 1996 was estimated using subsamples of the
substrates before the deployment of the substrates. All of the sub-
samples used were considered the ideal for that substrate type:
whole (with some partially fragmented) oyster and surfclam shells
and ash pellets = 5 cm in diameter. Interstitial volume was calcu-
lated using the volumetric displacement of the substrate packed to
the top of a container (=5.85 L). This displacement value was then
subtracted from the container volume to give interstitial volume.
All interstitial volumes were corrected to reflect the substrate type
within a 1-L container. This process was carried out five times for
each substrate in order to generate mean and standard deviation
values. These values were then compared using a one-way analysis
of variance ANOVA.

Statistical Analysis

The 95 clam reefs were not compared statistically with the
1996 reefs because of the dual confounding effects of temporal
difference in deployment and considerable differences in surface
areas of the reefs. Summary statistics generated for oyster densities
and sizes by reef type are reported in graphical form.

Over the course of the study, some tidal height strata on some
of the 1996 reefs were much reduced as a result of settling and/or
erosion, thus we were unable to complete sampling from all tidal
heights for the duration of the study. Therefore, for the purpose of
comparing the abundance of oysters by substrate, we confined our

analysis to the subtidal samples, for which there is a complete set
of samples. Abundances were log transformed |ln (x-i-l)] to con-
form to normality assumptions as required. A two-way ANOVA
was carried out (with substrate type and time as the main effects)
to ensure that there was no interaction term. Upon satisfaction of
this criterion, a randomized complete block design ANOVA was
conducted using substrate type as the main effect blocked by time
(Sokal and Rohlf 1981, pp. 345-352). The 96 Clam reefs had
returns from all tidal heights for all time periods bar one (one reef
in November 1997). Therefore, we were able to compare oyster
abundances from all tidal heights of the 96 Clam reefs. The values
from these tidal heights were compared accordingly. The Oyster
reefs had complete returns from the MLW and subtidal samples for
the two replicate reefs, resulting in valid comparisons of these tidal
heights blocked according to time.

RESULTS

At the initial sampling of the 95 Clam shell reefs in October
1995. high oyster numbers were recorded at all tidal heights (Fig.
2). The intertidal samples had the highest oyster numbers through-
out, followed by the MLW and subtidal samples, respectively.
Subsequently, oyster abundances declined precipitously at all tidal
heights. By November 1996, the elevations of the reefs were re-
duced through subsidence, compaction, and/or erosion to the point
that intertidal samples could not be retrieved (Fig. 2). Despite some
fluctuations, the numbers of oysters on these reefs tended to re-
main stable in the following sampling periods. Throughout this
period, the abundance of oysters remained fairly constant, mean
values for the MLW and subtidal samples were 834 oysters/m" and
345 oysters/m", respectively. There were no appreciable differ-
ences in size distribution among the tidal heights through the sam-
pling periods. Therefore, the size frequencies from each tidal
height within each sampling period were pooled, and these are
graphically represented in Figure 3. A unimodal population distri-
bution is apparent for the first year of the monitoring (October
1995 to September 1996). Following a small recruitment event in
December 1996 (Fig. 3). a bimodal population distribution was
evident. Between August 1997 and November 1997, mortality
among larger animals and an influx of small, newly recruited
individuals was apparent. Thereafter, the size distribution on these
reefs remained relatively stable, with small, newly recruited indi-
viduals dominating in terms of over-all abundance (Fig. 3).

Relatively low numbers of oysters were present in the Ash reef
samples from December 1996 through August 1997 (Fig. 4). In
November 1997. the young-of-the-year animals were detected on
the reef and increased the over-all number of oysters sampled. The
recruitment event in each year sampled was followed by a rapid
decline in the numbers of oysters found on the reefs. Also,
throughout the sampling of the Ash reefs, the intertidal stratum
consistendy contained higher oyster densities than the other tidal
heights. The MLW stratum for the most part, had greater oyster
densities than the subtidal stratum. The size distribution of oysters
on the ash reefs was highly variable, with smaller oysters (< 25
mm) dominating throughout and larger oysters rare (Fig. 5).

The 96 clam reefs displayed patterns similar to the Ash reefs in
terms of over-all recruitment patterns and abundances (Fig. 6).
Again, relatively low densities were found each sampling period.
Recruitment events were followed by a sharp decline in oyster
densities. Intertidal stratum had greater oyster densities than the
other two tidal heights in all but two sampling periods (November

390

O'Beirn et al.

I I I I I I in I I I I I I I I I I I I

0 15 30 45 60 75 90

Size (mm)

December '96

30—1

January '96

25 —

so-

I

ls—

10 —

i

li

5 —

,

III.

" 1

1 1 1 III

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

September '96

jliUu

0 15 30 45 60 75 90

Size (mm)

I III 11 1 I I ll~ri I I I I I I
0 15 30 45 60 75 90

Sao (mm)

May '97

I" B B

so-
ts —

mtii

„--:=Jlll,D.^a0

rJ,Vn

mJm^

Si;e (mm)
November '97

30 —

25 —

Size (mm)
February '98

Mill

0 15 30 45 60 75 90

Size (mm)

I I 111111111111111111 I
0 15 30 45 60 75 90

Size (mm)

August '97

ll- - III...

May '98

i,B,yiia,y, ,^,B,..

111111111 1111 1 1 1 1 11 1 1

0 IS 30 45 60 75 90

Size (mm)

ttttttttttttttttttttti

0 15 30 45 60 75 90

Size (mm)

I I I I I I I I I I I rnn I i i i i

0 IS 30 45 60 75 90

Size (mm)

August '98

15 —
10 —

iAJUoxvaJiw-

1111111 i I I rn~["rrri~n i

0 15 30 45 Ml 75 W

Size (mm)

JfiBj

I I I I rni r rrn n i i i i i

0 15 30 45 60 75 90

Size (mm)

30 —
Il-

November '98

ls —
10 —

5 —

■ 1

lllhllh ..

1 1 1
0 15

1 1 1 M 1 1 I 11 1 II 11 1
30 45 60 75 90
See (mm)

February '99

m i^~! 1 I 1 :.. 1 II I I I I I

I 15 30 45 60 75 90

S^Q (mm)

Figure. 3. Oyster size frequency distribution over the course of the study from the 95 Clam shell reefs. Size distributions were all animals
combined from the three tidal heights.

1997 and August 1998). The size distributions within each sam-
pling period was indicative of a population dominated by small
oysters (< 20 mm; Fig. 7). However, in later sampling periods,
there was a greater proportion (albeit small) of larger oysters on
the 96 Clam reefs than found on the Ash reefs.

In 1996, in contrast to the low recruitment of oysters found on
the reefs of coal ash and clam substrate, the Oyster reefs had a
modest recruitment in December 1996 (Fig. 8). Survival on the
oyster reef was greater than on the other substrate types, and again
oyster densities were greater intertidally than at the other two tidal
heights, The si/e distribution of oysters on the oyster shell reefs
was approaching a unimodal normal distribution by May 1997
(Fig. 9). Recruitment events detected in November of subsequent
years resulted in a bimodal si/e distribution. However, relatively
large numbers of larger oysters persisted on the reefs.

Interstitial volumes differed significantly among the substrate
types (Table 1). The oyster shell interstitial volume (0.7 L inter-
stitial volume/I L of substrate) was significantly greater than the
volumes of both the clam (0.58 L) and coal ash (().4.S L) substrates.
Analysis of variance of oyster densities from Subtidal samples
detected significant differences among the Oyster. Ash, and Clam
substrates (Table 2a). The Oyster substrate had significantly
greater numbers of live oysters than the other reef types (Table 2a).
The Inlerlidal samples from the 96 Clam reefs had significantly
greater densities of oysters than the .Subtidal samples (Table 2b). In
addition, the densities of oysters found in the MLW samples were
significantly greater than those found in the Subtidal samples on
the Oyster shell reels (Table 2c).

DISCUSSION

The reef bases at Fishemian's Island, Virginia, have all per-
sisted, but quite different oyster populations have developed de-
pending upon both the year of deployment and the substrate type
used. Reduced elevations were observed in all reef bases, likely the
result of some combination of subsidence, compaction, and ero-
sion. Although interstitial volume estimates differed among the
substrate types used on the 1996 reefs (Table I ), subsequent (rnis)
handling of the clam shells and large-scale production of the ash
substrates (hence, poor quality control) resulted in additional com-
paction. These factors served to further the disparity between the
oyster shell and the other substrates in terms of interstitial volume.
This variation, we believe, had very significant consequences for
the development of resident oyster populations as discussed below.

Oyster recruitment levels varied across the region over the
duration of the study. As part of the ongoing yearly monitoring of
oyster reproduction in the lower Chesapeake Bay. the Virginia
Institute of Marine Science (VIMS) uses spatfall collectors (shell-
strings) to determine patterns and levels of oyster recruitment (un-
published data, Virginia Oyster Spat Survey, 1970 to 1998,
VIMS). During 1996 and 1997, recruitment estimated from the
shellstrings al Fisherman's Island was lower in magnitude and
later in each year compared with the I99.'i shellstring results. This
pattern was consistent with observations throughout the lower bay
(Morales- Alamo and Mann 1996. Morales-Alamo and Mann
1997). Sampling on the reef surfaces was not timed specifically to
record early postseltlemenl abundance. Other studies have shown

Oyster Habitat Restoration Substrate Suitability

391

Ash

1200
. 1000

I 800
I 600
O 400 ^
* 200

0 J

^±>^

k-

\.

Time
Figure. 4. Oyster abundance (number per m", mean ± SD) from three
tidal height>^ throughout the study on the Ash pellet reefs planted in
1996. Legend as in Figure 2.

coal ash pellets (Alden et al. 1996, Andrews et al. 1997) and
suifclam shells (Luckenbach unpublished data) are suitable sub-
strates for oyster settlement. We would expect that early postsettle-
ment densities, scaled to available substrate area, were comparable
across reef type, but we lack confirming data.

Postsettiement survival of oysters varied in relation to tidal
elevation, but the patterns were partially confounded by the loss of
some tidal elevations from some reefs. The general trend observed

was one of greater survival of oysters in the intertidal (Figs. 2, 4,
6, and 8), which is consistent with other studies conducted in the
mid and southern Atlantic states of the U.S. (Kenny et al. 1990,
Michener and Kenny 1991, O'Beirn et al. 1995, O'Beirn et al.
1996, Roegner and Mann 199.5). Despite some variations in this
pattern, significant differences were apparent for 96 Clam reefs,
for which we have all tidal elevations present (Table 2b). In ad-
dition, oyster densities varied on the Oyster reefs between the two
tidal heights evaluated (Table 2c). However, in the case of the Ash
reefs, this trend was reversed on the final sampling period, with
oysters virtually absent from intertidal samples (Fig. 4). These
findings serve to highlight the importance of vertical relief when
constructing oyster reefs in such environments as Fisherman's Ls-
land.

Variation in oyster abundance across substrate type was evident
at all tidal heights (compare Figs. 4, 6, and 8), but because of
missing levels on some reefs, statistical comparisons by substrate
type were made only for the subtidal level (Table 2a). The signifi-
cant trend of greater abundance of oysters on the Oyster reefs
compared to the Ash reefs and 96 Clam reefs at this tidal level was
evident throughout. Over-all mean density on the Oyster shell reef
(935/m") exceeded that on the 96 Clam shell reef (149/m") and the
Ash reef ( 141/m") roughly sixfold. Visual comparisons of the reefs
are even striking. The Oyster shell reefs supported an uninter-
rupted layer of live oysters, which was not apparent on the other
substrates, both of which had only sporadic clusters of oysters. In
addition, the clam shell and coal-ash pellets reefs mostly retained

December '96

80—1
60
40 —

lu

i~i~i~i~r I I I I I I I I I I I I I I

5 15 25 35 45 55 65 75 85 95

Size (mm)

August '97

JllU.

I { I I I I I I I I I I I I I I

5 15 25 35 45 55 65 75 85 »5

Size (mm)

60 —

40-

20-

80— I
60-
40 ■

February '97

I I I I 1 I I n I 1 \ I I i~i \ I i I

5 15 25 35 45 55 65 75 85 95
Size (mm)

November '97

Jl

u

Mm.

5 15 25 35 45 55 65 75 85 95

Size (mm)

80-
60-
40-
20-

May '97

0 I !~l~l~l I I I I 1 I I I I I ! I I I 1 I
5 15 25 35 45 55 65 75 85 95

Size (mm)

February '98

liu

T I I I I i I I \ \ \ \ \ I [ 1 I
5 15 25 35 45 55 65 75 85 95

Size (mm)

80 -^

May '98

60 -

40-

20-
0-

4

1 i*i*i~i"i II'

S 15 25 35 45 55 65 75 85 95
Size (mm)

80 -

August '98

60 -

40-

1

20-
0 —

1 1 1*1 i~i i"i i"i 1 1 1 1 1 1 1 1 1 1

5 15 25 35 45 55 65 75 85 95
Size (mm)

80 —

November '98

60 —

40 —

20^

ill

■

II II B^ —

I
5

IS 25 35 45 55 65 75

85

95

Size (mm)

Figure. 5. Oyster size frequency distribution over the course of the study from the Ash reefs planted in 1995. Size distributions were all animals
combined from the three tidal heights.

392

O'Beirn et al.

96 Clam

Time

Figure. 6. Oyster abundance (number per m", mean ± SD) from three
tidal heights throughout the study on the 96 Clam shell reefs. Legend
as in Figure 2.

their original bleached white and dark gray colors, respectively,
throughout the study, which is indicative of little or no biotic
development on the reefs.

The dominance of the oyster shell substrate was further under-
scored when examining the size data of oysters from each of the
substrate types. Small oysters (< 20 mm) dominated both the Ash
and 96 Clam substrates (Figs. 5 and 7) throughout the entire moni-
toring period. There was no persistence of larger (older) oysters in
either of these reef types. The 95 Clam reefs and the Oyster shell
reefs had relatively greater proportions of larger oysters represent-

ing multiple year classes (Figs. 3 and 9). In August 1998, 22%
(138 oysters/m"^) of the standing stock of oysters on the Oyster
shell reefs had shell height s 60 mm. This represented a substan-
tial number of larger oysters that could contribute considerably to
future reproductive events (Cox and Mann 1992) and. therefore,
realizes a primary goal of the restoration efforts. In addition, the
higher density of oysters resulted in a reef matrix that is likely to
ensure the maintenance and stability of the valuable interstices.

We suggest that several factors related to the availability of
interstitial space account for the observed differences in oyster
abundance across the reefs. First, the reduced interstitial volume in
the ash pellets and clam shell relative to oyster shell may have
reduced the amount of surface area available for settlement. Bartol
and Mann (1999) have reported oyster settlement onto shells 10-
15 cm below the surface in a constructed reef in the Piankatank
River, Virginia, and J. Nestlerode and F. O'Beirn (unpublished
data) have made similar observations in substrate baskets buried in
these reefs at Fisherman's Island. The density estimates we report
here include oysters collected to a depth of 15 cm scaled to a flat
surface area of the reef and do not account for subsurface area that
might be available for oyster attachment. Thus, oyster settlement
onto the Oyster shell reefs may have exceeded those on the Ash
and 1996 Clam shell reefs. Becau.se recruitment levels were low,
however, and attachment surface was not in limited supply, it is
unlikely that settlement differences accounted for most of the
variation across reef type.

Differential inortality of oysters at the surface and below the

60 -

December '96

40 -

20 -
0 -

.III

I I 1 1 1 1 1 M 1 1 1 1 1 1 1

5 15 25 35 45 55 65 75 85 95
Size (mm)

February *97

1 I I I I i I I I I 1 I I I [ I 1 I

5 15 25 35 45 55 65 75 85 95
Size (mm)

May '97

nn

1 1 1 1 1 1 1 1 1 1 1 1 1

5 15 25 35 45 55 65 75 85 95
Size (mm)

August '97

~ I 1 I I r~rn I r~\ \ i rn n i

5 15 25 35 45 55 65 75 85 95
Sizr(mm)

November '97

I III I I I I I I I I I I I I I I

5 15 25 35 45 55 65 75 85 95
Size (mm)

February '98

■[■■■■L- 1

T i I I I \ rn rn i i i i i i i i i

5 15 25 35 45 55 65 75 85 95
Size (mm)

60 -1

May '98

40 -

20-

i h li'i'i'i i"

0 -

5 15 25 35 45 55 65 75 85 95
Size (mm)

August '98

1 I I I I I I I I I I I I

5 15 25 35 45 55 65 75 85 95
Size (mm)

November '98

Hn

0 — iLn.rti Lh tpi,t«i ra.t-a UJ.m.H.m,

I I I ; I i I I I I I I I I I I I I I I
5 15 25 35 45 55 65 75 85 95
Size (mm)

Figure. 7. Oyster size frequency distribution over the course of the study from the 96 Clam shell reefs. Size distributions were all animals
combined from the three tidal heights.

Oyster Habitat Restoration Substrate Suitability

393

Oyster

.MKIII

2500
^ 2000
H 1500
O 1000 S
500 I

ll^^

.1

\ \ \ \ K

Time

Figure. 8. Oyster abundance (number per ni", mean ± SD) from three
tidal heights throughout the study on the Oyster shell reefs planted in
1996. Legend as in Figure 2.

surface of the reefs is a likely explanation for the abundance pat-
terns we observed. Bartol and Mann (1999) have demonstrated the
value of interstitial space in aiding the survival of young oysters.
The refuge afforded by the interstices protects the young oysters
from predation and buffers them from climatic extremes. The con-
siderably lower levels of interstitial space located on the clam shell
and ash reefs most likely resulted in increased exposure of the
young oysters to potential predators and other detrimental envi-

ronmental factors (see reviews by Shumway 1996. White and Wil-
son 1996).

Finally, we expect a degree of positive density dependence in
the development of oyster populations on constructed reefs. If the
initial settlement and survival of oysters is sufficient (in part be-
cause of factors above), living oysters come to dominate the sur-
face features of the reef and contribute to further interstitial space.
In effect, the oysters themselves provide a refuge in numbers. In
addition, the presence of large numbers of resident oysters in sub-
sequent years may enhance settlement through the release of wa-
ter-soluble settlement-inducing peptides (Tamburri et al. 1992,
Turner et al. 1994). For example, the large recruitment event in
1995 (Fig. 2) was sufficient to result in a veneer of living oysters
covering most of the clam shell substrate. Thus, when a smaller
recruitment event occurred in 1996. the 95 Clam reefs and the 96
Clam reefs presented quite different habitats for new recruits and
both recruitment and survival were greater on the older clam shell
reefs (compare Figs. 2 & 3 with Figs. 6 & 7). Similarly, the
abundances of oysters and spatial complexity of the oyster shell
reefs have been increasing since their planting in 1996. Both the 96
Oyster shell reefs and the 95 Clam shell reefs developed abundant
oyster densities, with multiple year classes present and reef sur-
faces dominated by living oysters. In contrast, the Ash reefs and
the 96 Clam reefs have failed to develop abundant oyster popula-
tions, and generally only supported small size classes, which di-
minished in abundance after recruitment events.

Our findings suggest that in areas and years with high oyster

30 -
20 -
10

December '96

5 15 25 35 45 55 65 75 85 95
Size (mm)

40-1

30-

20-

February '97

I I I I I I I 1"! I I I I I I I i I I
5 15 25 35 45 55 65 75 85 95
Size (mm)

May '97

1 I I I \ I i I I [ \ I I \ \ I I I I
5 15 25 35 45 55 65 75 85 95

Size (mm)

40-

August '97

30-

20-

1

10-

.l.lllllll..

1 1 1 1 1 1 1 1 1 1 111

5 15 25 35 45 55 65 75 85 95

November '97

5 15 25 35 45 55 65 75 85 95
Size (mm)

40 -
30 -
20-
10-
0-

lijgliin

zLilflnBiLnM.

I I II 1 I I 1

5 15 25 35 45 55 65 75 85 95
Size (mm)

August '98

5 15 25 35 45 55 65 75 85 95
Size (mm)

40-

February '98

30 -

20 -
10-
0 -

1 lillllll.-

5 15 25 35 45 55 65 75 85 95
Size (mm)

November '98

30-
20 -
10 -

n nnHnnn^n-

5 15 25 35 45 55 65 75 85 95
Size (mm)

Figure. 9. Oyster size frequency distribution over the course of the study from the Oyster shell reefs planted in 1996. Size distributions were all
animals combined from the three tidal heights.

394

O'Beirn et al.

TABLE 1.

ANOVA and Tukey HSD tests on interstitial space obtained from
the three substrate types.

TABLE 2.

Results of the ANOVAs and Tukey HSD tests on (a) oyster

abundance according to substrate type, (b) oyster abundance at

tidal heights on clam reef, and (c) oyster abundance at tidal heights

ANOVA:

Interstitial Volume by Substrate Ty

pe

on oyster reefs.

Source

df

SS

F Value

/•-value

(a) ANOVA: Oyster Abundance by Substrate Type
(Subtidal Elevations Only)

(SD)

2
12

0.156

0.178

Oyster

0.7 L (0.04)

42.8

Clam
0.58 L (0.06)

0.0001

Ash
0.45 L (0.02)

Substrate
Error

Source df SS F Value

P-Value

Tukey test
Mean volumes

Substrate 2 74.39 28.09

error 43 56.94
Tukev test: Oyster Clam Ash

.0001

Interstitial volume given as

interstitial volume in liters per

1-L substrate.

i«cruitment rates, the nontraditional substrates used here can serve
as suitable base materials tor restoring oyster reefs if mounded to
provide sufficient vertical relief. In low recruitment environments,
however, it is important that adequate interstitial space be present
to support oyster survival. In the present study, only oyster shells
provided adequate interstitial space for the development of an
oyster population in low recruitment years. Given our initial con-
cern that oyster shells are in short supply throughout much of the
mid-Atlantic region of the U.S. and the unpredictable nature of
recruitment in many areas, we are led to ask how to best use
available substrates for reef restoration. Repeated handling of
surfclam shells — from the shucking house to reef construction —
seems assured of resulting in fragmentation and the tight packing
on reefs described above. Mixed shell plantings using surfclam
shells in combination with other shell (e.g.. whelks and hard
clams) may support better development of oysters by reducing
compaction and increasing available interstitial space (J. Wesson.
Virginia Marine Resources Commission, pers. comm). Improved
quality control in the production process of coal ash pellets could
result in more uniform-sized pellets, similar to those used by An-
drews et al. (1997). which had a mean diameter = 5 cm, provided
greater interstitial space, and supported good oyster survival. Per-
haps the greatest impediment to the use of coal-ash pellets in future
oyster reef restoration efforts results from the U.S. Federal High-
way Act of 1995. which mandated the use of recycled material in
roadbed construction: thereby, changing coal ash from a waste
product into a commodity and increasing its cost.

A variety of alternative substrates for oyster settlement have
been tested in other studies including slate (Haven et al. 1987).
expanded shale, shredded tires (Mann et al. 1990), gypsum. Ran-
gia cuneutu shells, limestone, concrete, and gravel (Soniat et al.
1991, Haywood and Soniat 1992, Haywood et al. 1999). Varying
degrees of suitability were observed for the different substrate
types. In North Carolina. limesti)ne marl is a routinely used settle-
ment substrate in a fishery enhancement program (Marshall et al.
1999). The applicability of these substrates for large-scale endeav-
ors may have to be re-evaluated in light of the findings presented
in this study, particularly as they relate to substrate stability and
interstitial volume.

The construction of reef .structures in order to promote shellfish
restoration represents a significant investment of public and pri-
vate resources. Developing protocols that help maximi/.e ecologi-
cal return on this investment will he important for future efforts to
restore oyster reef, as will evaluating these design and construction
protocols on sufficiently large spatial and temporal scales. We

(b) ANOVA: Oyster Abundance by Tidal Height
(Clam Shell Reefs Only)

Source

df

SS

F Value

P-Value

Tidal height

error
Tukey test:

2

38

Intertidal

14.85
70.35

4.01
Mean Low Water

.0263
Subtidal

) ANOVA: C

>yster Abund
Oyster Reef

(c

ance by Tidal Height
5 Only)

Source

df

SS

F Value

P-Value

Tidal height

error
Tukey test

1

26

Intertidal

8.99
7.83

29.86
Mean Low Water

.0001

Tukey Test given in descending order of magnitude.

have observed an interaction between the substrate used in the
construction and oyster recruitment levels in the development ot
oyster populations on large-scale constructed reefs. During periods
of low natural recruitment, only substrates that provide adequate
interstitial space (oyster shell in the current study) are sufficient to
support the development of a viable reef During periods of high
recruitment, poorer quality substrate (i.e.. that providing less in-
terstitial space) may prove sufficient as the newly recruited oysters
themselves serve as ecosystem engineers (Jones et al. 1994) pro-
viding physical refuge. In temperate, polyhaline environments, the
provision of vertical relief is important in ensuring oyster survival.
Again, the combination of substrate placement and oyster recruit-
ment, survival, and growth interact to affect restoration success.
Therefore, restoration design criteria (e.g.. the actual configuration
of interstitial space and degree of vertical relief) must account for
both geophysical (e.g., siltation and ice scour) and biological (e.g..
subtidal and intertidal predators) mechanisms. Gi\en these poten-
tial constraints, we appreciate that the many factors influencing
oyster survival and growth, and hence a successful start to resto-
ration efforts, have yet to be elucidated.

ACKNOWLEDGEMENTS

The authors thank the numerous individuals that helped in the
field sampling. This work was supported by the U.S. E.P.A.
Chesapeake Bay Program. This is contiibution no. 2288 from the
Virginia Institute of Marine Science.

Oyster Habitat Restoration Substrate Suitability

395

litp:ratl're cited

Alden. R.. M. Luckenbach, A. Dombrowski. V. Harlow, A. Abbgy. L.
Ramirez & I. Weber. 1996. An evaluation of the feasibility and envi-
ronmental acceptability of using pelleti/ed coal lly ash as a substrate
for oyster reef development. ARML Techn. Rept. 301,^, Old Dominion
University. Norfolk. Virginia.

Andrews. R. S., R. W. Alden. M. W. Luckenbach & J. A. Wesson. 1997.
The use of coal combustion by-product as substrate for oyster reef
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Bartol. I & R. Mann. 1999. Small-scale patterns of recruitment on a con-
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W. Luckenbach. R. N4ann and J. Wesson (eds.). Oyster Reef Habitat
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Burreson. E. M. & L. M. Ragone Calvo. 1996. Epizootiology of Perkinsiis
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Coen, L. D., D. M. Knott. E. L. Wenner, N. H. Hadley & A. H. Ringwood.
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133-158. In: M. W. Luckenbach, R. Mann and J. A. Wesson (eds.).
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proaches. Virginia Institute of Marine Science, Gloucester Point, Vir-
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Coen, L. D. & M. W. Luckenbach. in press. Developing success criteria
and goals for evaluating shellfish habitat restoration: Ecological func-
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Cox, C. & R. Mann. 1992. Temporal and spatial changes in fecundity of
eastern oysters. Crassostiea rirginica (Gmelin 1791). in the James
River. Virginia. J. Shellfith Res. 11:49-54.

Frankenberg. D. 1995. North Carolina Blue Ribbon .Advisory Council on
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Journal of Shellfish Research. Vol. 19. No. 1. 397^00, 2(K)().

THE GULF COAST OYSTER INDUSTRY PROGRAM: AN INITIATIVE TO ADDRESS

INDUSTRY'S RESEARCH NEEDS

JOHN SUPAN

Office of Sea Grant Development
Louisiana State University
Baton Rouge, Louisiana 70803

ABSTRACT The Gulf Oyster Industry Program (GOIP) was created in response to petitions from the Louisiana Oyster Task Force
and Gulf Oyster Industry Council. These organizations initially sought long-term, research-ba.sed assistance through the Louisiana Sea
Grant College Program. Subsequently, they worked with the Gulf region's Sea Grant network, the National Fisheries Institute, and the
congressional delegations of several Gulf states to have the GOIP established as part of the National Sea Grant College Program in
the National Oceanic and Atmospheric Administration. Implementation of this new program in a competitive funding environment
necessitated reconciling two, sometimes conflicting, management goals: ( 1 ) projects having the greatest utilitarian benefits as judged
by the oyster industry stakeholders; and (2| projects having the greatest scientific merit as determined by expert peer reviewers.
Program development, implementation, and outcome of the first-year proposal solicitation and selection process are presented.

KEY WORDS: Gulf oyster, research, programming

INTRODUCTION

The Gulf region leads all other regions of the U.S. in oyster
production (NMFS 1998); however, the nation's total annual pro-
duction of molluscan shellfish has been declining steadily. As in
other areas, oyster producers in the Gulf states face myriad prob-
lems associated with ( 1 ) the presence of opportunistic bacteria,
especially vibrios, in oyster-growing waters; (2) multiple-use con-
flicts in traditional oyster grounds, especially those associated with
coastal restoration projects; (3) pollution from upstream urban and
industrial development, recreational camps, and oil production fa-
cilities; (4) the depletion of harvestable stocks by oyster predators
and diseases, especially the parasite Perl<insus marimis; (5) uncer-
tainty about long-term stability of oyster leasing policies; and (6)
declining profitability caused by declines in seasonal meat yields,
a changing work force, stringent new regulations, and limited tech-
nological options.

In response to a request from the legislatively appointed Loui-
siana Oyster Task Force (LOTF) and the Gulf Oyster Industry
Council (GOIC). the Louisiana Sea Grant College Program devel-
oped and implemented the Gulf Oyster Industry Initiative, a long-
term, research-based plan to assist the industry achieve full eco-
nomic recovery. In 1997, the industry successfully petitioned Con-
gress to support the initiative with $1 million per year for 5 years.
Administered by the National Sea Grant College Program, the
money is to fund competitive grants through Sea Grant universities
around the country. The resulting program combines the scientific
knowledge of highly qualified academic researchers with the ex-
perience of industry and management agency personnel in a co-
ordinated, comprehensive search for viable solutions to the most
pressing problems of the Gulfs oyster industry. Although the Sea
Grant programs of the Gulf region have supported many oyster-
related projects during the last 25 years, this particular undertaking
is novel in its scope, management, and the close working relation-
ships between scientists and industry stakeholders.

The program's objectives are (I) to assist the oyster industry
with needs analyses and prioritization; (2) to seek credible scien-
tific viewpoints and responses to those needs; (3) to educate the
Gulf oyster industry about the scientific status of their issues; and
(4) to facilitate the development and funding of research proposals
to address those issues.

APPROACH

The GOIP was designed to have continuous involvement of the
oyster industry. In the program's first year, the research priorities
were established by an Industry Advisory Panel (lAP) and de-
scribed in the Request for Preliminary Proposals (RFP). The panel
consisted of an oyster harvester and a processor from each Gulf
state, ensuring that both viewpoints would be recognized. The
panelists were selected by the directors of Gulf coast Sea Grant
programs, with recommendations from the GOIC and the LOTF.

After preliminary proposals were received from academic re-
searchers, the lAP determined which ones should be selected for
solicitation as full proposals. The full proposals were subsequently
evaluated by a Scientific Review Panel, which comprised repre-
sentatives from the oyster industry, the National Sea Grant Office,
and a Gulf state shellfish management agency, and scientists fa-
miliar with contemporary oyster research. This panel selected pro-
posals for possible funding on the basis of their scientific merit and
cost effectiveness, and provided a general ranking of the proposals
based on peer reviews and the panelists' individual knowledge of
the subject matter.

RESULTS

Primary consideration for funding was given to proposals
addressing research topics previously identified as critical to the
continued viability of the oyster industry. In the GOIP's inaugural
year (FY 98), 18 full proposals totaling $1.1 million were solicited
after a review of 35 preliminary proposals totaling $3.9 million
in requested funds. Ten proposals were ultimately funded, totaling
approximately $868,000. The grant recipients were from Virginia,
North Carolina, Florida. Alabama. Mississippi, Louisiana, Texas,
and California. The research topics and examples chosen by
the lAP follow; projects receiving funding in that topic are de-
scribed.

Pathogenic Organisms

A perception that the consumption of raw oysters is hazardous
to public health is reducing the demand for shellstock oysters and

397

398

SUPAN

inflicting severe economic distress on oyster growers, harvesters,
and processors. Suggested research topics include:

• develop means of treating shellstock and/or raw oyster meats to
eliminate human pathogens;

• develop and evaluate strains of transgenic oysters that are ca-
pable of destroying pathogenic organisms;

• develop an international database on Vibrio parahaemolyliciis
to centralize information on the organism;

• develop depuration methods for removing vibrios from oysters;
and

• develop quick depuration methods using probiotics

1998 Projects

Project Title: Use of GRAS compound, Diacetyl, for the re-
moval of Vibrio vulnificus from shellstock and shucked oysters.

The objectives of this project are to determine the effectiveness
of diacetyl for the reduction and/or elimination of naturally occur-
ring populations of V. vuhiificus within shellstock and shucked
oysters and to investigate the conversion of opaque morphotypes
of V. vulnificus to the translucent morphotype in both oyster prod-
ucts.

Funding level: $39,564 GOIP and $20,480 match for Year 1.

$38,763 GOIP and $21,424 match for Year 2.

Consumer Attitudes and Preferences

Consumption of oysters has fallen significantly during the past
decade. Lack of knowledge concerning the attitudes, preferences,
and other characteristics of potential oyster consumers is perceived
as an obstacle to recovering traditional markets and expanding
demand for new and traditional oyster products. Suggested re-
search topics include:

• determine oyster consumer demographics, consumption pat-
terns, attitudes, and preferences;

• develop news media protocols for researchers and state regula-
tory personnel;

• develop and market-test new oyster products; and

• determine the market characteristics, including sale (i.e., region,
size of establishment, average sales, etc.), distribution, and pre-
ferred product forms.

Oyster Disease

Incidences of oyster disease are rising dramatically in Gulf
oyster grounds, and much of the region's oyster industry is at risk
of severe economic loss through mortality of harvestable oysters.
Possible research topics include:

• develop and evaluate genetic strains of oysters with superior
growth and disease (pathogen) resistance; and

• develop technology to produce quantities of oyster larvae with
superior growth and disea.se resistance adequate for commercial
operations.

1998 Projects

Project Title: Predicting time to critical levels of the oyster
disease Perkinsus mcirinus: A new tool for increasing oyster pro-
duction.

The objectives of this project are to determine monthly lc\ els
off. marinus in Galveston Bay oysters, predict the time needed to
reach a critical level of disease, evaluate the reliability of the
predictions, and use the information to increase oyster production
by moving or harvesting oysters before they arc killed by disease.

Funding level: $55,229 GOIP and $27,365 match for Year 1.

$54,229 GOIP and $27,365 match for Year 2.

Project Title: Optimization of gene delivery for improved oys-
ter health.

The objectives of this project are to compare commercial lipo-
fection reagents to determine which is most effective; evaluate
lipofection of sperm, eggs, and embryos; and evaluate the expres-
sion and function of transferred genes by disease challenging lar-
val and adult oysters with Perkinsus marinus.

Funding level: $60,307 GOIP and $30,153 match for Year 1.

Project Title: Creation of an oyster cell line for Crassostrea
virginica (Gmelin).

The objectives of this project are to produce retroviral vectors
that express different reporter genes and proto-oncogenes from
appropriate promoter elements in oyster cells; to optimize condi-
tions for the infection of primary cultured cells from C. virginica.
to infect primary cultures established from both C. virginica hearts
and from enzymatically disrupted early embryos with the trans-
forming retroviral vectors, to characterize the growth characteris-
tics and phenotype of immortalized cell lines, and to test cultiva-
tion of the oyster pathogens Haplosporidium nelsoni and Perkin-
sus marinus in the oyster cell lines.

Funding level: $76,098 GOIP and $37,195 match for Year 1.

$88,889 GOIP and $56,930 match for Year 2.

Coastal Restoration, Freshwater Diversion

Coastal land loss, deterioration of estuarine habitat, and coastal
restoration programs (e.g., freshwater diversions, and sedimenta-
tion projects) are causing widespread dislocations and conflicts
with established oyster-producing operations. Suggested research
topics include:

• educate oyster farmers, public officials, and citizens regarding
the economic role of the oyster industry and the economic costs
of displacing and relocating oyster bedding operations;

• develop and test freshwater diversion and oyster farming strat-
egies that take account of temperature and salinity conditions
likely to promote fouling of bedded oysters by the hooked mus-
sel Uschadium recurvum [Rafinesque]).

1998 Projects

Project Title: The Caernarvon Freshwater Diversion Project
and oyster farmers: what happened and what it means.

The objectives of this project are to describe the Louisiana
oyster relocation program after recent court cases and political
activity, allowing oyster farmers to make pro-active decisions
about opting into the lease relocation program as it currently exists.

Funding level: $30,666 GOIP and $15,989 match: I year proj-
ect.

iMbor and Mechanization

The traditional labor base that supports oyster growing, har-
vesting, and processing is shrinking rapidly, with consequently
declining production and increased costs. Suggested research top-
ics include:

• investigate and de\elop cost-effecti\e mechanized approaches
to oyster har\esting. processing, and packing; for example, oys-
ter shucking and harsesting machinery.

1998 Projects

Project Title: Technical and economic evaluation of a freeze-
licat-cool process that facilitates oyster shucking.

The objectives of this project are to de\elop and e\aluale pro-

Gulf Oyster Industry Program

399

totype equipmenl to test the freeze-heat-cool process thai will fa-
cilitate shucking of Gulf oysters.

Funding Level: $61,390 GOIP and $31,000 match for 1 year.

Genetics, Hatchery Production

Seasonal monetary losses from the processing of oysters with
poor meat yield hinder economic stability and growth of the in-
dustry. Some slates have areas suitable for growing oysters, but
these are not used because of traditional or regulatory restraints
against moving seed oysters from public to private beds. These
areas have high potential for oyster culture using hatchery-
produced oyster seed. Suggested research topics include:

• develop cost-effective hatchery, nursery technology to augment
wild oyster production by producing specialized strains: and

• address practical problems, such as fouling, predation. disease,
that may be common to oyster production in general but are
more acute in a farming situation.

1998 Projects

Project Title: Natural dermo resistance and its role in the de-
velopment of hatcheries for the Gulf of Mexico.

The objectives of this project are to use putatively resistant
broodstock from both the Gulf of Mexico and Chesapeake Bay and
determine the inherent resistance of their progeny to Dermo dis-
ease to provide clear evidence for the existence of 'naturally re-
sistant populations' of American oysters and to justify the contin-
ued development of these stocks by industry.

Funding level: $41,591 GOIP and $20,972 match for Year 1.

$42,693 GOIP and $21,772 match for Year 2.

Project Title: Optimum size for planting hatchery-produced
oyster seed.

The objectives of this project are to determine the optimum size
of hatchery-produced oyster seed for planting on bay bottoms and
to relate optimum size to costs for producing various size seed.

Funding level: $31,737 GOIP and $17,249 match for Year I.

$31,993 GOIP and $17,766 match for Year 2.

Hooked Mussel Fouling

Hooked mussel fouling on oyster growing areas has drastically
increased harvesting costs by requiring the laborious removal of
mussels from marketable oysters or further transplanting oysters to
areas with higher salinity. Suggested research topics include:

• determine predator-prey relationships:

• determine the effects of varying haline and aerial exposures to
mussel attachment; and

• determine mussel life cycle and/or recruitment.

Harmful Algal Blooms

The first reported occurrence of red tide in the central Gulf
region caused a lengthy public health closure, halting production
for weeks and causing severe economic hardship in the affected
area. A possible research activity is to develop rapid detection
methods for red tide.

1998 Projects

Project Title: Ozone-assisted depuration of red-tide-con-
taminated shellfish and seawater.

The objectives of this project are to determine the optimal
amount of ozone needed to inactivate the brevetoxins in contami-
nated clams and oysters in recirculating seawater depuration tanks;

determine the length of time required at the optimal amount of
ozone for the desired reduction of toxins to safe levels in shellfish:
and determine the effect on shelf-life of red-tide-contaminated
clams and oysters subjected to such depuration.

Funding level: $52,575 GOIP and $20,307 match for Year 1.

$52,575 GOIP and $20,307 match for Year 2.

Point-Source Pollution

Known point sources of pollution negatively affect certain po-
tential oyster-growing waters, with consequent public health risks
and loss of revenue to growers. Public health closures of oyster
grounds in restricted areas are costly to the Gulf states in terms of
lost resources, employment, and revenues. Possible research topics
include:

• evaluate alternative uses of recalled oysters;

• develop a process for identifying pollution sources and linking
clean-up to closure of beds.

1998 Projects

Project Title: Determination of design and operational criteria
for a marshland upwelling system to treat domestic wastewater
from coastal camps.

The objectives of this project are to characterize influent waste-
water parameters, to determine effectiveness of a marshland up-
welling system to remove fecal coliform and E. coli from raw
wastewater, to determine the extent and movement of the fresh-
water plume that develops over time, and to develop an educa-
tional program for marine advisory agents and potential users.

Funding level: $78,548 GOIP and $25,569 match for 1 Year.

Project Title: Legal authority to clean up oyster beds closed
because of pollution.

The objectives of this project are to examine both the applica-
bility and inapplicability of state and federal Clean Water Act
programs to compel clean up of shellfish beds contaminated by
point-source pollution.

Funding Level: $30,666 GOIP and $15,989 match: 1 year proj-
ect.

Black Drum Predation

Restrictions on use of gill nets as harvesting gear for finfish
have enabled black drum populations to flourish, with a conse-
quent increase in oyster predation by that species. A possible re-
search activity is to develop novel methods of deterring black
drum predation.

New Priority for FY99-00

The lAP added an additional topic for the FY99-00 preliminary
proposal solicitation: economic impacts of regulatory action. The
regulation of molluscan shellfish is unique compared with all other
foods. Regulatory action either by state or federal public health
agencies and subsequent news media responses can have severe
economic impacts on the harvesting, processing, and marketing of
shellfish, such as Gulf oysters. Suggested research topics include:

• analyses of de-listing of a processor or state from the Interstate
Certified Shellfish Shippers List;

• analyses of inaccurate media reports on sales; and

• analyses of ramifications from product disparagement.

400

SUPAN

DISCUSSION

The scope of the projects awarded for 1998 is somewhat sur-
prising considering the oyster industry's involvement in the pro-
gram, because some of the research is basic in nature. Of the
approximately $868,000 awarded for FY98-99 projects. 39% sup-
ported oyster disease research. 17% addressed oyster hatchery and
genetics research, 24% investigated point-source pollution and
harmful algal blooms. 7% was for labor and mechanization needs,
and 3% focused on coastal restoration topics. Most surprising is
that only 9% of the total awards addressed pathogenic organisms,
such as Vibrio vulnificus, an issue perceived widely as having the
greatest negative impact on the Gulf oyster industry. Despite the
desperate problems Gulf oyster farmers are having with hooked
mussels and black drum predation, no proposals were awarded in
those categories during the program's first fiscal year. These re-
sults reflect how the GOIP solicitation and selection processes
focused researchers on the industry's needs, and selecting the most
highly ranked proposals based on their scientific merit.

It is apparent that the GOIP is addressing several important
topics identified in earlier studies of oyster industry problems.
Several of the research topics identified in the Gulf states' regional
oyster management plan (GSMFC 1991 ) and a plan addressing the
restoration of the American oyster industry (Anonymous 1990),
which engendered the National Oyster Disease Program adminis-
tered by the National Sea Grant Office, are being addressed by
GOIP projects. The GOIP is distinguished by the way in which it
addresses industry's annual needs assessment and prioritization.
The greatest programmatic challenge is to fund proposals that not

only address industry's needs but also have high scientific merit
and educate the industry.

ACKNOWLEDGMENTS

Mr. Ronald Becker, Associate Executive Director of the Loui-
siana Sea Grant College Program, led the development of the Gulf
Oyster Industry Initiative that was used by industry to gamer Con-
gressional support for the program. His leadership in program
development, and assistance with implementation and evaluation,
as well as review of this manuscript, are greatly appreciated. The
review by Elizabeth Colemen is also appreciated. Many research
topics from the LOTF Research and Development Committee's
White Paper were incorporated into the GOIP. The GOIP would
not have come to fruition without the time and effort spent by
many members of the Gulf oyster industry in program develop-
ment and the solicitation of Congressional support, without whose
partnership this program would not have happened.

LITERATURE CITED

Anonymous. 1990. A plan addressing the restoration of the American
oyster industry. Virginia Sea Grant Publication VSG-90-02. Virginia
Sea Grant College Program. Chariottesville. Virginia. 64 pp.

Gulf States Marine Fisheries Commission. 1991. The oyster fishery of the
Gulf of Mexico, United States: A regional management plan. Publ. 24.
Gulf States Fisheries Commission. Ocean Springs, Mississippi. 184 pp.

National Marine Fisheries Service (NMFS). 1998. Personal communica-
tion. NMFS. Fisheries Statistics and Economics Division, website:
http//www. nmfs.gov.

Jrmriwl i>f SlwUfish Research. Vol. 19. No. 1. 4()1-40S. 2000.

A REVIEW OF SHELLFISH RESTORATION AND MANAGEMENT PROJECTS

IN RHODE ISLAND

MICHAEL A. RICE,- APRIL VALLIERE," AND
ANGELA CAPORELLI'

^Department of Fisheries, Animal and Veterinaiy Science

University of Rhode Island

Kingston. Rhode Island 02881
'Rhode Island Department of Environmental Management

Division of Fish and Wildlife

Coastal Fisheries Laboratory

1231 Succotash Road

Wakefield. Rhode Island 02879

Rhode Island Seafood Council

212 Main Street

Wakefield. Rhode Island 02879

ABSTRACT Shellfish management and restoration efforts in Rhode Island date back to the late 1 9th century. From the late 1890s
to the Second World War the Rhode Island Fisheries Commission operated a lobster hatchery in Wickford Harbor in response to a
perceived decline in lobster catches in Narragansett Bay. Berried lobsters were collected, eggs hatched, larvae reared, and postlarval
fifth stage juveniles were released to the bay. The project was discontinued primarily because of costs and a failure to demonstrate the
efficacy of juvenile seeding in improving lobster catches. From the 1930s to the 1980s, there have been several similar efforts to
establish hatcheries to produce juvenile bivalve mollusks for public and private reseeding efforts, but none of these efforts were
economically sustainable. The longest running efforts to improve shellfisheries have been state programs to relay northern quahogs,
Mercenaria mercenaria. from dense population assemblages in waters closed to shellfishing. Large-scale relays began in the 1950s in
response to heavy fishing pressure but ended in the 1960s when commercial power dredging for .shellfish was banned in Narragansett
Bay. A small-scale state program existing since the late 1970s pays a modest fee to supervised shellfishers for hand digging quahogs
in closed waters and planting them in management areas for depuration and eventual harvest. The amounts of shellfish relayed annually
has varied widely since 1977, ranging between 7 and 322 metric tonnes, with an average of 98 metric tonnes per year. A new relay
program has been underway since 1997. It involves assessing the shellfish stocks in the closed Providence River and hiring dredge
boats to relay shellfish into down bay management areas. Based on maximum sustainable yield (MSY) considerations, annual relays
should not exceed 10.3% of the standing crop (or 2721 metric tonnes) in the Providence River. An effort to restore lobsters onto
monitored artificial reefs is underway using settlement funds from a 1989 oil spill in Narragansett Bay. Finally, the Rhode Island Public
Benefit Aquaculture Project, a joint educational effort with commercial fisheries involvement, is involving secondary level students in
the nursery culture of shellfish (though marina-based upwellers) for seeding of public shellfish beds.

KEY WORDS: Shellfish restoration, Rhode Island, shellfish relay, shellfisheries, Narragansett Bay

INTRODUCTION that the Narragansetts would ". . . wade deepe (sic) and dive . . .""

for oysters and quahogs. Shell middens found along the shore of

Since the King Charles Charter of 1663 uniting the Rhode Narragansett Bay, notably in the Potowomut area of what is now

Island Colony of Newport to the mainland colony of Providence Warwick, are testament to the importance of shellfishing in the

Plantations, there has been a codified recognition of the impor- pre-Colonial era. Even the scientific name of the northern qua-

tance of fish and fisheries to all the citizens of Rhode Island. hog — Mercenaria mercenaria (Linnaeus, 1754) — is testimony to

Although the charter is best known for its early establishment of the fact that the white and purple beads made from their shells

religious freedoms, it also set forth the first principles of a public were an important trading currency. From the Colonial period and

trust doctrine by entrusting the stewardship of coastal waters to the early statehood until the Civil War, shellfisheries were essentially

colonial assembly. All citizens were assured of free access to the subsistence or small-scale commercial operations as authorized

waters for fishing and the collection of seaweed. In 1842 these under the King Charles Charter or under the Article 1, Section 17

public trust principles were incorporated directly into Article 1, provisions of the 1842 Rhode Island State Constitution. Oysters

Section 17 of the state constitution, and they now form the basis of were harvested as feed for swine and for storage as a personal food

all fisheries and coastal management efforts undertaken in Rhode item during winter. The shells were burnt to produce lime (Kochiss

Island's tidal waters. Nixon (1993) provides an overview and 1974).

analysis of Rhode Island's public trust doctrine as it relates to The second major period in Rhode Island's shellfisheries began

shellfisheries and aquaculture in coastal waters. with the passage of the Oyster Act of 1864 and the establishment

The history of Rhode Island's shellfisheries can be broadly of the Rhode Island Shellfisheries Commission. This act of the
characterized as having three distinct periods. During the first legislature allowed, for the first time, the leasing of tracts of sub-
period, which began in pre-Colonial times with the Narragansett merged public trust lands for the purpose of cultivating oysters
Indians, shellfishing was usually a summer activity. Roger Wil- (Nixon 1993). The early oystermen in Rhode Island readily rec-
liams (1643) the founder of Providence Plantations Colony noted ognized that the waters in Narragansett Bay were very good for

401

402

Rice et al.

growout, or maturation, of oysters (Crassostrea virginica). But.
the seed beds in the state were not particularly productive and
recruitment was very sporadic, so tons of seed oysters were
brought annually into Narragansett Bay from Long Island Sound
and as far away as the Chesapeake Bay (Hale 1980). This massive
transplantation of oysters onto extensively managed aquaculture
beds might be considered Rhode Island's first successful program
of shellfish restoration, albeit the direct beneficiaries were the
oyster leaseholders. In 1910. during the height of Rhode Island's
oyster aquaculture industry. 8100 ha of Narragansett Bay was
leased to private companies for oyster culture and 7000 metric
tonnes of oysters were harvested annually (NMFS landing statis-
tics as cited in Olsen and Stevenson 1975).

Throughout the period of massive oyster aquaculture leases in
Narragansett Bay. state efforts to boost shellfish production in-
cluded programs to monitor and control shellfish predators. The
former Rhode Island Shellfisheries Commission and the oyster
companies initiated an annual starfish census. These are reliable
estimates of starfish populations in Narragansett Bay from 1880 to
1940 (Pratt et al. 1992). From time to time — when the predator
populations became particularly high — there were starfish
"bounty" programs (Hale 1980). and as part of regular oyster bed
maintenance the oyster vessels were rigged with starfish mops
similar to those still used on oyster beds in Connecticut (Olsen et
al. 1980). The old-time oystermen recognized that predator control
programs were a very effective way to increa.se shellfish produc-
tion, but there was a lack of understanding that starfish could
regenerate from body parts. One common practice of starfish "con-
trol" was to cut landed starfish in half and throw them overboard
(Luther Blount, former President of Warren Oyster Company,
pers. comm. 1993).

Beginning in the 1920s, the oyster aquaculture industry began
a slow decline, culminating in a near collapse following the Great
Hurricane of 1938. A number of causes have been attributed to the
decline of the oyster industry in Rhode Island. These include
changes in upland land uses and increased sedimentation of prime
beds (Hale 1980), increased metal pollution due to a burgeoning
metal-plating industry (Nixon 1995). and increased eutrophication
and hypoxia in the upper reaches of Narragansett Bay due to
sewage disposal (Desbonnet and Lee 1991). The Great Hurricane
of 1938 was certainly a major blow to the oyster industry. Most of
the shoreside docking and processing facilities were severely dam-
aged by the storm (Olsen et al. 1980). and the recovery of the
industry was hampered by the lack of a readily available workforce
due to the onset of Worid War II (Hale 1980).

Another reason for the decline and eventual demise of Rhode
Island's oyster industry may lie in the major socio-political
changes that occurred in Rhode Island during the mid-l93()s. In
many ways, Rhode Island's oyster industry was a product of the
"mill town" social system that grew up during the Industrial Revo-
lution and the heyday of Rhode Island's textile industry in the late
19th and early 2()th centuries. The oyster industry, as it was con-
stituted, was extremely labor-intensive, very much like other in-
dustries of the era. Beginning in 1935, political shifts in the state
government (known locally as Gov. T. F. Green's bloodless revo-
lution) toward more populist policies may have had some impact
upon the oyster industry. McLoughlin (1978) argued that this
change in political philosophy liati a profound impact on the textile
industry and other industries thai failed to adapt in a changing
political climate.

The third period of Rhotle IsUuhI's shellfisheries. reviewed by

Hale (1980) and Boyd (1991), arose immediately after Worid War
II. Many of the troops returning from Europe or the Far East in the
1940s began tonging for quahogs. largely because the old oyster
beds were no longer tended and the oyster companies were not
hiring. In the late 1940s the keyport bullrake was invented in the
Mid-Atlantic region and was quickly introduced to Rhode Island.
After several technical refinements, the bullrake became the most
widely used commercial shellfishing implement by the 1960s
(Boyd 1991).

The two key controversies in the shellfisheries during the 1940s
and 1 950s were fees paid by fishermen to oyster leaseholders for
the privilege of fishing on the beds, and the introduction of power
dredges for harvesting quahogs. When oyster production on the old
leases declined, there was little or no effort by the state to revoke
the leases and return the grounds to the open fisheries. Only after
the demise of the last Rhode Island oyster company, the Warren
Oyster Company in 1 952 ceased culture operations, were all of the
old oyster leases eventually revoked. The creation of a Coastal
Resources Management Council in the late 1970s and changes in
the aquaculture laws (General Laws of Rhode Island 20-10-1) in
the eariy 1980s included provisions against aquaculturists retain-
ing leases in public trust waters when active aquaculture opera-
tions cease (Olsen and Seavy 1983).

Throughout the 1950s and 1960s, there were countless discus-
sions about how the quahog fishery should be managed. The main
issue was whether the fishery would consist of a large number of
small-scale operators using hand tongs and bullrakes or a relatively
small number of operators using power dredges. The rakers and
tongers argued strongly to management officials that power dredg-
ing was environmentally damaging. This prompted a number of
studies, including that of Glude and Landers (1953) which showed
that while dredging did allow individuals to harvest more shellfish
in a shorter period of time, it was no more damaging than the
cumulative impacts of large numbers of handrakers. By the eariy
1960s, state management officials set into statute the banning of
power dredges in most of Narragansett Bay (General Laws of
Rhode Island 20-6-7). Thus the strategy was to allow greater num-
bers of fishermen through limits on individual effort. Under this
system of limiting individual effort, the fishery grew and nour-
ished. When the Rhode Island quahog fishery reached its peak in
1985. there were an estimated 1000 full-lime commercial shell-
fishermen, landing 2200 metric tonnes (meat weight) of shellfish,
worth ,$15 million dockside. representing about 25''/f of all quahog
produclion nationally (Boyd 1991; PraU et al. 1992).

During the decade of the 1990s there has been a steady decline
in quahog catches and a decrea.se in the number of active full-time
shellfishermen. According to the Rhode Island Department of En-
vironmental Management statistics in 1997. there was an estimated
500 full-lime fishermen landing about 651 metric tonnes (meat
weight) of shellfish. Rhode Island's national market share in qua-
hogs has dipped to about 89}. This erosion of the fishery since the
1980s has caused concern in the industry and generated calls for
means to rebuild ihe lishery through expanded relays, seeding, and
other public aquaculture projecls.

OVERVIEW

Concern about declining shellfisheries is certainly not new in
Rhode Island. Tlirough ihc years there have been numerous at-
tempts to use aquaculture techniques to enhance fisheries. As pre-
viously noted, oysters were transplanted onto lease beds for matu-
ration, but this was more of a directed pri\ate enterprise practice

Shellfish Restoration and Management Projects

403

on privately held lease beds. The first genuine public aquaculture
for a marine species was a lobster hatchery established in Wick-
ford Harbor in 1898 by the Rhode Island Inland Fisheries Com-
mission in response to declining lobster fisheries (Meade 1901).
As one contemporary account put it, "It is no exaggeration to say
that in practically every known natural region of the North Atlantic
coast, the lobster fishery is either depleted or in a state of decline"
(Herrick 1909). Personnel from the hatchery would gather berried
female lobsters from the Narragansett Bay trap fishery, and care-
fully incubate the eggs until hatching. They maintained larvae in
floating tine mesh net cages and fed them a diet consisting of
ground beef liver and cooked chicken eggs. While in the floating
cages, larvae were gently agitated with a mechanical apparatus to
keep them suspended to reduce cannibalism (Meade 1908). After
reaching fourth or fifth postlarval stage, they were released into
Narragansett Bay. In the first rear of lobster releases, 1901, only
9000 juveniles were released into Narragansett Bay. But in 1908.
the Wickford station was releasing 400.000 lobsters, and by 1920
the facility was releasing over 1 million lobsters yearly. Peak
production of the facility was in 1934 when lobster releases
reached over 1 .5 million (IFC 1934). The lobster hatchery program
continued by the Inland Fisheries Commission (IFC) until 1935,
when the agency was reorganized into the Department of Fish and
Wildlife. The Wickford lobster hatchery continued under Fish and
Wildlife until the 1940s (Carlson 1954). After nearly a half century
of operation this remarkable pioneer program was eventually dis-
continued as a cost cutting measure. The site of this first state
lobster hatchery is now used by the Rhode Island Department of
Environmental Management's Division of Fish and Wildlife as a
fisheries laboratory and a base for the patrol craft of state fisheries
conservation officers.

The first Rhode Island oyster hatchery was established by Paul
Galtsoff in the late 1930s at the end of South Ferry Road in
Narragansett, on what is now the campus of the University of
Rhode Island's Graduate School of Oceanography (GSO). The
intent was to establish a facility similar to the successful oyster
hatchery in Milford. Connecticut, operated by Victor Loosanoff of
the Bureau of Commercial Fisheries (the forerunner of the Na-
tional Marine Fisheries Service) to aid the Long Island Sound
oyster industry. Galtsoff operated the hatchery for a time but it had
little impact on the then moribund Rhode Island oyster industry;
however, it became the forerunner of the National Marine Fisher-
ies Service Narragansett Laboratory. The hatchery building itself
remains on the GSO campus as the Mosby Center, the campus
cafeteria building. The other legacy of the period is Galstoff s
valuable reprint collection used as reference material in his classic
(1964) treatise on the American oyster. The collection is housed
currently at the nearby Pell Marine Science Laboratory.

As the quahog fisheries began to develop in the late 1940s and
eariy 1950s, much of the quahog stocks in Narragansett Bay were
located behind pollution closure lines. The first baywide wide
assessment of quahogs in Narragansett Bay showed that greater
than 60% of the quahogs in the bay resided in the closed Provi-
dence River and Mount Hope Bay sections of the bay (Stringer
1959). There was intense fishing pressure on the remaining open
areas by both hand-diggers and dredge boats. In 1954. the Division
of Fish and Wildlife initiated a shellfish relay program to dredge
quahogs from the closed areas and deposit them in management
areas in certified waters that would eventually be open for fishing.
Between 1954 and 1968. the relay program typically moved an
average of 1140 metric tonnes of shellfish annually (Table \).

TABLE L

Quahog relays in Narragansett Bay in metric tonnes whole shell
weight (Source: Division of Fish and Wildlife Annual Reports).

Method of Harvest

Year State Dredge Vessels Hired Dredge Boats Handraking

S774 —

4697 —

1725 —

2767

1954

—

1955

—

1956

5163

1957

4998

1958

4374

1959

4695

1960

3125

1961

2932

1962

4027

1963

1169

1964

—

1965

—

1966

—

1967

—

1968

—

1971

329

1977

—

1978

—

1979

—

1980

—

1981

—

1982

—

1983

—

1984

—

1985

—

1986

—

1987

—

1988

—

1989

—

1990

—

1991

—

1992

—

1993

—

1994

—

1995

—

1996

—

1997

—

1998

238

—

54.4

—

20.4

820

138

—

83.6

7016

—

7487

361

7702

361

6412

—

2916

99.2

66.9

—

85.7

—

71.8

—

86.5

—

87.7

—

81.6

—

49.9

—

108.2

—

59.8

—

103.4

—

88.5

—

133.5

—

101.7

—

322.4

—

25.1

—

108.1

—

7.0

—

78.3

—

7.5

—

60.3

—

122.2

—

290.5

After 1968 and the banning of power dredging in Narragansett
Bay. the transplant program changed character. Beginning in 1977,
under the leadership of Arthur Ganz, the state-conducted relay
program began paying a modest fee to supervised shellfishermen
to dig quahogs and move them to the management areas in certi-
fied waters. On average over 22 y, 98 metric tonnes of shellfish
were moved annually (whole shell weight; ranging from 7 to 322
metric tonnes per year). This program remains popular with the
shellfishing community and has been ongoing until the present.

In spite of their popularity in the shellfishing community, the
value of relay programs has long been in dispute in Rhode Island.
The reluctance by state officials to dedicate permanent funding for
a long-term shellfish transplant program has led to intermittent
stocking attempts, primarily into already highly productive areas
such as Greenwich Bay. Critics have classified the program as a
"put & take" subsidized fishery, while shellfishermen contend they
are denied access to highly productive areas due to long standing

404

Rice et al.

sewage treatment deficiencies and argue for just compensation.
Since the 1980s, shellfishermen have become dependant upon the
Greenwich Bay transplant area for economic survival during the
winter months. While a limited number of shellfishermen tradi-
tionally participate in transplants, hundreds of fishermen are ob-
served harvesting upon the reopening of management areas.

Another effort to restore shellfisheries in Rhode Island included
an effort by the Shellfish Commission of the Town of New Shore-
ham (Block Island) between 1987 and 1990 to rear hatchery seed
quahogs in plastic-mesh-covered trays (Littlefield 1991). In 1989.
about 120.000 Merceuaria inercenaria (notata strain) 15-20 mm
in size were seeded into the Andy's Way section of the Great Salt
Pond. In 1990. about 130.000 more were seeded into the same
area. Littlefield ( 1991 ) reported that legal-size (about 48 mm valve
length) notata quahogs were showing up in the 1990 fall fishery,
but he did not estimate a percentage recovery rate. This project was
discontinued in 1991 when Mr. Littlefield resigned from town
government.

In addition to quahogs. scallops have been an iinportant shell-
fishery in Rhode Island. Beginning in the early 1970s there were
three major pushes to restore scallop (Argopecten hradians irra-
dians) fisheries using hatchery reared seed. Scallop fisheries were
historically abundant in the shallow Greenwich Bay region of Nar-
ragansett Bay and the barrier beach lagoons and estuaries along the
south shore of the state (Olsen and Stevenson 1 975 ). By the late
1960s and early 1970s, scallop catches were small and sporadic.
As a result the Division of Fish and Wildlife established a hatchery
for scallops at the old ferry terminal in Jamestown in 1973. The
operation was moved to better facilities at the Division of Fish
and Wildlife Coastal Fisheries Laboratory in Jerusalem in 1978.
During its 7 y of operation, several thousand scallops were dis-
tributed to coastal barrier beach ponds. The hatchery ceased op-
erations in 1980 for lack of continued state funding (John Karls-
son. Division of Fish and Wildlife retired, persona! communication
1998).

The next scallop restoration effort, in 1988-1991. was the
Rhode Island Scallop Restoration Project. This effort arose in to
response to massive scallop and other bivalve mortalities as a
result of blooms of the picoplankton Aitrcococciis anophugefcrens
in Narragansett Bay and coastal estuaries during the summers of
1985 and 1986 (Tracey 1988). This 'brown tide' event occurring
on two successive years decimated scallop (Argopectcn irradians
irradians) populations in the coastal sail ponds and estuaries. The
aim of the restoration project was to solicit funds as a nonprofit
corporation and to work cooperatively with the Division of Fish
and Wildlife and Spatco Ltd., a private hatchery, to hatch and rear
juvenile scallops for release in the coastal ponds (Burns 1991 ). As
a result of this collaborative project. 60.000 seed scallops ( 15-20
mm size) were released into Point Judith Pond in 1989. In 1990.
20.000 seed (15-20 mm) were released in the Great Salt Pond of
Block Island, and 5 million seed (1-3 mm) were released into
Point Judith Pond due to lack of adequate nursery facilities. Also
that year, 250,000 seed (15-20 mm) purchased from a Maine
hatchery were distributed to Quonochontaug, Ninigret, and Win-
nepaug ponds (Robert Rheault, Spatco Ltd., personal communica-
tion. 1998; Division of Fish and Wildlife records). The project was
discontinued in 1991 because the nonprofit corporation could not
raise sufficient funds to continue, and the intricacies of state bid-
procurement regulations made advance seed orders from the pri-
vate hatchery participating in the project a challenging process.

CURRENT SHELLFISH RESTORATION

Scallop

The third scallop restoration effort, the Restoration and En-
hancement of Bay Scallop Populations in Narragansett Bay Proj-
ect, has been underway since 1995 through the Rhode Island De-
partment of Environmental Management Aqua Fund. The aim was
to reintroduce bay scallops. Argopecten irradians. to areas of his-
torical abundance in Narragansett Bay. using both hatchery-reared
scallops and scallops gathered from mesh spat collector bags simi-
lar to those recently used for scallop restoration in nearby West-
port. Massachusetts (Tammi et al. 1998). Seed averaging 20 mm
(range 15-24 mm) was both free planted and placed in protective
cages for overwintering at sites with suitable habitat. The scallops
were monitored for growth, survival, and predation rates. A total of
1 million seed had been planted in Narragansett Bay as of Fall
1998, producing small sets of bay scallops in adjacent areas of
some of the sites. While overall recovery of planted scallops has
been minimal, several areas of the bay supported a recreational
harvest of bay scallops for the first time in decades.

In 1997. additional funding was received to add scallop stock-
ing of the south shore coastal ponds, utilizing the same method-
ology as the Narragansett Bay study. The coastal pond study pro-
vided control sites for comparison with the existing sites in the
bay. A total of 80.000 hatchery-reared scallops were planted in the
fall of 1997 and again in 1998. Growth and survival rates of
scallops placed in the coastal ponds were significantly higher than
at any of the sites in Nan^agansett Bay. despite observations of
equally high predation and fouling. Additionally, populations of
seed scallops have been observed in the vicinity of caged animals,
as well as being found in two of the five ponds where spat col-
lectors were deployed. The project is in its final year of monitoring
and as of December 1998 there is no dedicated funding on the
horizon to continue scallop enhancement efforts.

Lobster Fishery

After nearly a 50-y hiatus, there has been a revival of the idea
of restoring and enhancing Narragansett Bay lobster fisheries be-
ginning in 1996 in a cooperative study between the University of
Rhode Island and the National Marine Fisheries Service (Cobb et
al. 1998). Funded by restoration and remediation funds from a
1989 oil spill in Narragansett Bay. the aim of this modem effort is
to increase lobster habitat, assess the survival of lobsters naturally
recruiting onto artificial reefs, and assess the sur\ival of hatchery-
reared lobsters on the artificial reefs. This approach of focusing on
habitat differs from the earlier approach of releasing juveniles
directly into the bay without protecti\e habitat. Six artificial reefs
( 10 X 20 m) consisting of either cobbles or boulders were placed
in Narragansett Bay. Soft bottom and naturally rocks bottom con-
trol areas served as control areas. In 1996 and 1997. natural lobster
recruitment into the areas was monitored. In 1998, about 2400 fifth
stage, tagged ju\enile lobsters (4.3/nr) were released into the test
sites (Kathleen Castro. University of Rhode Island, personal com-
munication 1998). This enhancement program should run until
2001 and yield valuable data about habitat enhancement as a
means for reducing predation on released hatchery-reared stock.

Quahog

In response to declining quahog fisheries in the 1990s, the
Rliiidc Island Department of Hn\ ironmental Management (DEM).

Shellfish Restoration and Management Projects

405

Division of Fish & Wildlife (DFW) began a population and utili-
zation study of the uncertified shelltlsh resources in the Providence
River (funded by a grant from the U.S Department of Commerce).
Evaluating shellfish relay programs as a tool for enhancing Rhode
Island's quahog fishery is essential to the development of a state-
wide shellfish management program. Judicious utilization of shell-
fish resources in uncertified waters for either relay stocking or
depuration may provide revitalization of Rhode Island's shellfish
industry. Project activities included a survey to determine current
quahog biomass in the Providence River, calculation of estimates
of maximum sustainable yields, and development of a rational plan
for the transplanting of uncertified shellfish stocks. One of the key
work elements of the project was a pilot project to test the feasi-
bility of transplants. The DFW is evaluating different methodolo-
gies of transplanting for optimum benefit for the resource and the
industry.

During 1997. DFW conducted shellfish dredge surveys in the
Providence River to assess quahog population densities. This area
had not been surveyed since 1977. The survey was conducted from
onboard the 29' RfV Inspector Clambeaux utilizing the same ran-
dom stratified procedures pioneered in a DFW study of quahog
populations in the West Passage of Narragansett Bay (Russell
1972). These techniques have been used annually since 1993 for
assessing quahog populations in other areas of Narragansett Bay
(Lazar et al. 1995). The Providence River stations were divided
into 500 X 500 m grids and a 30-m tow was made using a hydraulic
dredge. From these samples the DFW determined quahog abun-
dance, size structure, and densities, and the maximum sustainable
yield (MSY) that could be transplanted without depleting the
stock. The total of 51 tows completed had a mean density of 9.37
± (1.34 se) quahogs/m". Total standing stock biomass for the
Providence River was 26.400 metric tonnes. The biomass was 86%
top necks (60 mm valve length or larger); few sublegal or count
necks were observed over the course of the survey. Subsamples of
the quahogs were measured and morphometric comparisons cal-
culated between shellfish in certified and uncertified waters. A
baywide MSY was calculated using a biomass dynamic model that
integrates catch per unit effort (CPUE). landings, and survey data.
Data analysis indicated a maximum of 272 1 metric tonnes could be
removed annually from the Providence River for relay purposes
without impairing stock production. For year I (1998). DFW rec-
ommended a minimum of 238 metric tonnes be harvested by
dredge vessel for transplanting during this prototype project.

Prior to commencement of the transplant, several areas were
evaluated for appropriate bottom types, existing shellfish densities
and current fishing effort, predation, and proximity to depleted
areas. The "High Banks" Shellfish Sanctuary was established as a
management area and closed to commercial harvesting of shellfish
for a period of 2 y (Fig. 1). The expectation is that this dense
concentration of large adult clams will repopulate adjacent areas.
The RV Captain Bert from the University of Rhode Island was
contracted to dredge from July to November 1998. A total of 238
metric tonnes of shellfish were moved from the Providence River;
all tow positions were logged using global positioning system
(GPS) coordinates. Data collected onboard included tow coordi-
nates, depth, bottom type, density, and shell measurement. These
data will be utilized to update the 1997 biomass assessment. Ad-
ditionally, the donor area and the adjacent highly productive "Area
A" (or Upper Narragansett Bay area) will be monitored through
annual dredge surveys to provide estimates of the impacts of the
large-scale relays and of quahog recruitment in those areas. The

1998 PROVIDENCE
RIVER
RELAY
PROJECT
Dredge!

RIDFW/CFL/iarae

Apponaug
Ca«e

Figure I. The source area of quahogs for the 1998 Providence River
Relay Project and the recipient High Banl^s Sanctuary Area in Nar-
ragansett Bay Rhode Island. Also shown are the shallow coves of
Greenwich Bay that serve as the source areas of quahogs for handrak-
ing relays into the Greenwich Bay Management Area.

High Banks relay area will also be monitored over 1999-2000 to
address the efficacy of large-scale relays of the Providence River
stock. To address recovery, growth, predation, fishing pressure,
and mortality, a portion of the relayed stock has been tagged and
their location logged by GPS coordinates. They will be monitored
by SCUBA and DFW's dredge research vessel in 2000.

Cost analysis of the dredge relay program indicates $0.09/kg
was expended to move shellfish, compared to a range of $0.08 to
$0. 1 3/kg typically expended by contracting handrakers. Expected
costs for utilizing handrakers in the Providence River would likely
have been well in excess of $0.1 3/kg. due to the added expense of
daily contracting of transport vessels and the additional personnel
costs for monitoring and enforcement. Also, logistically it is un-
workable to provide adequate supervision of individual rakers in a
relatively large area in the midst of a major shipping channel.
However, contracting handrakers to move shellfish from smaller
coves immediately adjacent to recipient areas appears to be an
economical option, as is the case for the current Greenwich Bay
relay program.

In addition to the relay efforts, there has been interest in using
nursery-reared quahog seed stock for replenishing public fishing
grounds. In 1996. John Williams of Warwick Cove Marina seized
upon the idea of nursery culture of shellfish in marina waters
(Rheault and Rice 1989) using existing floating upweller tech-
niques (Hadley & Manzi 1984) being developed and employed by
Robert Rheault of the Moonstone Oyster Company. At the incep-

406

Rice et al.

tion of the Rhode Island Public Benefit Aquaculture Project, Mr.
Williams' concept was to incorporate secondary education into the
physical activities of raising shellfish. The project would be a
irieans of teaching science, math, and language arts, and the prod-
uct would be used to restore fishing beds in Narragansett Bay. The
project, which began in 1997 with some seed monies from local
foundations, had as its a mission to develop a true public benefit
aquaculture project that would integrate well with current uses of
the public waters of Rhode Island, without compromising the in-
tegrity or quality of the state's aquatic resources. The Rhode Island
Seafood Council began assisting in the development and coordi-
nation of a team to guide this effort. The Project team realized
early on that in order to be fully beneficial to the state, all stake-
holders needed to be educated about the project goals.
The Project goals include:

• Strong skills-based curriculum development for secondary level
education that is tied to natural resources, and the application of
aquaculture principles to inject applied learning into the cur-
riculum.

• The inclusion of commercial and recreational shellfishermen in
order to build their understanding of the potential of public
aquaculture to rebuild shellfish stocks, and to utilize their ex-
perience in guiding educational activities and assisting in the
determination of survival of seeded stock

• The development of a self-sustaining resource restoration plan
for Rhode Island incorporating aquaculture techniques to restore
declining shellfish resources.

To meet the goals, a strong team of state, local, and educational
representatives committed to guide the development of the Project.
Initial advisors to the project were university personnel with ex-
pertise on scientific issues and independent commercial fishermen
and personnel from marine trades organizations to assist in devel-
oping an economically viable plan of work that minimized user
conflicts. The advisory team grew to include representatives from
the following organizations:
Rhode Island Department of Labor and Training
Rhode Island Manufacturers Extension Service
Rhode Island Legislative Commission on Aquaculture
Rhode Island Economic Development Corporation
Rhode Island Department of Environmental Management
Coastal Resources Management Council
Rhode Island Seafood Council

University of Rhode Island-Department of Fisheries and Veteri-
nary Science
Roger Williams University Center for Economic and Environmen-
tal Development
Newport, Warwick. Chariho, and Cranston .^rea Career and Tech-
nical Centers
Middletown Alternative Learning Program
Rhode Island Quahog Company
Warwick Cove Marina
Ram Point Marina
East Passage Yachting Center
Newport Yacht Club
The Rhode Island Shcllfishermen's Association

Students from four technical high schools began building up-
wellers in the spring of 1998. These schools were partnered with
marinas in the local area, and high school students were partnered
with college-level mentors from the Marine Biology Program al
Roger Williams University in Bristol. Three of the high schools
built five upweller units that were deployed early summer at part-

ner marina facilities. The five units were stocked with a total of
100.000 seed of Menenaria mercenaria. Due to an unusually high
rainfall in the spring, quahog seed was very difficult to obtain from
local suppliers. As a result, quahog seed (2.5 mm) was purchased
from Virginia after satisfying strict state importation guidelines. In
addition, one unit was stocked with local oyster seed donated by
the Rhode Island Sea Grant Marine Advisory Service and another
unit was partially stocked with scallop seed donated by National
Marine Fisheries Service Milford Laboratory.

As the quahog seed reached the presumed "predator resistant"
size of 20 mm they were given to the DEW and planted in a
management sanctuary had been opened for public harvest in De-
cember 1999. Current Rhode Island shellfish regulations define
shellfish seed as a shell dimension of 20 mm or less for quahogs
and 32 mm for oysters (RIDEM 1998). To assure public health,
shellfish seed can be grown in uncertified waters of marinas, but
they must be moved to certified waters for final growout and
depuration once they reach the prescribed size limits. These seed
definitions allow a minimum 1-y depuration period based on local
growth rates.

Shellfish growth was monitored weekly by students from the
partner schools and all data on growth, salinity, and temperature
were recorded. As the shellfish grew students were able to apply
math, science, writing, and public speaking skills to the project
through presentations at their respective schools and at the Third
Annual Rhode Island Aquaculture Conference, held in October
1998. This was a perfect way for students to start building a
portfolio and to create a network of professional mentors. After the
conference, there have been several other schools and marinas that
expressed interest in participating in the project.

This collaboration has also allowed students to work alongside
state biologists and to be involved in data collection for the tagging
and transplant-restoration project at the High Banks Management
Area being carried out by Df^. All students that have taken part
in the tagging and relay-restoration effort have a better understand-
ing of occupations within DEW, and have more direction in their
education and future career choices. Additional statew ide partner-
ships are being built: the Rhode Island Department of Health has
become eager to expose students to the workings of a USFDA-certi-
fied shellfish testing lab and is making the lab available for inter-
active tours for the students involved in the aquaculture program.

As of December 1999 there are two proposed plans with po-
tential to assist ongoing shellfish restoration projects in Rhode
Island. One proposal is to start a commercial shellfish hatchery by
the Hope Shellfish Company LLC at the Quonset Point Industrial
Park, which would be partially funded from private investors, the
Rhode Island Economic Development Corporation, and the Rhode
Island Economic Policy Council. Part of the stated mission of the
proposed hatchery is to provide shellfish seed for public aquacul-
ture and mitigation projects in the state. The other proposal is a
plan to mitigate damages caused by the January 1996 grounding of
the barge North Cape and the devastating oil spill that resulted on
the south shore beaches of Rhode Island. The National Marine
Fisheries Service in cooperation with Rhode Island state agencies
is proposing an ambitious multi-year plan to rc-seed molluscan
shellfish beds in the coastal barrier beach salt ponds and estuaries
near the spill site.

CONCLUSIONS

Shellfish restoration efforts in Rhode Island have a long his-
tory. It is very instructive to examine past projects to discover the

Shellfish Restoration and Management Projects

407

reasons for either success or failure, and to apply these lessons to
current and planned efforts. There are a number of attempted proj-
ects and a number that got started but did not prove to be sustain-
able in the long run. Some of the projects that have arisen out of
a sudden "windfall" of funds or out of short-term public concern
have been particularly susceptible to not building long-term sus-
tainability. Some projects have flourished due to individual initia-
tive, but failed to continue when the principals either "got tired" or
moved from the area. The most successful projects in terms of
longevity are those that have had perennial support by commercial
and recreational fisheries clientele, local communities, and state
government agencies. For successful shellfish restoration projects,
there must be melding of good science, consensus on policy, pub-
lic acceptance, economic feasibility, multisector cooperation, and
a measure of good luck.

A cautionary note, however, is needed. In recent years there has
been an increasing trend in academia and government toward col-
laborative, multi-agency, and multisector projects for greater cost
effectiveness, sharing of talent, and sharing of resources. Although

the potential benefits of collaborative multisector projects are
great, there are some risks. It is short-sighted to underestimate the
amount of time and effort required to bring people and organiza-
tions together and to maintain continued collaboration toward a
common goal. This problem of coordination and management is
not intractable; it simply needs to be recognized and planned for.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the following individuals
for kindly sharing data and other infonnation to make this review
of Rhode Island shellfish restoration projects possible: Najih
Lazar, Arthur Ganz, and Mark Gibson of the Rhode Island De-
partment of Environmental Management, Division of Fish and
Wildlife; Dr. Robert Rheault of Moonstone Oyster Company;
Kathleen Castro of the Fisheries. Animal and Veterinary Sciences
Department at URI; John Williams of Warwick Cove Marina; and
Ralph Boragine of the Rhode Island Seafood Council. This is
publication 3684 of the College of the Environment and Life Sci-
ences, University of Rhode Island.

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Rhode Island Shellfisheries Conference. Rhode Island Sea Grant. Uni-
versity of Rhode Island. Narragansett, Rhode Island.

Bums, W. G. 1991. The Rhode Island scallop restoration program, pp.
89-91 In: M. A. Rice, M. Grady & M. L. Schwartz (eds.l. Proceedings
of the First Rhode Island Shellfisheries Conference. Rhode Island Sea
Grant. University of Rhode Island. Narragansett. Rhode Island.

Carlson, F. T. 1954. The American lobster fishery and possible applica-
tions of artificial propagation. Y'ale Consen\ Stud. 3:3-7.

Cobb. J. S.. K. Castro. R. A. Wahle & J. Catena. 1998. An artificial reef for
lobsters {Homanis ameiicanus) in Rhode Island. USA. pp. 75-78. In:
Proceedings of a Workshop on Lobster Stock Enhancement, held in the
Magdalen Islands (Quebec) October 29 to 31, 1997. Canadian Industry
Report of Fisheries and Aquatic Sciences, Department of Fisheries and
Oceans. Ottawa, Canada.

Deshonnet. A. & V. Lee. 1991. Links between water quality and shellfish-
eries in Narragansett Bay. Rhode Island, pp. 1 1-18. In: M. A. Rice. M.
Grady & M. L. Schwartz (eds.l. Proceedings of the First Rhode Island
Shellfisheries Conference. Rhode Island Sea Grant, University of
Rhode Island. Narragansett. Rhode Island.

Galtsoff P. S. 1964. The American oyster. Crassoslrea virginica. U.S. Fish
Wild!. Sen: Bull. 64:1^80.

Glude. J. B & W. S. Landers. 1953. Biological effects of bullraking vs.
power dredging on a population of hardshell clams. Venn.': mercenaria.
Nail. Shellfi.'ili. A.'isoc. Addr. 1951:47-69.

Hale. S. O. 1980. Narragansett Bay: A Friend's Perspective. Rhode Island
Sea Gram. University of Rhode Island. Narragansett. Rhode Island. 1 30
pp.

Herrick. F. H. 1909. Natural history of the American lobster. Bull. Bur.
Fhh. 29:149-108.

Inland Fishery Commission (IFC). 1934. Si.xty-fourth Annual Report of the
Commissioners of Inland Fisheries of Rhode Island for 1934. Provi-
dence, Rhode Island.

Kochkss, J. M. 1974. Oystering from New York to Boston. Wesleyan Uni-
versity Press, Middletown, Connecticut. 251 pp.

Lazar. N., A. Valliere & A. Ganz. 1995. Quahog stock assessment and
implementation of an interim management plan in Greenwich Bay.
Rhode Island, pp. 5-29. In: M. A. Rice & E. Gibbs (eds.). Proceedings
of the Third Rhode Island Shellfisheries Conference. Rhode Island Sea
Grant, University of Rhode Island. Narragansett, Rhode Island.

Liltlefield. C. N. 1991. Growth of seed quahogs (Mercenaria mercenaria)
in nursery trays in the Great Salt Pond, Block Island, Rhode Island, pp.
81-88. In: M. A. Rice. M.Grady & M. L. Schwartz (eds.l. Proceedings
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McLoughlin, W. G. 1978. Rhode Island: A Bicentennial History. W. W.
Norton. New York. 240 pp.

Meade. A. D. 1901. Habits and growth of young lobster, and experiments
in lobster culture, pp. 61-80. In: Thiny-first Annual Report of the
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Meade. A. D. 1908. A method of lobster culture. Proc. Fourth IntI Fish.
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Ni.xon, D. 1993. A legal history of shellfish regulation in Rhode Island, pp.
19-22. In: M. A. Rice & D. Grossman-Garber (eds.). Proceedings of
the Second Rhode Island Shellfish Industry Conference. Rhode Island
Sea Grant, University of Rhode Island, Narragansett, Rhode Island.

Nixon, S. W. 1995. Metal inputs to Narragansett Bay: A History and As-
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Olsen, S. & G. L. Seavy. 1983. The State of Rhode Island Coastal Re-
sources Management Program as Amended. Rhode Island Coastal Re-
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Olsen. S. B & D. K. Stevenson. 1975. Commercial marine fish and fish-
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quahog in Rhode Island. Rhode Island Sea Grant Report no. RIU-T-
92-001. University of Rhode Island. Narragansett, Rhode Island.
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Rhode Island.

Rhode Island Department of Environmental Management (RIDEM). 1998.
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Part XIX. aquaculture of molluscan shellfish in Rhode Island Tidal
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Wildlife Service, Washington. D.C. tality of mussels (Mytihis eduHs) during the 1985 'brown tide' in Nar-

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recruitment of bay scallops, Argopecten irradians (Lamarck 1819), to Williams, R. 1643. Chapter XIX, of fish and fishing, pp. 122-125. In: A

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Jourmtl of Shellfish Research. Vol. 19. No. 1. 409-412. 2000.

A PLAN FOR REBUILDING STOCKS OF OLYMPIA OYSTERS (OSTREOLA CONCHAPHILA,

CARPENTER 1857) IN WASHINGTON STATE

ANITA E. COOK, ' J. ANNE SHAFFER,' BRETT R. DUMBAULD,^*
AND BRUCE E. KAUFFMAN"

' Washington Department of Fish and Wildlife

Point Whitney Shellfish Lab

1000 Point Whitney Road

Brinnon, Washington 98320
'Washington Department of Fish and Wildlife

Willapa Bay Field Station

P.O. Bo.x 190

Ocean Park. Washington 98640

ABSTRACT The Olympia oyster ( Osrrt'o/o conchaphila) is native to the state of Washington. Once the basis for a thriving, statewide
oyster industry, its numbers were drastically reduced by the mid 1940s. Water quality and overharvesting are thought to be the major
factors causing its near demise. The Pacific oyster (Crassoslrea gigas) has since replaced the Olympia oyster in Washington and world
markets. Concern over the much reduced status of the stocks of native oysters led the Washington Department of Fish and Wildlife
to develop a plan to rebuild them. The goal of the strategy, to restore the Olympia oyster within its historical range, is quite simple,
but offers many challenges. Key elements of the draft strategy presented here include a description of the historical and current
distribution, habitat requirements, and current problems associated with restoring stocks of the native oyster in Washington State.
Partnering with Tribal co-managers, local commercial shellfish interests, and the general public provides new opportunities for
restoring the Olympia oyster, a top priority for state management of this species. Suggested priorities for strategy implementation are
also discussed.

KEY WORDS: Olympia oyster. Ostreola conchaphiUi. Washington, estuary, plan

INTRODUCTION

The Olympia oyster (Ostreola conchaphila Carpenter, 1857;
formerly Ostrea lurida) is native to the Pacific coast of North
America and occurs in marine waters from Bahia de San Quentin,
Baja California, to Sitka, Alaska (Ricketts and Calvin 1968, Baker
1995). It primarily inhabits sheltered waters or estuaries. Once
common in Washington state, the Olympia oyster now has a re-
stricted and very patchy distribution in Willapa Bay, Grays Har-
bor, and southern Puget Sound.

The Olympia oyster has been the focus of human harvest for
several thousand years. The Washington tribes used the oyster
extensively and often based settlement locations on its harvest
(Swan 1857, Washington Secretary of State 1935, Steele 1957,
Elmendorf and Kroeber 1992). With European colonization, the
Olympia oyster supported a large commercial industry. Olympia
oyster beds in Puget Sound, Hood Canal, and Willapa Bay were
harvested extensively, and later cultivated with an elaborate sys-
tem of dikes (Steele 1957, Brown 1976. Westley et al. 1985).
Overharvesting in the late 1800s and severe water quality prob-
lems in the 1930s to 1950s caused Olympia oyster stocks to crash,
and the industry to terminate in Willapa Bay and Puget Sound,
respectively.

Recent interest and concerns about the status of native Olympia
oyster stocks in Washington State waters, widely recognized to be
reduced from historical levels, led the Washington Department of
Fish and Wildlife (WDFW) to begin developing a stock rebuilding
plan. General goals of the plan are wise stewardship, maintenance
of genetic integrity, and ecosystem restoration. As managers, how-
ever, we struggled with the scope of the goal statement, particu-

* Author to whom all correspondence should be sent. Email: dumbabrd
@dfw. wa.gov

larly whether or not to incorporate restoration to a level where
fisheries could occur. The resulting, more generic statement we
accepted, "to restore and maintain Olympia oyster populations on
public tidelands in their native range." defers that discussion to a
later date.

STATUS OF THE POPULATION

Historical Population Size and Distribution

Historically, Olympia oyster stocks were very large in Wash-
ington State, with reported annual landings of over 130,000 bush-
els (4581 m-') around 1890, principally from Willapa Bay
(Townsend 1 896, Fig. 1 ). Landings were substantially higher in the
1870s (possibly as high as 200,000 bushels), but landings were not
well documented. Olympia oysters are very small so this repre-
sents a substantially greater number of individuals (approximately
1500 per bushel) than are found in current harvests of Crassostrea
gigas (Thunberg 1793).

Within the inland waters of Washington, the Olympia oyster
had a scattered distribution throughout Puget Sound and Hood
Canal (Westley 1976, Fig. 1). Samish Bay once supported a large,
naturally occurring Olympia oyster population, which was se-
verely depleted in the 1800s as a result of overharvest (Brown
1976). The most abundant natural Puget Sound populations his-
torically occurred around Olympia, primarily in Mud and Oyster
bays (Steele 1957).

Current Population Size and Distribution

Native oysters are currently present on state oyster reserves in
Willapa Bay on the coast, and North Bay and Case Inlet in south-
em Puget Sound. Dense natural sets have recently been observed
in the reserves in southern Puget Sound and an annual spawning
event has been noted in Willapa Bay. but very few juvenile oysters

409

410

Cook et al

QuUcroe

Olympia Oyster Production in Wasliington State

^

Figure 1. Map of Washington state showing the general distribution of
historic and current Olynipia oyster {Ostreola concaphila) stoclts.

appear to have survived at the latter location. Shellfish growers in
south Puget Sound had noticed similar sets in the mid 1980s, but
these sets were subsequently destroyed by severe winter weather.
Comparison of historical documents and local knowledge indicates
that current numbers are. at best, a mere fraction of. and possibly
more ephemeral than, historic populations.

Manageinciil

The Washington Department of Fish and Wildlife (WDFW)
co-manages Olympia oysters with the Tribes and other government
agencies in Washington state according to the provisions in the
federal district court's orders and judgments {United Stales v.
Washington. No. 921.1. subproceeding 89-.1).

Non-Tribal Commercial Fishery

WDFW harvest reports from 1S97 through 1990 (Fig. 2) show
a general decline in commercial production of Olympia oysters,
from a high of over 200,000 gallons (757,072 L) in the early part
of the century to an annual production of less than a 1000 gallons
(.178.5 F) since 1979. Preliminary data for 1991 through 1996 show
an annual commercial harvest of approximately 500 gallons (about
4000 pounds = 1814 kg of shucked oysters), most of which is
from private tidelands in south Puget Sound.

Currently there are three coniniercial Olympia oyster growers
in Puget Sound that operate solely on private tideland. The private

m

V

-Willapa Bay
Puget Sound

175,000
150,000

125.000

g 100.000

o

O 75,000
50,000
25.000

Figure 2. Olympia oyster production in Willapa Bay along the outer
coast and Puget Sound in Washington State. Production in Willapa
Bay declined rapidly in the early 1900s while that in Puget Sound
remained higher through the mid 1930s. Production in Willapa Bay
prior to 1896 was likely higher, but records are difficult to locate.

grower controls management of stocks on private tidelands; how-
ever. Washington State Department of Health requirements must
be met. and quarterly harvest reports are required by WDFW.

The Washington state oyster reserves were established in 1890
for the preservation and growth of Olympia oysters (Woelke
1969). Reserves were set aside to provide both "seed for the oyster
farmer and an exploitable stock for the fisherman." At the time
these laws were enacted, the typical practice in Willapa Bay was
to long or handpick the native oysters from low intertidal areas and
move them to privately held tidelands elsewhere in the bay. They
were held until they were shipped to markets in San Francisco,
California, and Portland, Oregon. The reserves originally com-
prised 4548 ha in Willapa Bay and 1821 ha in Puget Sound. Some
lands have since been sold by the state legislature. Reserves cur-
rently encompass 4047 ha in Willapa Bay and 405 ha in southern
Puget Sound.

With the decline in the Olympia oyster population, reserve laws
were changed in 1947 to reflect the growing importance of the
Pacific oyster, Crassostrea gigas, which was imported into Wash-
ington state in the mid 1920s and rapidly established itself on
reserve tidelands, particularly in Willapa Bay. Willapa Bay re-
serves are now actively managed for commercial harvest of the
Pacific oyster. An average of 54,000 bushels ( 1903 m') of Pacific
oysters are sold each year from managed intertidal tracts, as a
result of which $79,000 is returned annually to the state general
fund (Dumbauld and Kauffman 1996). Growers are required to
return 40'/f of the live oyster volume in shell to the tracts to
maintain stocks via natural spawning and settlement.

No commercial oyster harvest occurs on the Puget Sound oys-
ter reserves. While Olympia oysters exist on both the Willapa Bay
and Pugel Sound reserves, no active management has occurred for
Ihis species, and the last commercial harvest of Olympia oysters on
reserve tidelands occurred in Pugel Sound in 1*^)29.

Non-Tribal Recreational Fi.shery

The Olympia oysler has been managed passively on public
beaches in Washington state for many years. Olympia oysters are
included in the regulations that apply to all classified oysters.
Cunent harvest limits include a combined daily limit of 18, and
oysters must be shucked on the beach and the shells left at the

Stock Rebuilding Plan for Olympia Oysters

411

same place and tide height where they were taken. They may be
harvested only by hand or with a hand-held manually operated
prying tool (no hammers, etc.). Beginning May 1. 1998. regula-
tions were changed for all areas except Hood Canal and the outer
coast. These regulations included a minimum size restriction of
6.35 cm (2.5 inches), which was designed to minimize recreational
harvesting of Olympia oysters.

All oyster reserves continue to be closed to recreational harvest
of interlidal clams and oysters with several exceptions where
clams and Pacitlc oysters may be taken. Seasons for non-reserve
beaches are set based on the population and projected harvest of
Pacific oysters. With the exception of one beach in North Bay.
Puget Sound oyster reserves currently are not actively managed for
oysters. Oyster dikes in Oakland Bay. once designed for Olympia
oysters, have created excellent habitat for Manila clams {Tapes
phillipinanim. Adams & Reave. 1850). Although the majority of
the Puget Sound reserves are closed to recreational clam and oyster
harvest due to access issues, these Manila clam beds do provide
stock to trade with the tribes to enhance other recreational oppor-
tunities.

Tribal Commercial Fisheries

The tribes of inland and coastal Washington historically have
played a dominant role in the commercial harvest of Olympia
oysters (Steele 1957) and at least one tribal war was fought over
rights to harvest Olympia oysters (Swan 1857. Esveldt 1948.
Steele 1957). There are no current Tribal commercial fisheries
targeted for Olympia oysters. However, commercial harvest of
Olympia oysters is not prohibited in the State/Tribe Interim Man-
agement Agreement except in areas the state has declared as arti-
ficial beds. The Point No Point Treat) Council Tribes are the only
tribes that have issued regulations for the commercial harvest of
oysters (species not specified) on public tidelands. Their annual
commercial regulations have a clause prohibiting the harvest of
oysters less than 6. .^5 cm in length for single oysters, which would
eliminate virtually all harvest of Olympia oysters. The majority of
tribal oyster bed openings are for single oyster harvest, but some
harvest of Olympia oysters could occur when clusters are har-
vested, which has occurred recently at a few beaches. Olympia
oysters may be harvested in ceremonial and subsistence fisheries.

Genetic Integrity of Olympia Oyster Stoclis

Research suggests that the rate of natural genetic exchange is
low among distinct coastal populations of native oysters in Wash-
ington, Oregon, and northern California (Baker 1995): however,
no information exists on genetic exchange within Washington wa-
ters. This is particularly important when considering historic
Olympia oyster farming practices, which included seed transport
both within and between regions.

Conserving the natural genetic integrity of Olympia oyster
stocks is an important component of the stock recovery strategy.
Artificial enhancement of Olympia oyster stocks should meet ac-
ceptable standards for maintaining the genetic stock integrity for
indigenous species. These standards include ( 1 ) using brood stock
for seed production from the same geographic area where seeding
will take place, and (2) establishing and maintaining the minimum
number of brood stock necessary to maintain genetic \ariability
while maintaining stock identity. Genetic integrity will be a topic
for funher dialog in effons to rebuild Olympia oyster populations.

FACTORS AFFECTING THE POPULATION

Habitat and Water Quality

Pollution has been the primary factor in the demise of the
Olympia oyster throughout lower Puget Sound and Hood Canal.
Sulfur waste liquor (SWL) from the Rayonier pulp mill, which was
built on Oakland Bay in 1927. was identified as the cause of the
demise of all Olympia oyster stocks in south Puget Sound (Gunter
and McKee 1960). Tidal currents carried effluent to Oakland Bay
beds within a tidal cycle and throughout lower Puget Sound within
a matter of days. Dramatic crashes were witnessed throughout the
Olympia oyster beds, and the Olympia oyster industry was de-
stroyed by the mid 1940s. The Rayonier mill was closed in 1957.
Unfortunately, monitoring of the Olympia oyster populations of
Puget Sound and southern Hood Canal had ceased following the
crash (Steele 1957, Gunter and McKee 1960).

Water quality impacts in Washington's waters have shifted
over the last 40 years from those caused by point source industrial
effluent to nonpoint source pollution. The impacts of contempo-
rary water quality degradation to residual Olympia oyster stocks
have not been studied. Possible contemporary sources of pollution
and water quality impacts affecting Olympia oysters include low
dissolved oxygen (DO), chlorine from sewage outfalls, nonpoint
pollution and associated eutrophication, sedimentation and silt-
ation, and herbicides (McMillen 1978, Couch and Hassler 1989,
Dumbauld 1996).

Honest

Overharvesting has been identified as the leading cause of
Olympia oyster stock crashes in Samish Bay (Puget Sound) and
Willapa Bay in the 1800s. Harvesting of other commercially val-
ued species may also impact Olympia oysters where they co-occur.

Interspecific Interactions

After the initial population declines, additional factors have
contributed to preventing the recovery of Olympia oysters. Intro-
duced predators (the Japanese oyster drill Cerateotoma iiiornatiim
Reduz, 1851, flatworms Pseiulostyloduis ostreaophagiisMyman,
1955, and the copepod Mytilicola orientalis. Mori, 1935) have
resulted in poor oyster conditions, and in the case of drills have
caused high mortalities (Peters 1993). Natural predators, including
starfish and diving ducks, are also thought to suppress recovery of
Olympia oyster populations. Other disturbances, including substrate
disruption by ghost shrimp and mud shrimp, smothering by slipper
shells, and competition with Pacific oysters for space and setting
habitat, are suspected of negatively affecting Olympia oyster recovery
(Steele 1957, Brown 1976. Westley 1976. Dumbauld pers. comm.).

STOCK REBUILDING ACTIONS

Restoration of this species may include both natural and arti-
ficial enhancement strategies. Natural restoration techniques, such
as water quality and habitat improvements will be the primary
focus. Primary objectives and actions necessary to rebuild Olym-
pia oyster stocks in Washington state vary with region but include:
( 1 ). Working with local experts, including the Tribes and shell-
fish growers, to define the historic and current distribution
of the Olympia oyster.
(2). Conducting population surveys to define current popula-
tion levels and establish a benchmark for long-term moni-
toring and management.
(3j. Defining water quality and inter-species interactions at a

412

Cook et al

regional level, and identifying priority areas for restora-
tion, at least in part, based on these interactions.

ACKNOWLEDGMENTS

Funding for this work was provided by the Washington De-
partment of Fish and Wildlife. The authors thank Randy Butler for
invaluable computer assistance. Steve Bloomfield, Bruce Brenner.

Ernie Dauman. Dave McMillian. Glen Rau. Justin Taylor. Lee
Weigardt. and Brian Kemmer graciously shared their history and
knowledge of Olympia oysters in Puget Sound and Willapa Bay.
Lauren Cole Warner and the U.S. Army Corps of Engineers made
travel to ICSR possible. Finally the authors thank Morris Barker
and Dick Burge for their extensive help and comments provided on
the stock rebuilding plan and this manuscript.

LITERATURE CITED

Baker, P. 1995. Review of ecology and fishery of the Olympia oyster.
Ostrea turida. with annotated bibliography. / Shellfish Res. I4:.'i01-
518.

Brown, J. 1976. Olympia oy.ster. Unpublished manuscript submitted to the
Washington Department of Fisheries, Olympia, Washington.

Couch. D. & T. J. Hassler. 1989. Species profiles: Life histories and en-
vironmental requirements of coastal fishes and invertebrates (Pacific
Northwest). Olympia oyster. U.S. Fish Wildl. Serv. Biol. Rep. 82
(11.124) U.S. Army Corps of Engineers. TR EL 82-4. 8 pp.

Dumbauld. B. 1996. Olympia oyster. Priority habitat and species summary.
Washington Department of Fish and Wildlife. Olympia. Washington. 6 pp

Dumbauld. B. & B. Kauffman. 1996. The Willapa Bay oyster reserves: a
brief history and resource survey proposal. Washington Department of
Fish and Wildlife, Willapa Bay Field Station, Nahcotta, Washington.
17 pp.

Elmendorf, W. W. & A. L. Kroeber. 1992. The structure of Twana Culture.
Washington State University Press, Pullman. Washington.

Esveldt. G. D. 1948. A review of the oyster industry of the state of Wash-
ington.Washington Dept. of Fisheries. Olympia. Washington. 28 pp.

Gunter. G. & J. McKee. 1960. On oysters and sulfite waste liquor. A
special report to the Washington State Pollution Control Commission,
Olympia. Washington. 93 pp.

McMillen, D. 1978. Letter from president, Olympia Oyster Growers As-
sociation to Washington Department of Fish and Wildlife, Olympia
Washington.

Peters, R. 1993. Past and current conditions of marine resources. Big

Quilcene Watershed Analysis. U.S. Forest Service. Olympic Region.
Olympia. Washington.

Ricketts. E. & J. Calvin. 1968. Between Pacific Tides. Stanford University
Press. Stanford, California. 614 pp.

Steele. E. N. 1957. The Olympia Oyster. Fulco Publications. Elma, Wash-
ington. 126 pp.

Swan, J. G. 1857. The Northwest coast or three years residence in Wash-
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Townsend, C. H. 1896. The transplanting of eastern oysters to Willapa
Bay. Washington, with notes on the native oyster industry, p. 193-202.
In: Report of the U.S. Fish Commissioner of Fisheries for 1895.

Washington Secretary of State, 1935. Oyster culture: A natural resource
revived. Office of the Secretary of State, Olympia, Washington.

Westley, R. E. 1976. Letter to J. Brown. Washington Department of Fish
and Wildlife, Olympia, Washington.

Westley. R. E.. A. S. Scholz & R. T. Burge. 1985. The Puget Sound oyster
reserves: a history and inventory with recommendations for the future.
Report to the Washington State legislature. Olympia. Washington. 25
pp.

West. J. E. 1997. Protection and restoration of marine life in the inland
waters of Washington state. Puget Sound/Georgia Basin Environmental
Report 6. Puget Sound Water Quality Action Team. Olympia. Wash-
ington. 144 pp.

Woelke. C. E. 1969. A history and economic evaluation of Washington
Stale oyster reserves. Report to the Washington Department of Fish-
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Journal of Shellfish Research. Vol. 19, No. 1. 4 13-+: I, 2000.

MUSSEL MYTILUS EDULIS (L.) FILTERING OF THE BALTIC SEA OUTFLOW THROUGH

THE ORESUND— AN EXAMPLE OF A NATURAL, LARGE-SCALE

ECOSYSTEM RESTORATION

JOEL HAAMER' AND JOHAN RODHE^

'National Board of Fisheries

Institute of Coastal Research

Nya varvet, byggnad 31

S-42 671 Vdstra Frohmda

Sweden
'Goteborg University

Department of Oceanography

Box 460

S-405 30 Goteborg

Sweden

ABSTRACT Investigations were undertaken to quantity the filtering capacity of mussels at the sill in Oresund. one of the straits
connecting the Baltic Sea with the Kattegat. The investigations included observations of hydrography and currents and measurements
of nutrients, chlorophyll-o. and phytoplankton. Tracing the water over the vast mussel banks at the sill, we found that about 75% of
the phytoplankton biomass was removed from the water. The clearance rate, defined as the water volume cleared of organic matter per
unit time and unit ash-free dry-weight of soft tissues of the mussels (AFDW), was estimated at 7 L/h/g. Based on the mussels present
at the sill (Madsen and Hojgard Petersen 1996), we determined that the mussels could clear the outflow from the Baltic Sea through
Oresund almost completely of phytoplankton biomass. We also found that the plankton biomass recovered after the passage of the sill.

KEY WORDS: Mussel filtering, clearance rate, ecosystem restoration. Oresund

INTRODUCTION

Benthic suspension-feeders like mussels iMytilus edulis) can
have a dominant influence on the flux of nutrients. This has been
found in various environments of Scandinavia: the Oslo Fjord in
Skagerrak ( Kirkerud and Bjerkeng 1994), the Roskilde Fjord in the
Kattegat (Mohlenberg 1995), and the Asko archipelago in the Bal-
tic Sea (Kautsky and Wallentinus 1980). These examples represent
regions with salinities ranging from more than 30 to 5 psu. Cloem
(1982) suggested that bivalves controlled the phytoplankton bio-
mass and prevented plankton blooms in the shallow estuary of
South San Francisco Bay, which receives effluent from 20 mu-
nicipal sewage treatment plants. All these examples are from areas
experiencing some level of eutrophication. Filtering by bivalves
decreases the negative effects of eutrophication in three ways: ( 1 )
by improving light conditions, (2) by decreasing the flux of or-
ganic matter to deeper layers with limited water exchange, and (3)
by leveling primary production. The flrst two ways are direct
effects of filtering. In the third case, filtering decreases primary
production during plankton blooms and remineralized nutrients are
supplied to the water over prolonged periods.

In situ measurements of nutrient uptake and remineralization
by mussel beds have been made in tunnel and fluine experiments
by Dame and Dankers (1988). Prins and Smaal (1990). and Asmus
and Asmus ( 1991 ). The common mussel was the dominant species
in these experiments, contributing to approximately 90% of the
total macrobenthic biomass. In the study by Asmus and Asmus
(1991), the uptake of chlorophyll-« (Chl-o) and the release of
inorganic nutrients were determined in water that passed through a
20-m long tunnel enclosing a mussel bed. In their experiments
phytoplankton biomass was reduced by 37±20'7f .

In the present study, Oresund, one of the three straits connect-
ing the Baltic Sea with the Kattegat and the North Sea (see Fig. 1 ),
was chosen as the site to measure the influence of mussels on

inorganic nutrients and phytoplankton fluxes. The sill is situated
between K0benhavn, Denmark, and Malmo, Sweden (Fig. 1 ). and
is covered with a large mussel bank. Here, grazing by other con-
sumers of seston could be neglected in comparison with the fil-
tering by mussels. In this area it was possible to trace the water
several kilometers over the mussel bank, corresponding to several
hours, which is a much larger scale than in the tunnel experiments
cited. The biomass of the phytoplankton community during and
after the water passed over the mussel banks could also be studied.

The strategy for the investigation was to trace the water over
the sill. Observations of hydrography and currents were performed
together with water sampling for the determination of nutrients,
Chl-n. and phytoplankton.

In the following sections the topography and the mussel popu-
lation in the sill area, the large-scale hydrographic conditions, and
methods of data collections are discussed. A simple model that
quantifies filtering by the mussels is presented, and conclusions
about the effects of filtering in general and on the out-flowing
Baltic Sea surface water in particular are drawn. The detailed
investigation of the plankton community is presented in a separate
study, Noren et al. (In press).

THE SILL AREA: TOPOGRAPHY AND MUSSEL POPULATION

The sill in Oresund, the Drodgen sill, has a minimum cross-
sectional area of about 60 • 10' m~. The minimum width is 14 km
and the depth ranges from 3 to 10 m. The horizontal area of the sill
is about 170 knr and the length of the sill in the main current
direction is about 14 km (Fig. 2). The ground of the sill can be
characterized as a transport bottoin. It consists of limestone, hard
clay, boulders, stones, and coarse sand. This substrate is preferred
by the red algae (Furcellaria liimljricalis. J. V. Lamouroux) and
when mussel larva first settle, often it is on the seaweed. When the
mussels are larger they move to the hard bottom substrate and as
a result, vast areas of the sill are almost totally covered by mussels.

413

414

Haamer and Rodhe

Figure 1. The location of Oresund, between the Baltic Sea and the North Sea (100 m depth contour indicated).

Due to low and highly variable salinity (see Hydrography),
mussels grow slowly and seldom reach a size of more than 40 mm.
Most mussels are in the size range 5-13 mm (Madsen and Hojgard
Petersen 1996). Due to the low salinity, starfishes are absent in the

12° 30"

E13°00'

ORESUND

N

56°

00'

50'

40'

55°
30'

Figure 2. ropouraphv of the Oresund with depth contours lor 6, HI.
211, and 30 m. Mussel hunks in the vicinity of the sill (shaded areas) and
sampling stations are indicated.

area and crabs are few; otherwise, these are usually the most
common mussel predators. However, a third predator, the eider
duck, is common in the Oresund sill area, with about 450.000 bird
days per year at Saltholni (Noem and Christensen 1997).

The investigations by others of the possible impact on the ma-
rine environment caused by the construction of a link across the
Oresund included video and echo sounding surveys and samplings
of the mussel population in the sill area. During the period 1990 to
1996, divers sampled 800 stations to determine coverage, biomass.
and size distribution of the mussels. Mussels occur throughout the
area (Fig. 2). with the densest population in the Flintriinnan be-
tween Saltholni and the Danish coast. There, the coverage of mus-
sels is TO-lOO'/f by area. In 1996. the areas with more than 40*^
coverage of mussels were estimated to be 128 km" (Fig. 2) and the
average biomass, in wet weight of soft parts and shell, was 7.08
kg/m- (Madsen and Hojgard Petersen 1996). Wet weight (WW) of
mussels with shell is converted to ash-free dry-weight of soft tis-
sues (AFDW) using a conversion factor of 0.03 (the tissue content
In these mussels is extremely low due to poor growing conditions).
The mussels in the sill area were estimated at about 900.000 tons
WW. which coiresponds to 27.(X)0 tons AFDW or 200 g AFDWMr.

HYDROGR.\PHY

The large-scale hydrographic conditions in the Baltic and the
North seas are discussed in Rodhe ( 1998). The connection between
these seas is through three parallel straits, one of which is Oresund.
The flow over the sill in Oresund is determined mainly by the
alternating difference in water level between the southern part of
the Baltic Sea and the Kattegat, on the North Sea side of Oresund.
The difference in salinity between the surface waters of the south-
ern part ol the Baltic Sea and the Kattegat, about 10 psu, is of
minor impoitance to the flow rate due to the shallowness of the sill.
On the Baltic Sea side of the sill a halocline separates the Baltic
Sea's low salinity surface water from the more saline deep v\ater;
the halocline is situated far below the depth of the sill. In Oresund
to the north of the sill, the water is stratified and the halocline is at
a depth of 10 to 20 m. Below that, the salinity exceeds 30 psu. The
salinity in the sill area varies between 8 and 24 psu. However,
intense vertical mixing at the sill keeps the water vertically homo-
geneous, except in cases of very weak current.

Filtering by Mussels in the Oresund

415

The average annual outflow from the Baltic Sea through Ore-
sund is 30.3 • 10'"m"' (9600 mVs). with an average salinity of 9.5
psu. which is close to the average salinity of the Baltic Sea surface
water inside the sill. The average annual inflow to the Baltic Sea
is 17.4 • 10'"nv' (5500 mVs), with an average salinity of 13.3 psu
(Svensson at al. 1994, Mattsson 1996).

A long time-series (1931-1976) of current registrations at the
Drogden light vessel showed two dominating current directions:
50" and 230'\ which represent inflow and outflow, respectively.
During this 45-y period, flow was northward 52%- of the time, with
an average speed of 27 cm/s, and southward 32% of the time, with
an average speed of 40 cm/s (the direction of current velocities <
5 cm/s was not noted). The highest observed speed during north-
ward flow was 175 cm/s, with an estimated transport of 105,000
mVs. The maximum velocity during southward flow was 225 cm/
s, which corresponds to a discharge of 125,000 m'/s. The average
duration of northward flow and southward flow was 1 .5 days and
1.2 days, respectively. The longest observed period of northward
flow was 23.3 days and of southward flow 1 1.5 days. The salinity
variations from these alternating flow directions affect the metabo-
lism of the mussels, resulting in decreased growth.

OBSERVATIONS AND METHODS

We were interested in two processes: (I) filtering by mussels
when water passes over the mussel bank, and (2) the development
of the phytoplankton community after the water's passage over the
sill. Our main observations were made during a persistent outflow
from the Baltic Sea. The water leaving the sill area continues as
surface water over the deep and stratified part of Oresund to the
north of the sill, and the phytoplankton community develops in the
surface layer without contact with the bottom. On the other hand,
during flow toward the Baltic Sea, water subducts below the low-
salinity surface layer after passing over the sill, due to its higher
salinity.

Our main investigation was carried out onboard the R/V Skag-
erak. of Goteborg University, during 2 days in May 1997. In
addition, the Swedish Meteorological and Hydrological Institute
has supplemented the investigation by sampling some of the sta-
tions during different seasons in 1997 and 1998 (sampling was
performed from onboard the R/V Argos).

The observations in May 1997 were carried out along a track
passing over the sill (Fig. 2). The stations were chosen so that
stations Tl to T4 were situated on the shallow part of the sill
(depth about 8 m). where we expected the water to be well mixed
vertically. These stations were all well within the mussel-bank
region. Station TO was situated a short distance to the south, and
T5 a short distance to the north of this region. Station T6 was
situated in the deep and stratified part of Oresund. The track es-
sentially followed the direction of the current. However, the time
to make the observations along the track was about half of the time
needed for the water to be advected along the track, indicating that
we did not follow exactly the same water mass.

During the 2 days of sampling in May 1997, three profiles were
made. On May 13 the stations Tl to T4 were sampled twice within
5.5 h. On May 14 stations TO to T6 were sampled once within 7 h.
The R/V Argos sampled the stations within about 3 h.

Temperature and salinity were measured with a CTD (Niel
Brown MK 5). Nutrients (total N, total P) were analyzed with an
autoanalyzer ( Alpkem/RFA2) according to methods recommended
in The New Baltic Manual (Carlberg 1972). Water samples for
Chl-a analysis were taken with Niskin bottles at one or two depths

(see Table la and b). The lOO-ml samples were filtered through
GF/F filters, and the filtrate was extracted in 90% acetone fori 2 h
at 4°C. Chl-o determination was inade fluorimetrically according
to standard methods. Currents were measured at 0.5, 1, 2, 3, 4, 6,
8, and 9 m above the seabed with pendulum current meters (Ced-
erlof et al. 1996) at every station except T6, where no current
observation was made. In addition, current data were provided
from fixed cun'ent meters at Flinten SV (close to station T3 in Fig.
2), 5.1 and 8.1 m above the seabed.

RESULTS

As expected, the water temperature and salinity was almost
vertically homogeneous in the shallow stations of the sill area.
Also, the horizontal variations were small. Exceptions were April
20, 1998, June 23, 1998, and August 27, 1998. when both vertical
and horizontal gradients were found, showing that the observations
were made in different water masses. However, the current was
unidirected in the vertical in all observations. The observations are
presented in Table 1 .

General conclusions from the observations are:
( 1 ). The changes in total phosphorus and nitrogen concentra-
tions were relatively small, and showed no systematic de-
crease or increase following the water's passage over the
sill. This was expected because the majority of the nutri-
ents were dissolved, and thus would not be removed from
the water as a result of the mussels" filtering. Also, nutrient
inputs due to excretion from the mussels were too small to
be of any appreciable influence on total nitrogen and phos-
phorus, see below.
(2). In some cases, large vertical and horizontal changes in
ammonium concentrations were found. This indicates
strong local sources or sinks. Otherwise, it would have
been homogeneous, as were temperature and salinity. In a
majority of the observations the concentration of ammo-
nium increased, following the passage of the water over
the sill, both in the northerly and the southerly direction.
This increase in ammonium could be due to excretion from
the mussels. Note that in two of these samples there were
differences in salinity, indicating that the observations
were made in different water masses.
(3). Large, systematic changes in the Chl-a concentration were
observed on all occasions, except in the one case with
weak currents and a large horizontal salinity gradient. A
majority of the observations showed a somewhat lower
value at the bottom. Figure 3a shows observations of
NH4-N versus Chl-a, from which it is obvious that there is
a negative correlation between the two parameters. This
supports the idea that the mussels remove phytoplankton
from the water and. at the same time, excrete ammonium.
The fact that neither NH4-N nor Chl-a shows systematic
variation with salinity (Fig. 3b and c) indicates that the
inverse correlation between NH4-N and Chl-a is related to
a local source-sink and is not an indication of different
water masses that are advected over the sill.
Figure 4 shows the change in chlorophyll concentration along
the track in May 1997 (see also Table la). The effect of the
mussels" filtering is striking. An approximately 75% decrease in
the Chl-a concentration was observed when the water passed over
the mussel bank, changing from about 1.1 mg/m' at station TO to
about 0.3 mg/m' at station T4. After the water passed over the
mussel bank the concentration again increased, to about 1.4 mg/m

416

Haamer and Rodhe

TABLE la

.

Date

Depth

T

s

Tot P

Tot N

Chl-fl

Dir (degree)

Station

YYMMDD

m

°C

psu

Hmol/I^

(imol/L

mg/m'

Xelocity (cm/s)

Tl

y7()513

3

74

84

0.5

19

0.87

50/28

Tl

970? 13

9

74

84

0.4

18

0.63

t:

970513

1

7.6

84

0.4

15

0.55

50/28

t:

970513

7

7.6

8.4

0.4

15

0.47

T3

970513

1

7.6

8.4

0.4

17

0.30

50/45

T3

970513

6

7.6

8.4

0.4

17

0.47

T4

970513

1

7.9

8.4

0.4

15

0.30

50/31

T4

970513

1

7.9

8.4

0.5

15

0.22

Tl

970513

4

7.5

8.4

0.4

16

1.20

50/45

T3

970513

4

7.7

8.4

0.4

16

0.47

50/35

T4

970513

4

7.6

8.3

0.4

15

0.28

50/32

TO

970514

1

7.9

84

0.5

18

1.20

50/28

TO

970514

6

7.9

84

0.6

17

1.00

Tl

970514

1

7.8

8.3

0.5

15

0.82

50/42

Tl

970514

6

7.8

8.3

0.5

16

0.63

T2

970514

1

7.9

8.3

0.5

15

0.65

50/40

T2

970514

6

7.9

8.3

0.5

15

0.53

T3

970514

1

7.9

8.3

0.5

15

0.48

50/60

T3

970514

6

7.9

8.3

0.4

15

0.48

T4

970514

1

7.9

8.3

0.5

15

0.33

50/51

T4

970514

6

7.9

8.3

0.4

15

0.31

T5

970514

1

8.3

8.4

0.4

15

0.84

50/23

T6

970514

3

84

8.6

0.5

15

1.40

Temperature (T). salinity (S). total phosphorus (Tot-Pl, total nitrogen (Tot-N). chlorophyll-ii (Chl-<il. and vertical average of the current (Direction/
Velocity) as observed during May 1997 (IW Skagerak).

at station T6. (In the calculations later changes in Chl-(( concen-
tration were assumed to reflect changes in the phytoplankton bio-
mass. This is a reasonable assumption when we track a water mass
over several hours in the middle of the day.)

Was the observed increase in Chl-a the indicative of recovery
of the phytoplankton community within the same water mass, and
with the available nutrients'? We do not know for sure, but the
indications are strong. A rough estimate of the time to advect water
from station T4 to T6, based on the current measurements, is 2
days. Two doublings of biomass during that time is quite realistic.
Also, based on temperature, salinity, total phosphorus, and nitro-
gen measurements from the 2 days of sampling in May 1997 (see
Table la), we conclude that the observations were made within the
same water mass and that mixing with the underlying water was
negligible. Our conclusion is that the observations at stations T4 to
T6, shown in Fig. 4, indeed show the development of the phy-
toplankton community in a water mass exposed to the effective
filtering by mussels and, possibly, supplied with ammonium.

The detailed current observations from May 1997 were used to
calculate the bottom stress, forttiulated as friction velocity (u.).
and the drag coefficient (Cjl. relating the \clocity to the bottom
stress. The definitions of these quantities are

(u.)- = T|,/p
where t,, is the bottom stress and p is the vvalcr density, and

Cj = (u*/u,„)"

where ti,„ is the velocity, averaged in the vertical direction. The
friction velocity was estimated by tilting the current observations

to a logarithmic profile at each station. Results of the calculations
are shown in Table 2 together with the depth-averaged velocity at
each station. The small variations in the direction of the current
were not considered in the calculations. Although the calculations
were made using single observations, we believe that estimated
friction velocities are the correct order of magnitude. The com-
paratively large drag coefficients (the average is 7.0 * 10" ) seem
to be realistic since the ground is very rough (see the section The
Sill Area). In addition, the mussel beds at the bottom increase the
bottom friction by increasing the bottom roughness. Further, the
mussels" pumping activity increases the bottom friction (Wester-
berg unpubl.). The current observations and the friction velocities
are used in the calculations that follow.

QUANTIFICATION OF FILTERING USING AN
INTERPRETATION MODEL

An interpretation model was formulated to apply values to the
filtering by the mussels. The model description follows: as the
water flows over the mtissel bed. turbulence keeps the biomass
evenly distributed verticallv . The mussels Alter a fraction of the
water coltinin per unit length in the flow direction. The size of this
fraction depends on the velocity, the depth of the flow, and filter-
ing by the mussels. Because observations of changes within a
water mass are made over a few hours. Chl-<( concentration was
treated as being proportional to the biomass and changes in Chl-a
were used to calculate the amotint of biomass filtered. The obser-
valions with high horizontal resolution and simultaneous current
measurements (those in 1997) were used. To make the model as
simple as possible, we assumed the flow properties were horizon-

Filtering by Mussels in the Oresund

TABLE lb.

417

Date

Depth

T

S

Tot P

NH,-N

Tot N

Chl-a

Dir (degree)/

Station

YYMMDD

m

°C

psu

pmol/L

Mmol/L

(iniol/L

mg/m'

Velocity (cm/sec)

TO

9712L';

1

5.3

7.9

0.36

0.2

22.6

0.9

50/75

TO

9712L'i

8

5.4

7.9

0.36

0.27

18.9

0.9

50/75

TI

971215

1

5.4

8.2

0.59

0.27

22.7

1.1

50/75

TI

9712L'i

7

5.4

8.4

0.62

0.31

23.9

1.0

50/65

T2

971215

T

5.4

8.2

0.60

0.28

21.9

1.0

50/75

T2

971215

8

5.4

8.2

0.60

0.63

21.2

1.0

50/65

T3

971215

2

5.4

8.5

0.62

0.37

33.3

1.1

50/75

T3

971215

7

5.4

8.5

0.61

0.28

21.5

0.9

50/65

T4

971215

2

5.3

8.4

0.57

0.75

23.3

0.8

50/68

T4

971215

7

5.3

8.5

0.61

0.73

20.7

0.6

50/60

T5

971215

->

5.1

8.6

0.65

0.62

20.4

0.8

TO

9S0319

0

3.6

9.3

0.86

1.05

25.4

0.5

50/30

TO

980319

8

3.6

9.3

0.65

1.00

25.4

0.4

40/30

T4

980319

0

3.6

9.8

0.36

0.73

21.3

0.6

40/30

T4

980319

7

3.6

9.8

0.37

0.89

21.8

0.7

40/30

TO

980420

0

5.6

7.5

0.56

0.14

17.8

1.2

230/5

TO

980420

7

6.5

10.3

0.50

0.13

18.9

1.2

230/5

T4

980420

0

6.4

11.4

0.57

0.13

19.6

1.5

230/10

T4

980420

7

5.5

22.4

0.93

0.59

19.2

1.9

230/10

TO

980603

2

12.1

7.4

0.10

0.14

18.4

1.5

50/30

TO

980603

7

12.1

7.5

0.10

0.13

18.7

1.5

50/30

T4

980603

2

13.3

8.1

0.02

0.15

21.0

1.3

40/35

T4

980603

7

13.2

8.2

0.15

0.12

18.6

1.3

40/35

TO

980623

~>

13.7

7.5

0.48

0.10

21.1

2.5

230/10

TO

980623

1

13.6

7.8

0.47

0.22

23.6

1.9

230/10

T4

980623

1

14.3

8.8

0.53

0.14

22 "*

2.0

230/10

T4

980623

1

14.8

10.1

0.48

0.20

19.3

1.3

230/10

TO

980721

-f

14.4

7.8

0.54

0.12

19.8

1.8

50/50

TO

980721

1

14.4

7.8

0.59

0.12

21.0

1.8

50/50

T4

980721

T

15.0

9.2

0.62

0.69

19.3

0.8

20/40

T4

908721

7

15.0

9.3

0.61

0.78

19.2

0.6

20/40

TO

980823

T

15.6

15.9

0.25

0.67

16.8

0.5

220/85

TO

980823

7

15.6

16.0

0.26

0.68

17.3

0.4

220/65

T4

980823

~)

15.7

17.2

0.18

0.21

15.7

1.7

220/85

T4

980823

1

15.7

17.2

0.20

0.26

17.2

1.4

220/85

TO

980827

->

15.1

12.3

0.19

0.10

17.9

3.7

40/40

TO

980827

7

15.3

13.7

0.21

0.08

17.3

2.9

40/35

T4

980827

■)

15.0

16.6

0.27

1.16

17.5

1.2

40/45

T4

980827

7

15.0

16.6

0.28

1.21

17.6

1.1

40/35

TO

980922

-)

14.6

7.6

0.02

0.12

20.6

2.8

40/31

TO

980922

7

14.6

7.6

0.04

0.09

21.2

6.8

40/31

T4

980822

-I

14.4

7.9

0.30

1.02

19.9

0.5

40/41

14

980922

7

14.4

7.9

0.28

1.22

20.3

0.5

40/41

Temperature (T). salinity (Si. total phosphorus (Tot-P). ammonium (NHj-N). total nitrogen (Tot-N). chlorophyll-o (Chl-(j|. and vertical average ot the
current (Direction/Velocity). Five observations from December 1997 to August 1998 (R/V Argos).

tally homogeneous over the sill. This assumption is based on the
relative homogeneity of the depth distribution and the bottom
structure. Hov\ever. the degree to which this assumption is fultllled
does not change the result in a qualitative way. it only effects
its quantitative accuracy. Another assumption is that the local
change in biomass with time is small compared with the change
over time following water flow over the sill. This assumption is
critical for the result, and the similarity among the observations
from the three crossings of the sill indicates it is well supported
(see Fig. 4).

Before proceeding with the calculations, the question must be
answered whether the vertical mi.xing is vigorous enough, that is.
if all the water between the observation sites potentially can be
filtered by the mussels. If not. then the mussels' filtering will only

affect water in the vicinity of the bottom. Fulfilling the flow cri-
terion requires that the vertical turnover time be considerably
shorter than the time for the water to be advected between the sites.
In the present situation the vertical turnover is determined by the
turbulence generated by the bottom friction. Thus, the friction
velocity can be used as an estimate of the turbulent velocity. The
condition to be fulfilled is:

h/u'<l/U

(1)

h, u', I, and. U are the depth, the turbulent velocity scale, the
advective length, and the advective velocity, respectively.

Using information from Table 2 and Fig. 2 to estimate the
magnitude of the terms in Eq. ( I ). h = 10 m. u' = 0.02 m/s. U =
0.5 m/s. and I = 3000 m. Using these values, the vertical turnover

418

Haamer and Rodhe

E

0.5

a)

oo o
o o

o o o o

S° « ° ° O °o
-I I I I I I I I L.

1.5

S 1.0
O

E

0.5 -

2 3

Chl-a (mg/m'

10 20

Salinity (psu)

-

c)

-

o

o

0

-

0 o

-

0

-

°8°

©

o

-

o

s

8 o

J

0

o

1

10 20

Salinity (psu)

30

Figure 3. (A) Observations of NHj-N versus Chl-a., (B) Chl-a versus
salinity, and (C) NHj-N versus salinity.

time (lel'l side of Eq. I ) is 500 s and the advection time is 60()() s.
We conclude that the condition (Eq. I) is fultilled.

Wc also assumed that the water filtered hy the mussels is "im-
mediately" mixed in the water column. This is supported if the
flow of water through the mussels at the bottom, converted to a
vertical velocity, is much sinaller than the turbulent velocity. This
will be confirmed a posteriori. The model set up is illustrated in
Figure 5. We formulated a conservation equation for ehlorophvll in

20 30 40 50 60

Distance (km)

Figure 4, Observations of Chl-a concentration along the transect
crossing the sill in Oresund. Data from the observations in May 1997
(Table la). The line connects the ensemble average at each station. The
sampling stations are shown in Figure 2,

a one unit wide channel, where the horizontal transport of chloro-
phyll units by the current is determined by the vertical average of
the velocity (U) multiplied by the depth (h) and by the vertical
average of the chlorophyll concentration (Cl. (This is reasonable
becau.se the vertical gradient of the concentration was small.) The
loss of chlorophyll, per unit area, due to the filtering by mussels is
formulated as an area-average velocity (w,„). representing the fil-
tering by mussels multiplied by the local chlorophyll concentra-
tion. The change over a short distance, and per unit length in the
x-direction, of the horizontal transport can be formulated:

d/dx(UhC) = -w„,C

(2)

To solve the equation. U and h will be treated as constants. (To do
this we assumed that the flow is horizontally homogeneous.) The
solution to Eq. (2) then is

C/C„ = exp[-(w,„/Uh)x]

where C,, is the concentration at x = 0.
Solving for w„,:

w„ =-ln(C/C„) ■ Uli/x

(3)

(4)

Equation (4) is used between successive observational sites to
estimate w„,. The average value of Uh is used at each pair of sites.
The results are presented in Table .'<. The scatter in the calculated
w,„ is in the authors' opinion astonishingly small. The estimated
mean is 0.4 • 10"' m/s. Note that this is about two orders of
magnitude smaller than the turbulent velocity. We also see that
there seems to be no systematic variation of w,„, neither with the
a\erage velocity nor with the velocity at the bottom. This fact
supports the calculations.

The clearance rate (CR) is defined as the water volume cleared
of organic matter per unit time and unit AFDW of mussels. We can
use the velocity. w,„. to estimate CR. The average mussel density
at the sill is about 200 g/nr, measured as AFDW. Thus, the esti-
mated CR is about 2 • 10"'' mVs/g AFDW, which is approximately
equal to 7 L/h/g AFDW. This compares well with values given by
Jorgensen ( UAM)) for small mussels. A rough estimate of the clear-
ance rate by the entire mussels population on the sill. 27. ()()() tons

Filtering by Mussels in the Oresund

419

TABLE 2.
Flow properties calculated using the detailed curent measurements from May 1997.

Date
YYMMDD

Station

Depth
m

U

cm/s

u.

cm/s

*10^

970513/1

970513/2

970514

TO
Tl
T2
T3
T4
TO
Tl
T2
T3
T4
TO
Tl
T2
T3
T4

12
10

9.5

9

9
12
10

9.5

9

9
12
10

9.5

9

9

no

no

27.6

2.3

26.9

2.1

44.9

1.6

31.0

2.6

no

no

33.5

3.0

no

no

37.1

4.1

25.5

2.0

24.6

2.3

39.4

2.6

37.7

3.4

55.3

4.2

46.2

4.4

no = not observed).

no
6.9
6.1
1.3
7.0
no
8

no
12.2
6.2
8.7
4.4
8.1
5.8
9.1

U is the depth-averaged velocity, u, is the friction velocity, and c^ is the drag coefficient (no

AFDW. then becomes 5 ■ lO"* m''/s. This figure is more than two
times the average flow rate over the sill during outflow from the
Baltic Sea. Even though this is a rough estimate, we conclude that
the mussels at the sill have the capacity to filter the outflowing
water from the Baltic Sea.

DISCUSSION OF LOCAL AND LARGE-SCALE EFFECTS OF
FILTERING BY MUSSELS

In our study we have assigned numerical values to the filtering
by the mussels in Oresund. The local effect of the mussels is
obvious to everyone who visits the sill area: the water is extremely
clear. Therefore, one can find flourishing fields of eelgrass
(Zostera marina L.) down to 7 m deep and Laminaria sacclarina
(L.) down to 14 m in Oresund. A large supply of nutrients, remin-
eralized from the mussels during summer, when a shortage of
nutrients normally occurs, could be an additional contributor to the
good growth of SAV.

The input of ammonium from mussel banks due to the nietabo-

u(z)

U

it

■hue

,w„C

Transport

Large scale Small scale

velocity velocities

Figure 5. Conceptual model used to calculate filtering by mu.ssels.
Water flows with velocity u(zl over the mussel bed. The total depth is
h. Bottom friction induces turbulence, characterized by a velocity, u'.
The filtering by mussels induces a vertical velocity, w„,, at the bottom.
The biomass. measured as Chl-o concentration. C, is advected with
mean current, U, in the x-direction. The filtering by mussels at the
bottom acts as a sink for biomass. This sink is expressed as a vertical
velocity multiplied by the chlorophyll concentration.

lism of the mussels is considerable. The amount fluctuates due to
changes in temperature, salinity, nutrient supply, and the physi-
ological status of the mussels (Smaal et al. 1997). Our observations
also indicated a release of ammonium from the mussel banks. In
the observations from December 1997. the NHj-N concentration
increased from about 0.3 (xmol/L to about 0.7 jjtmol/L after the
water passed over the mussel bank. The water transport through
Oresund was about 36,000 m Vs (a rough estimate based on current
observations). Assuming that the observed increase in the ammo-
nium concentration is representative, we estimated the total release
of NH4-N to be 14 mol/s, or 0.7 ton/h. from the mussel banks at the
sill. During northward-directed flow such a release constitutes an
appreciable, accessible supply of nutrients to the central part of
Oresund (see Fig. 2). The total production in Oresund is estimated
at 91 g C /m"/year (Mattsson 1993). For assimilation of carbon to
take place, nitrogen is needed; in this case about 1.3 ton/h. on the
average. Using the Redfield ratio to relate C to N, the ammonium
released from the mussel banks can be expected to have a large

TABLE 3.

Calculations of the vertical velocity (w„) related to the filtering by
mussels between different station pairs.

Date
YYMMDD

Station
Pair

W„
mm/s

U
cm/s

Uci.5

cm/s

970513/1

970513/1

970513/1

970513/2

970513/2

970514

970514

970514

971215

Ensemble average

Std. deviation

T1-T2
T2-T3
T3-T4
T1-T3
T3-T4
T1-T2
T2-T3
T3-T4
T1-T4

0.7
0.2
0.3
0.5
0.3
0.3
0.3
0.4
0.3
0.4
0.2

36
38
35
31
39
46
51
69
41

17
26
26
16
14
19
20
31
no
21

U is the depth mean velocity, averaged between the stations, and u,, , is the
velocity 0.5 m above the bottom (no = no observation).

420

Haamer and Rodhe

effect on primary production, in a qualitative as well as in a quan-
titative way.

The concentration of inorganic nutrients is higher in Oresund
compared with what is found in the adjacent seas (Mattson 1993).
Despite that, the measured pelagic primary production is lower in
the vicinity of the sill than in the Kattegat, in the northern part of
the Oresund. and in the southern part of the Baltic Sea. Edler
(1977) suggested that the low production is a result of the rapid
salinity changes in the area. The reduction of the chlorophyll con-
tent in the water, by the mussels at the sill, might be an alternative
cause of low productivity in the sill area. Tracing the water close
to the sill, we found the beginning of a bloom: nutrient-rich water
but few phytoplankton.

Increased nutrient concentration in the Kattegat, in the Belt
Sea, and in the Baltic proper has led to a doubling of summer
primary production in these areas since the mid 1960s (Shultz et al.
1990). Vast bottom areas in the southern part of Kattegat have
suffered from temporary anoxia since the beginning of the 1 980s,
probably as a result of an increased tlux of organic matter from the
photic zone to the deeper layers (Anderson and Rydberg 1988).
The oxygen demand has also increased in the deep water of the
Baltic Proper. The opposite is true for the central part of Oresund,
where observations indicate a decrease in oxygen demand in the
deep water. Mattsson (1993) found a 30% decrease in oxygen
consumption between 1967 and 1986 in the Landskrona Basin
water, which is an isolated trough situated close to station T6 (see
Fig. 2). The maximum depth is 50 m and exchange with the deep
water of the Kattegat to the north is hampered by a 25-m deep sill.
Mattsson (1993) found this decrease surprising, considering the
increased nutrient concentration in the surface layer. His conclu-
sion was that the decrease in the oxygen consumption in the basin
water reflected a decreased net production in the surface layer.

However, there is another possible reason for the decreased
flux of organic matter to the deep water in Oresund. Kautsky and
Wallentinus (1980) suggested that mussel populations in regions
with few predators expand to the carrying capacity of the area. As
stated earlier the Oresund sill area is such a region. Consequently,
we assume that the mussel banks at the sill have grown during the
last decades due to eutrophication, and that the mussels" capacity
for filtering is determined by the peak in the supply of organic
matter. In fact, according to local fishermen the banks have grown
during the period, but there is little scientific documentation. The
supply of organic matter to the mussels varies with the water flow
and the season of the year. Consequently, during most times of the
year, when the nutrient supply is moderate, the starving mussels
can Alter the passing water more efficiently than before. The total
effect would probably be a net decrease in the amount of organic
matter that could escape from being filtered by the mussels. The
effect on the oxygen conditions in the Landskrona Basin would be
twofold: The filtering of seston from water flowing from the Baltic
Sea to the Oresund will decrease flux to the deep water. We can
also expect that "new" production, in the water Altered by the
mussels, implies a shift of the phytoplankton community toward
smaller species. Smaller plankton settles slower and decomposes
faster. This means that a smaller portion of the organic matter
produced in the surface layer settles before the water leaves the
Oresund.

There is reason to believe that the mussel banks in Oresund
accumulate large amounts of nutrients when there is excess of food
for the mussels during the spring phytoplankton blooms. Also,
they contribute lo the nulrienl accunuilalinn with a net release of

nutrients during the summer (Asmus and Asmus 1991). The im-
plied leveling effect on the nutrient fluxes should be important for
other species in the Oresund ecosystem.

The present study is a preliminary study for a project investi-
gating the potential improvements in water quality from mussel
cultivation in eutrophicated areas of the Swedish west coast (e.g.,
the Orust-Tjom fjord system to the north of Goteborg). The deep
basins in these fjords often suffer from low oxygen concentration
in late summer and autumn due to restricted water exchange and a
large supply of organic matter from the surface layers. The popu-
lations of filter feeders in these fjords have increased during the
last decades due to eutrophication. but they have not reached the
carrying capacity. There has been a small decrease in the tlux of
organic matter to the deeper layers in the inner parts of the fjord
system (Kajrup 1996). but it is not possible to isolate the effect of
increased water purification from that of an increased population
of filter feeders. Future work will focus on the potential for de-
creasing BOD in the deep basin water by shifting part of the flow
of organic matter to mussel culture. Long-line culture of mussels
has been practiced on the Swedish west coast since 1970 with
positive results for settling of larva and for growth in many loca-
tions. In the future we hope the results of the investigation from the
sill in Oresund will help us site mussel farms in areas where the
filtering activity of mussels can counteract the negative effects of
eutrophication.

CONCLUSIONS

We have found that the mussels at the sill in Oresund. through
their filtering, are capable of clearing phytoplankton almost com-
pletely from the passing water; and the remineralization by the
mussels can be expected to have a first-order effect on the eco-
system in Oresund. in a qualitative as well as in a quantitative way.

ACKNOWLEDGMENTS

The authors are indebted to Katarina Ahrahamsson for her par-
ticipation in the field work during the May 1997 expedition, and
for providing the chlorophyll data. The Swedish Meteorological
and Hydrological Institute (SMHl) is acknowledged for sampling
extra stations with R/V Art;(>s for this project. Oresundskonsortiel
is acknowledged for supplying the current meter data from the
Flinten SV. We are also grateful to the crew of R/V Skagerrak.
The figures were prepared by Agneta Malm and most chemical
analyses were made by Britt-Marie Widheden. This study is a part
of an investigation of the possibilities tor using mussel culture to
reduce the negative effects of eutrophication in Swedish fjords,
within the MISTRA project: Sustainable Coastal Zone Manage-
ment Project (SUCOZOMA). The study was also financed in part
by the Swedish Environment Protection Agency through project
539310-97-01.

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Jounwl of Shellfish Rfseorch. Vol. 19. No. 1. 423-424, 2()()().

TEMPORAL AND SPATIAL DISTRIBUTION OF ENTEROCOCCUS IN SEDIMENT, SHELLFISH
TISSUE, AND WATER IN A NEW ZEALAND HARBOUR

S. DE LUCA-ABBOTT. G. D. LEWIS, AND R. G. CREESE

Leigh Marine Laboratory

School of Environmental and Marine Science

University of Auckland

PO Box 349

Warknorth. New Zealand

ABSTRACT Enterococci, a group of faecal bacteria commonly found in stormwater discharges, were used to trace the spatial and
temporal impact of waste streams from an outfall in the Whangateau Harbour, northeastern New Zealand. A seasonal trend in levels
of enterococci in two infaunal bivalves. Austroveiuis snitclibuni (Gray in Wood 1828) and Macomomi lilUma (Iredale 1915) was
detected, with maximum contamination correlating with high winter rainfall. Rainfall events were also shown to affect stormwater and
harbor water significantly. Median enterococci levels in A. suirchhiiryi were higher at the putative impact site compared to the reference
site, and were higher than M. lilmna at both sites. Bacterial levels in surficial sediment and A. sutlchhiiryi tissue declined with distance
from the stormwater outfall and the stormwater channel. Enterococci were effective for determining the spatial and temporal patterns
of stormwater discharge in this harbor, and may have general applicability as an indicator of such discharges.

KEY WORDS: Stormwater, enterococci, Austrovenus srutchburyi. cockle. Macomona liliana

INTRODUCTION

There is a paucity of literature concerning temporal and small-
scale spatial effects of stonnwater discharges on the bacteriologi-
cal quality of the marine environment adjacent to small coastal
settlements in New Zealand. Typically, these settlements comprise
dwellings along harbor or estuary fringes, with stormwater flows
discharged directly onto the intertidal zone, which is inhabited by
many edible shellfish species. Long-term monitoring of bacterial
contamination is needed to establish average or background levels,
which in turn assist in the interpretation of short-term event-driven
episodes. It is also important to ascertain the distribution of bac-
terial contamination across the adjacent foreshore, in order to iden-
tify areas of potential high risk. Such information would highlight
areas likely to be contaminated with other stormwater pollutants,
as well as sites unsafe for harvesting of edible shellfish resources
and areas unsuitable for shellfish restoration efforts.

Snelder and Williamson (1997) define stormwater as rainwater
that runs off impervious surfaces and is usually discharged as a
point source into waterways such as estuaries and harbors. When
discharged into high-energy receiving environments there is im-
mediate dilution, whereas sheltered habitats such as harbors and
estuaries accumulate particulate matter contained in such dis-
charges (Snelder and Williamson 1997). The main contaminants
found in urban stormwater include heavy metals, polycyclic aro-
matic hydrocarbons (PAHs). organochlorine pesticides, hydrocar-
bons, nutrients, suspended solids, and microorganisms (Snelder
and Trimian 1995. Snelder and Williamson 1997). All have the
potential to compromise the sustainability of marine environments
(Morrisey 1997).

Stormwater discharges often affect the bacteriological quality
of the receiving water (Gannon and Busse 1989). Of concern to
environmental managers is the potential for microorganisms con-
tained in waste discharges to affect human health adversely
through bathing or the consumption of affected shellfish (Kebab-
jian 1994). Water and seafood contaminated with sewage have
been reponed to transmit gastrointestinal disorders, hepatitis, chol-
era, and a range of eye. ear. nose, and throat infections (Mclntyre
1995). The spread of disease via contaminated water and seafood

has prompted the use of indicator microorganisms as a warning of
unsafe conditions (Elliot and Colwell 1985). These indicator mi-
croorganisms are not themselves pathogenic. They generally occur
in high concentrations in the same environments as pathogenic
microbes but are simpler to detect and quantify (Fattal et al. 1984).
Therefore, they can be used to indicate a potential risk of disease
(Elliot and Colwell 1985).

Survival of both pathogenic and indicator bacteria in marine
waters is affected by many physical, chemical, and biological fac-
tors, including temperature, sunlight, salinity, predation. adsorp-
tion, sedimentation, and dilution (Borrego et al. 1983, Nicholson
1988). Biological parasitism and predation are also responsible for
destruction of faecal bacteria entering the marine environment in
untreated sewage (Roper and Marshall 1974). Survival character-
istics of indicator bacteria and the pathogens they are modelling
should be similar.

The coccoid bacteria Enterococciis. which naturally inhabits
the gut of humans and warm-blooded animals, has gained favour
in recent years over another bacterium. Escherichia coli. as an
indicator bacteria, as they survive longer in seawater, and have
good correlation with gastrointestinal symptoms (Miescier and Ca-
belli 1982. Elliot and Colwell 1985. Donnison 1992. Sinton et al.
1993).

Shellfish have the ability to concentrate bacteria and other con-
taminants from water or sediment via their mode of feeding, and
are therefore useful tools for investigating faecal pollution (Ayres
et al. 1978. Nicholson 1988. Prieur et al. 1990). Most bivalves are
filter-feeders, passing large volumes of water across their gills to
obtain food and oxygen. Microorganisms and food particles in
suspension are trapped in mucus on gill, mantle, and labial palp
surfaces and then transponed by ciliary action to the mouth (Per-
kins et al. 1980. Cook 1991). Deposit-feeding bivalves feed by
ingesting surficial sediment, which may have considerably higher
levels of bacteria than the water column (Elliot and Colwell 1985,
Kueh 1987). Accumulation of bacteria in sediments is due to their
sorption to particles suspended in water, which then settle out onto
the surficial sediment (Davies et al. 1995). Whether ingested via
filter-feeding or deposit-feeding, digestive processes are not

423

424

De-Luca-Abbott et al.

thought to inactivate all microorganisms (Hedstrom and Lycke
1964). Depuration occurs through the discharge of faeces and from
the pumping of water through the mantle cavity (Perkins et al.
1980). Factors affecting feeding, and therefore accumulation of
bacteria, include temperature, turbidity, salinity, and physiological
condition of shellfish (Bonadonna et al. 1990). Filtration rate may
decline when these factors are suboptimal and therefore less indi-
cator and pathogenic microorganisms would be accumulated
(Ayres et al. 1978, Nicholson 1988). Retention of microorganisms
by bivalves is a function of spacing of gill filaments, water flow
through the mantle cavity, and filtering behavior, all of which add
to the variability in accumulation of bacteria among species (Bona-
donna et al. 1990).

This research aims to identify the effects of stormwater dis-
charges on enterococci levels in bivalve shellfish, sediment, and
water. Seasonal and year-to-year bacterial levels are investigated
in experiment A. and event-driven episodes such as rainfall events
are analyzed in experiment B. The spatial distribution of bacterial
levels along the foreshore is presented in experiment C. This work
uses relatively simple, inexpensive microbiological techniques to
trace and determine spatial and temporal patterns associated with
low volume waste discharges from small coastal communities into
marine environments.

METHOD AND MATERIALS

Shellfish Description

Experiments in this study focus on the cockle, Auslrovenus
slutchbiiryi (Gray in Wood 1 828), and the wedge shell, Macomona
liliana (Iredale 1915). These two shellfish have overlapping dis-
tributions throughout many estuaries and harbors in New Zealand
(Larconibe 1968, Marsden and Pilkington 1995). Both are abun-
dant and widespread in the Whangateau Harbour. They have con-
trasting feeding modes (A. stiitchhiiryi is a filter-feeder, and M.
liliana Is a deposit-feeder) and therefore different exposure routes
to bacterial contamination.

Site Description

Three experiments were carried out in Whangateau Harbour,
northeastern New Zealand (Fig I ). This harbor is typical of many

^

Big -qL
Omaha J \ .

Lews r

t

i /Point\

/

/

X^ J Wells )

/

1

N

JP/lf /

/

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i

1

^

\ I

(

\ Omaha
\ Bay

-

WhangateauN

\,

>^

-^

V

^J

0 1

2

3 km

Figure 1. Location of saniplin)> .sites.

New Zealand estuaries, and is fringed by several small, rural com-
munities, with a combined population of c. 1400. Low levels of
waste enter the harbor from these communities (e.g.. Klein and
Gowing 1993). Samples were taken from an area adjacent to a
stormwater outfall at the putative impact site (Point Wells). This is
a small residential community, of 324 people in 137 dwellings,
with houses close to the foreshore and open stormwater drains. All
homes have on-site sewage disposal systems (septic tanks). The
comparison site at Lews Bay, across the harbor, has only three
residences close to the shore.

Sample Collection and Processing (All Three Experiments)

Shellfish

Bivalves were collected at low tide, placed in labelled plastic
bags, and transported to the laboratory on ice, where they were
processed within 6 h of collection. Shellfish were opened using
aseptic techniques, and the whole animal was extracted and placed
in sterile stomacher bags. For each sample, sufficient individual
shellfish were used to provide a sample weight of between 10 and
40 g. Generally, 15 A. stutchhuryi from both sites, 15 M. liliana
from Lews Bay, and 7 M. liliana from Point Wells were used.
Fewer M. liliana were required from Point Wells to achieve the
desired sample weight, as they were larger than those from Lews
Bay. Bags containing samples were individually placed in a Sea-
ward Stomacher laboratory blender (model 400) and macerated at
normal speed for 2 min. Samples were diluted tenfold with Gel-
dreich phosphate buffer (WHO 1982). A five-tube Most Probable
Number (MPN) series of azide dextose broth, as described by
Donnison (1992), was set up, with double-strength medium used in
the first row. Tubes were inoculated with 10, I. and 0.1 ml of
diluted sample, according to the MPN series, and then incubated in
water baths at 35'^C for 48 h. Positi\'e tubes (turbid) were plated
onto membrane filters, which had been divided into six segments
(one for each of the five tubes within a dilution and one control
segment). Filters were placed onto mE agar plates, which had been
brought to room temperature. Plates were inverted, placed in a
sealed plastic box and incubated for 48 h at 41 ± 0.5 "C. Filters
were aseptically transferred to esculin iron agar (EIA) plates
(which had been equilibrated at room temperature), and incubated
at 41 ±0.5 °C for approximately 20 min. Streaks of growth that
were pink-to-red with a brownish-black precipitate underneath
were scored as positive. MPN scores were determined from stan-
dard MPN tables and bacteria per 100 g of shellfish tissue calcu-
lated.

Sediment

Surficial sediment samples (approximately 50 g) were collected
by .scraping a sterile stainless steel scoop across the sediment al
low tide, capturing the top centimeter of sediment. The scoop was
washed and flamed in alcohol prior to the collection of each
sample in order to ensure no cross-contamination. Sediment was
placed in sterile plastic hags and transported on ice to the labora-
tory and processed within 6 h of collection.

Sediment samples were prepared according to the enterococci
MPN technique described by Donnison (1992). Approximately 10
g of each sample were weighed out and placed in a sterile plastic
bag. A tenfold dilution was made using sterile phosphate buft'ered

Enterococcus in Sediment, Shellfish and Water

425

diluent (WHO 1982). Diluted samples were shaken by hand for 2
inin to release bacteria from the sediment into suspension. Diluted
samples were then used as the inoculum for a five-tube MPN series
and processed as for shellfish.

Water

Water samples were collected in sterile glass bottles and trans-
ported to the laboratory on ice and away from sunlight. The mE/
EIA membrane filter technique was used to enumerate enterococ-
cus concentration (APHA 1992. Donnison 1992). Three replicates
of 10 ml and three replicates of 50 ml from each water sample
were filtered through a Sartorius 0.45-|j.m cellulose nitrate filter
paper, which was aseptically transferred to mE agar plates (previ-
ously equilibrated at room temperature). The plates were inverted,
placed in a sealed plastic box. and incubated for 48 hours at 41 ±
0.5 °C. Filter papers from plates showing positive growth were
transferred to EIA agar plates and incubated for a further .^0 min.
Colonies that were pink-to-red with a brownish-black precipitate
underneath were scored as enterococcus. The mean number of
colonies per 100 ml of sample was calculated.

Rainfall data were derived from daily information collected at
the University of Auckland. Leigh Marine Laboratory, appro.xi-
mately 15 km from sampling sites.

Experimenlal Protocols

Experiment A

The first experiment investigated the seasonal and year-to-year
variability of enterococci levels in the body tissue of cockles and
wedge shells. Three replicate composite samples were collected
from each site. 50 m from the foreshore, every 2 mo during 1996-
1998. and analyzed according to the schedule above. Median MPN
of enterococci per 100 g shellfish flesh were plotted against maxi-
mum daily rainfall over the preceding 4 days.

Experiment B

The long-term monitoring in experiment A revealed high vari-
ability in enterococci le\els. and peaks of enterococci appeared
to be related to high rainfall. This hypothesis was examined in
more detail in this second experiment, which investigated the
effect of specific rainfall events on the microbiological quality
of shellfish tissue, sediment, and water. Samples were taken from
late April to early May for a 14-day period in 1996 and a 12-day
period in 1997. Each day at low tide a single sample each of
sediment, A. stiiichburyi. and M. liliana was taken from the sites at
Point Wells and Lews Bay. Sampling sites were 50 m from the
foreshore at both sites. A stormwater sample and a harbor water
sample (from Big Omaha Wharf, see Fig. 1) were also collected
daily. Processing and analyses were carried out as detailed above.
Median number of enterococci/ 100 ml water were plotted against
daily rainfall and compared with MPN enterococci/ lOOg of shell-
fish tlesh and sediment. Median enterococci concentrations for
harbor water, sediment, and both shellfish species were calculated
using data from all experiments (A. B. and C) and plotted with
results of this experiment (B) to provide a comparison with typical
levels.

Experiment C

Data from experiment B regarding the temporal patterns of
bacteria in shellfish and sediment prompted investigation into

the spatial patterns of bacterial levels in sediment and tissue of
A. stutchhuryi around a stormwater outfall at Point Wells. To
delineate the effects a suite of experiinents was carried out using
three 8()-m transects extending seaward from the stormwater
drain outfall. The first transect was placed perpendicular to
the foreshore. The other two transects were placed on either side of
the first transect, at 45° and 135° to the foreshore {see Fig. 2).
Aitstrovenus suitchbwyi samples were taken in July and August
1998 every 10 m from 10 to 80 m along the central transect and
from 20 to 80 m along the two radials. In July 1998, sediment
samples were taken at 0 m, 5 m. and then every 10 m out to 60 m
on the central transect, and 2.5 m. 5 m, and every 10 m out to 60
m on the radial transects. MPN enterococci/ 1 OOg shellfish flesh
and sediment were plotted against distance from the stormwater
drain.

RESULTS

Long-term temporal variability in enterococci levels in A.
stutchhuryi and M. liliana tissue plotted against maximum daily
rainfall for the preceding 4 days is shown in Fig. 3 a and b.
Bacterial levels of 20 MPN/ 1 OOg were at or below the detection
limit of the procedure, which is shown as a horizontal dotted line
on each graph. Peaks in the levels of enterococci occurred over the
winter months (June to October) (Fig. 3 a and b), and these winter
peaks approximately corresponded with highest rainfall. Bacterial
levels were also raised in February and April for A. stutchhurxi at
Point Wells. Only three results over the entire sampling period at
this site for A. stutchhuryi were below the detection limit of 20
MPN/lOOg. Bacterial levels were below detection in most months
for A. stutchhuryi at Lews Bay. and for A/, liliaiui at both sites (Fig.
3 a and b). During the sampling program, enterococci levels in A.
stutchhuryi were above the detection limit 77% of the time at Point
Wells, compared with 24% at Lews Bay. The detection limit was
exceeded 41% of the time for M. liliana at both sites.

The effect of a rainfall event on stormwater and harbor water
quality was specifically investigated in the first period of the sec-
ond experiment. During intense monitoring in the autumn of 1996.
rainfall of 40 mm on day 5 resulted in an approximate 100-fold
increase in enterococci on day 5 for stormwater and on day 6 for
harbor water (Fig. 4a). Enterococci levels then declined and re-
turned to background levels (< 100/100 ml) by day 8. Enterococci

Point Wells Foreshore

Stormwater
Outfall

Temporal

Sampling

Sites

Stormwater
Channel

/

45°

Right

Transect

Centre
Transect

Figure 2. Position of temporal shelinsh and sediment sampling sites
and transect design for spatial sampling around stormwater outfall at
Point Wells.

426

De-Luca-Abbott et al.

■■ Point Wells
i Lews Bay
Detection Limit
• Maximum Daily Rainfall
T Total Rainfall (over 4 days)

Figure 3. Temporal pattern of enterococci levels in body tissue of (a)
A. stulchburyi and (b) M. liliana at Point Wells and Lews Bay.

levels in sediment sinowed a similar pattern (Fig. 4b), with in-
creased concentration on day 6 (the day after the rainfall event) to
approximately 1000 MPN/lOOg. followed by a slower decline over
the subsequent few days to background levels of less than 100
MPN/lOOg. Analyses of tissue from A. stutchhiiiyi showed the
same pattern (Fig. 4c), reflecting harbor water quality at 10,000
MPN/lOOg on day 6. Bacterial depuration by this bivalve was very
rapid, with levels declining to approximately 100 MPN/lOOg by
day 7. Macomoiia liliana were affected to a lesser extent, with a
slight rise above 100 MPN/lOOg on days 6 and 7, remaining el-
evated until day 8 (Fig. 4d). These patterns suggest that entero-
cocci levels in A. stutchhiiryi closely reflect the bacterial levels in
water, whereas those for M. liliana more closely mimic levels in
sediment.

The second intensive monitoring period, in 1997, was during a
period where rainfall did not exceed 4 mm on any day. Enterococci
levels in stormwater and harbor water were not above 100 MPN/
lOOinl during the sampling period, with a small rise above back-
ground levels in harbor water due to factors other than rainfall
(Fig. 5a). Levels in sediment did not rise above 100 MPN/lOOg
and were only marginally higher than median background levels at
any time (Fig. 5b 1. Bacterial levels in A. .stulclilniiyi were more
variable, with most results ranging between 100 and 1000 MPN/
lOOg (Fig. 5c), often above background levels. Again, enterococci
levels remained relatively constant at or below 100 MPN/lOOg for
M. liliana. with small increases above background levels occurring
(Fig. 5d).

The first in the suite of experiments investigating the spatial
distribution of enterococci around a stormwater outfall at Point
Wells was carried out after 4 days of very heavy rainfall (daily
maximum 118.9 mm) in ,Iul\ 1998. There was little chaiiiie in

81

m 9

OS*

o o

Point Wells Stomnwater
Harbour water
Rainfall

40
30
20
10
0

T T 1 T-

6 7 8 9 10 11 12 13 14

Days

Figure 4, Daily enterococci levels over 14 days in .\pril and May 1996
in (a) stormwater and harbor water, (bl sediment, (c) A. stiitchburyi,
and (d) M. liliana tissue. Median (background) enterococci levels are
shown as a dashed line.

bacterial levels with increased distance from the origin (i.e.. the
stormwater drain) (Fig. 6 a-c). Along the central transect entero-
cocci density declined from 300 MPN/lOOg at 0 m to 70 MPN/ 100
g at 70 m (Fig. 6a). A similar pattern is evident along the two radial
transects, with levels dropping from 230 to 270 MPN/ 1 00 g at 20
m to 20-90 MPN/ 100 g at 80 m respectively (Fig. 6b and c). This
experiment was repeated during a moderate rainfall event in Au-
gust 1998; the maximum daily rainfall for the preceding 4 days
was 13,2 mm. The overall pattern was different on this occasion,
with levels nearest to the origin not being the maximum for each
transect (Fig. 7a-c). Results for the left-hand transect (Fig. 7b),
which is directed away from the flow of the stormwater stream
(see Fig. 2), showed a relatively clear pattern of lower bacterial
levels with increased distance from the stormwater outfall. How-
ever, bacterial levels along the right-hand transect reached a maxi-
mum at 30 m (Fig. 7c). which is the point where the stormwater
streain crosses this transect (Fig. 2). Levels are high for the full
extent of this transect. The central transect"s lowest bacterial levels
are at the origin and highest levels at the 80 m mark, with high
variability among points (Fig. 7a).

Enterococci levels in sedirnent declined from 9000 MPN/lOOg
at 0 m to 270 MPN/lOOg al 60 m along the central transect (Fig.
8al. The pattern is similar for the left-hand transect (Fig. 8b).
although the first sample at 2.5 m ( 1 100 MPN/IOO g) is lower than
that at 5 m (3000 MPN/IOO g). However, the density of entero-
cocci declines to 340 MPN/IOO g at 60 m. Figure 8c indicates a
decline in bacterial levels with increased distance. Daily maximum
rainfall for the preceding 4 days was 46.3 mm.

Enterococcus in Sediment. Shellfish and Water

427

Point Wells Stomnwater
Harbour water
Rainfall

T T-

10 11 12

Days

Figure 5. Daily enterococci levels over 12 days in April and May 1997
in (a) stormwater and harbor water, (b) sediment, (c) A. stutchburyi,
and (d) M. liliana tissue. Median (background) enterococci levels are
shown as a dashed line.

DISCUSSION

There is a seasonal nature to the patterns of enterococci levels
in shellfish in the Whangateau Harbour. As both species at both
sites exhibited peaks during the wetter winter months, it is likely
that these increases in bacterial levels are due to the cumulative
effects of many stormwater outfalls, nonpoint source runoff, and
leaching from on-site sewage systems. Soils often become satu-
rated during winter months due to high rainfalls and flooded efflu-
ent leach fields associated with on-site wastewater treatment can
cause the discharge of untreated wastewater into the marine envi-
ronment (Cover 1993). Other researchers have found similar sea-
sonal effects. For example. Paille et al. (1987) detected a peak in
enterococci levels in oysters in Louisiana during late spring-early
summer plus a peak in early winter. However, as no rainfall figures
are given, it is difficult to ascertain the cause. LeMay et al. (1995)
found that in the first few months of winter, when there was high
rainfall, the levels of enterococci in marine water samples in Cali-
fornia were correspondingly high. Levels dropped to a background
level of around 100 Colony Forming Units (CFU)/100 ml in the
later winter months.

A confounding factor in winter is decreased water temperature.
as lower temperatures generally affect survival and detection of
bacteria (Ayres et al. 1978). Inhibition due to sunlight can also
decrease bacterial survivorship in summer (Borrego et al. 1983).
Aiislroveniis stutchhwyi at Point Wells exhibited high enterococci
levels in winter and summer months, and it is likely that A. stutch-
buni at this site have higher background levels overall. Macoinona
liliana at Point Wells did not exhibit high levels in summer, and
this may be related to both their location 10-15 cm below the
sediment surface, where they are more removed from waste dis-
charge flows, and to different feeding methods. The summer en-
terococci peak detected in A. siiiichbuni may be due to the influx

10000

1000-

100

10

o

" 10000

1000

J i 1 * i i

Detection
Limit

Centre Transect (9t>)

-1 1 1 1 1 1 1 1 1

100

b)

- Detection
Limit

1 1

1 1

i

i

i i J

■' I

1

1

I

Left Transect (135-)
1 1 ■ 1 1

e)

1000-

( 1

100-

Detection

i

i

1 1

* I i

10-

1 -

1

1

1

1

1

Right Transect (45-)

1 1 1 1

10 20

30

40 50

60

70 80

Distance from stormwater outfall (m)

Figure 6. Enterococci levels in A. stutchburyi tissue around stormwa-
ter outfall at Point Wells following hea^y rainfall (July 27, 1998).

of holiday visitors to this coastal settlement during the warmer
months, which would place a strain on the ability of on-site sewage
systems to adequately treat wastewater. This may lead to leach
fields becoming overloaded and surface or groundwater seepage
into the open stormwater drains or directly into the harbor. It can
be concluded that factors other than simply winter rainfall affect
the microbiological quality of cockles at Point Wells. Further, in
terms of human health risk, wet summers, where high rainfall and
increased human population occur concurrently, may represent the
"worst case" scenario. The experiments that investigated the effect
of rainfall events on water, sediment, and shellfish bacterial levels
unequivocally indicated that rainfall is associated with increased
levels of enterococci. This immediate effect on enterococci levels
in stormwater with increased rainfall (Fig. 4a-d) is intuitive, as
stormwater is defined as rainwater directed from impervious sur-
faces and stormwater is known to generally contain high levels of
fecal bacteria. Indeed, Pitman (1995) found > 10.000 fecal bacteria
per 100 ml in stormwater runoff samples taken in Goleta, Califor-
nia. The delayed effect on the harbor waters found during our
study is likely to be due to dilution of the input from many storm-
water drains and runoff from adjacent roads and farmlands, with
the cumulative effect not being detected until the day following the
rain event. However, LeMay et al. (1995) concluded that during
wet weather levels of enterococci in marine waters increased dra-
matically and subsequently fell to background levels within 2-3
days. The one-day delay in accumulation of bacteria by A. snitch-
buni, M. liliana, and sediment is also expected, as shellfish would

428

De-Luca-Abbott et al.

u

0000 -

a)

J

1 1

( 1

\> " ^

1000-

1,

( 1

100-

10-

Detection

Limit

1

1

1

1

I

Centre Transect (90)
■ 1 1 1 r

s
e

s

10000 -
1000-

b)

1 1

i

1 1

I

1 1

•)

100-

Detection
Limit
1

1

10-

I

1

1

1

Left Transect (1 35-)
J 1 1

10000

1000

100

10

I i h J i n

DetectioD
Limit

Right Transect (45-)

1 1 1 1 1 1 1 1 1

10 20 30 40 50 60 70 80

Distance from stormwater outfall (m)

Figure 7. Enterococci le>els in A. stutchbiiryi tissue around stormwa-
ter outfall at Point Wells following moderate rainfall (August 28.
1998).

take at least one tidal cycle to accumulate high levels of bacteria,
and as sediment samples were taken at low tide, it is unlikely that
bacteria from the stormwater outfall would have sorbed to surficial
sediment sampled on day one. Similar increases in bacterial levels
following heavy rainfall have been found in sediments (Goyal et
al. 1977) and shellfish (Paille et al. 1987. Pitman 1993).

Samples taken during a 12-day period of little or no rainfall
give an indication of the background variability in enterococci
levels for A. stiitchhuryi in the Whangateau Harbour (Fig. 5c). It is
clear that A. stiitchhuryi shows greater variability in enterococci
levels than M. liliana. and this could be due to its proximity to the
sediment surface (and therefore the discharge flow) and filter-
feeding mechanism.

The delineation experiments (Figs. 6-8) have complex inter-
pretations. Transects sampled after very heavy rainfall (Fig. 6)
give the anticipated pattern of decreased enterococci levels with
increased distance from the stormwater outfall. However, the lev-
els overall are relatively low, and this is likely to be due to the
Hushing effect of high rainfall (LeMay et al. 1995). After a more
moderate rainfall event (an order of magnitude lower than the peak
levels experienced) the pallern is unclear. [-.Micrococci levels along
the central transect increase to a maximum at 70-80 m. and this
may be due to pooling of water in this area, which can cause
bacteria to be retained in water from which cockles feed. The left
transect, extending away from the direction of the stormwater
channel shows the more expected decline in bacterial levels with
increased distance from source. Enterococci levels are high for the
length of the right transect, and al the .''0 m mark, due to Ihe

10000

1000

100

10

"J

(►

i

1 1

1 1

" ' T
( 1

Detection
Limit

1

1

1

1

Centre Transect (90°)
1 1 — 1 1

i

10000 -

b)

( 1

(1

c
o

E

1000-

i

i

i

U*

n

o

s

100-
10 -

Detection

u

s

Left Transect (135°)

s

1

I

1

1

1

1

1 1 1 1

s

c

Ul

<:)

0000-
1000 -

( 1

1 1

( 1

( 1

1 1

* 1 I

(1 H

100-

Detection
Limit

I

10-

1

1

1

1

1

Right Transect (45°)

— 1 1 1 r

0.0 2.5 5.0 10.0 20,0 30.0 40.0 50.0 60.0

Distance from stormwater outfall (m)

Figure 8. Enterococci levels in surficial sediment around stormwater
outfall at Point Wells (July 13, 1998).

transect crossing the stormwater channel, an increase is detected. It
is likely that levels remain high along this transect due to its
proximity to the stormwater channel, which overflows onto the
adjacent "flood-plains" and crosses the path of the transect.

Enterococci in sediment decline in an anticipated almost linear
pattern with increased distance along the central and right transect
(Fig. 8). Again the pattern along the left transect is different, with
distant samples also having high levels. The cause is likely to be
overflow from the stormwater channel at low tide, as the daily
maximum rainfall for the 4 days prior to this particular sampling
exercise was high (46 mm).

The identification of ecological impacts in harbors and estuar-
ies is problematic due to natural temporal and spatial variability in
populations (Sneldcr and Williamson 1997). A better approach
may he to focus on slormwater treatment rather than identification
of effects. In New Zealand, mitigation of effects of slormwater
discharges is based on the "Best Practicable Option" as defined by
the Resource Management Act 1991. Treatment generally involves
either sedimentation in ponds or nitration, both of which still allow
some conlamiiianls lo enter ihc recci\ing environmenl (Snelder
and Williamson 1997), However, Treworgy and Garrett (1989)
believe loss of coastal resources due to effects of waste discharges
often must incur a financial hardship in order to justify restoration
costs. A ncgalive impact on the economy of Ihe Ria Formosa area
in Portugal was attributed to an increase in anthropogenic dis-
charges and Ihe associated decrease in bivalve production (Bebi-
anno 1995). However, it is uenerallv verv difficult to calculate the

Enterococcus in Sediment, Shellfish and Water

429

financial cost of contaminated marine en\ironments. and it may be
this factor that hinders remediation and mitigation processes.

We conclude that enterococci can be used to trace the spatial
and temporal extent of stormwater discharges from small, urban
coastal communities. This information, once combined with
knowledge of the common toxic contaminants in such discharges,
can be used to identify coastal areas suitable for shellfish restora-
tion programmes, as well as detect and model potential effects on
the marine environment and shellfish resources.

ACKNOWLEDGMENTS

Thanks goes to the following for funding assistance for this
research: Northland Regional Council. Lotteries Grants Board.
Todd Foundation. Resource Management Law Association of New
Zealand. Grand Lodge of New Zealand. Freemasons, Soroptimist
International, and TVNZ Group. In addition, we would like to
thank Christopher B. Clarke and Megan Stewart for field and
laboratory assistance.

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Joiinuil of Shellfish Rcseaivh. Vol. 19. No. 1, 431-t3S. 2000.

RED AND GREEN ABALONE SEED GROWOUT FOR RESEEDING ACTIVITIES OFF POINT

LOMA, CALIFORNIA

DAVID LAPOTA, GUNTHER ROSEN, JAELYN CHOCK, AND
CONNIE H. LIU

Space and Naval Warfare Systems Center

Marine Enviroiimeiital Quality Branch, Code D362

53475 St rot he Road

Sun Diego. California 92152-6310

ABSTRACT California has a statewide closure on harvest of any abalone species from San Diego County north to San Francisco.
Prior to closure, harvests of the main species that compose the fishery (the red abalone. Haliolis rufescens Swainson 1822; the green
abalone. Haliolis fiilgens Philippi 1845; and the pink abalone. Haliolis corrugata Gray 1828) had plummeted to an all-time low of
several hundred thousand pounds per year. Overfishing and several diseases identified recently in abalone are creating concern within
the industry with respect to the recovery of the fishery. One method being considered to restore the fishery on a limited basis is
reseeding. Reseeding success is dependent on many factors, including planting location and techniques of seed placement. Other
important factors include the abundance and nutritive quality of macroalgae (kelp) available to the abalone. the seawater temperature
requirement of the species being seeded, the size of the seed being planted, and the presence of potential predators within the seed site.
In this study, we observed that shell growth and weight gain in juvenile red and green abalone are temperature-dependent. When
constant, elevated temperatures are maintained, growth rates improved as compared to growth rates in animals kept at the lower
temperatures normally found. More growth in juvenile red abalone was observed at 19 °C, with an average daily shell growth of 0.1
mm/day. Previous work suggested that Egregia laevigata, a brown kelp was a superior food source for juvenile abalone; however, the
growth rates of red abalone fed Egregia increased minimally compared to growth rates of juvenile red abalone fed another brown kelp
Macrocystis pyrifera. Maximum sustained shell growth of 0. 1 6 mm/day in juvenile green abalone was observed at a culture temperature
of 21 °C. Extrapolations of these growth rates for an entire year indicate growth of 36 mm/y for red abalone and 60 mm/y for green
abalone. Growth predictions based on laboratory-reared juveniles will vary from those observed in wild populations since seawater
temperatures are not static along the coast and may vary as much as 10-12 °C during the year. The nutritional value of kelp and other
algae may be as important as temperature in the effect on growth observed in the laboratory and in the field. Increased growth rates
exhibited by juvenile red abalone during the period from January through April 1977 may be reflective of changing nutrient ratios or
levels in kelp that are otherwise limiting during reduced or non-upwelling months. We are presently culturing hatchery-produced red
and green abalone seed for eventual planting in the U.S. Department of the Interior's Cabrillo National Monument. The seed are
approximately 45 mm in size and are fed a mixed kelp diet. They will be transplanted when they are approximately 75-100 mm in
size. Reseeding success assessed by annual dive surveys should help determine if planting larger animals increases survival of brood
stock and increases recruitment within the monument area.

KEY WORDS: Green abalone. red abalone. reseeding, growout, growth rates

INTRODUCTION pounds in 1952 and then a leveling off in total landings until 1966

(Heiman and Carlisle 1970). The fishery flourished between 1958

Historical Trends of the California Abalone Fishery and 1968, with average annual landings in excess of 4.5 million

pounds. Since 1968, however, landings have decreased. Only 1.3
The present day abalone fishery evolved from the intensive million pounds were taken in 1973. Landings in the state in 1994
harvesting activities of Chinese immigrants in the 1850s (Cox totaled only 322,000 pounds or 6% of that recorded 30 years ago
1960). Abalone is a revered food item in the Orient, and the Chi- ,pig i, jhe decline in landings have been attributed to intense
nese in California had developed a substantial fishery by 1879 harvesting procedures by commercial and sport divers, environ-
based upon the intertidal green abalone, Haliotis fiilgens. and the n^entai degradation of habitat, predation by sea otters, and in some
black abalone, H. ciacherodii. Commercial landings in California cases, competition for space and food from sea urchins (Cicin-Sain
at that time exceeded 4.1 million pounds (total weight equals shell gt ^i 1977 Teaner et al. 1981).
and meat weights combined) (Cox 1960). Soon thereafter, the Japa-
nese "sake barrel" divers replaced the Chinese-dominated fishery. Recent Strategy
followed by the "hard hat" divers. This change in harvesting meth-
ods (pumping surface air to submerged divers via air hoses) per- Reduced yields for both the commercial and sport fisheries
mitted harvesters to maintain landings while operating in deeper have prompted more stringent management regulations and have
offshore waters. After 1916, three major trends were observed stimulated interest in developing methods for enhancing natural
within the fishery: (1 ) a locational shift of the fishery center from populations. This involved limited entry to the commercial fishery
Monterey south to Morro Bay, Santa Barbara, and Los Angeles; and more stringent limitations on sport fisherman; however, land-
(2) a compositional change in the species of abalone harvested for ings still continued to decline. Most recently, the commercial and
the fishery; and (3) a drastic decline in total abalone landings after recreational abalone fishery, south of San Francisco, was closed
1965 (Cicin-Sain et al. 1977). in August 1997 so that a fishery management plan could be de-
Long-term trends of the fishery in the state depict an abrupt veloped. Recent work appears to indicate that the dispersal of
increase in landings from 146,462 pounds in 1942 to 4.784.033 abalone larvae may not be as widespread as once thought, indi-

431

432

Lapota et al.

1950 1955 1

1970 1975
Year

Figure 1.
the years

Commercial landings of abalone by species in California for
1954-1994.

Other studies indicated that growth in abalones was dependent
upon temperature (Sakai 1962, Leighton and Boolootian 1963,
Tomita and Saito 1966. Leighton 1972, 1974; McBeth 1972,
Lapota 1978. 1982). and perhaps to a lesser extent on the type and
quality of the diet (Kikuchi et al. 1967. Leighton 1968. 1976.
Leighton and Boolootian 1963. Tomita 1972). Our study examined
the influence of temperature and diet on the growth of juvenile red
and green abalones.

Previous studies seem to indicate that the release of larger
abalone (>25 mm) may substantially increase the chance of main-
taining and enlarging the number of brood stock for later recruit-
ment (Inoue 1976. Tegner and Butler 1985). Larger abalone are
less cryptic than smaller seed, which will make survival assess-
ment easier, and they also have thicker shells, which offer more
protection from predation by crabs, lobsters, and octupuses.

MATERIALS AND METHODS

eating that closure of a depleted fishing ground may not be suffi-
cient for recovery to occur. Our strategy for recovery emphasizes
replenishing previously depleted fishing areas by release of hatch-
ery-reared juveniles and establishing populations in presently un-
utilized or artificially improved habitats (Tegner et al.l98L Ebert
and Houk 1984, Tegner and Butler 1985, Schiel 1993, Kojima
1995).

Factors Affecting Abalone Growth

Reseeding success is dependent on many factors, including the
geographic location for the placement of seed and techniques of
seed placement. Other factors include the abundance of kelp, the
temperature requirement of the species being seeded, the size of
the seed or juveniles being planted, and the presence of potential
predators within the site. Growth rate data from earlier work and
this study on the red abalone and the green abalone as a function
of culture temperature and diet were reviewed. That is. what length
of time is required to grow larger (76-89 mm in size) H. nifesccns
(Fig. 2) and H. fulgens (Fig. 3) for field reseeding? A review of
previous research on growth rates in several species of abalone
indicated extreme variability related to age. species, food supply,
environmental conditions, geographic location, and season.

Tutschulte ( 1976) described growth rates for juvenile pink aba-
lone, Haliotis corrugate, green abalone. H. fiili;c'iis. and white
abalone, H. sorenseni. Juveniles of each were cultured in the labo-
ratory, reared in seabed cages, and marked and released into kelp
beds. The annual increase in shell length for all three species
ranged from 10.7 to 22.6 mm in the laboratory, 10.7 to 17.3 mm
in seabed cages, and 20.4 to 29.2 mm in the wild. An annual
increase in shell length of 25.9 mm was measured for laboratory-
reared juvenile red abalone, H. nifescens (Leighton 19681. How-
ever, growth rates of young H. rtifescens held in seabed cages off
La Jolla showed marked variation, with annual increases in shell
length from 9.8 to 35 mm. Similarly, juvenile red, pink, green, and
white abalones exhibited large variations in growth as a function of
seawatcr temperature (Leighton 1972. 1974). Variation in growth
rates of juvenile abalone has been observed in H. discits liannai
(.Sakai 1962). H. Iiihcrailalci (Foster 1967). and H. mUkw (New-
man 1968). Clearly, laboratory conditions must be strictly con-
trolled and monitored if the growth rate of a particular species of
abalone is to be determined accurately.

Culture System: Hubbs — Sea World Research Institute

Two flow-through seawater culture systems were used to cul-
ture both red and green abalones. In an earlier study in 1978,
conducted al the Hubbs-Sea World Research Institute (HSWRI)
(Lapota 1982), research was conducted in a seawater laboratory
equipped with automatic filtration and large heat exchanger sys-
tems. Three fiberglass tanks were modified and divided into six
water-bath compartments (Fig. 4). Tank dimensions were 241 cm
long. 73 cm wide, and 76 cm high. All three tanks were mounted
on a wood platform to insulate the bottom of the tanks and allow
for plumbing of seawater supply and drainage. The main wall
partitions of each tank were reinforced fiberglass wood cells 12 cm
thick. An aisle was left between the tanks to provide for access
during maintenance and survey activities. Polyethylene containers
(13-L volume) used to confine and culture the abalones were
placed on concrete bricks in each compartment of the water baths.
The containers were fitted with four drain fittings positioned equi-
distant around the circumference above the height of the water in
the controlled temperature bath. A single water input jet was
placed in the bottom of each container and powered by an air stone
to produce air lift for circulation. Water was introduced into each
container through the bottom, and returned to the controlled tem-
perature bath through the drain fittings at the top. Seawater was
added to each water bath at a rate of 0.3 L/min. which gave an
exchange rate of once per day. Seawater was supplied to each tank
through a 5 cm I.D. PVC pipe manitbld on the center wall partition
of each tank. Excess seawater was drained through the tank over-
flow in the controlled temperature bath.

Ambient seawater temperatures ranged from 15 to 19 C at
Perez Cow in Mission Bay. San Diego, during the study period,
and had to be adjusted to the prescribed culture temperatures. To
maintain the controlled temperature baths at 13. 15. and 17 °C.
ambient temperature seawater was cooled by circulating chilled
fresh water from a cold sump through plastic heat exchange coils
(16 mm O.D. by 13 mm I.D. l immersed in each of the three
controlled temperature baths. Magnetic drive pumps powered by
thermostatic controllers delivered the cold water to the coils as
required. A refrigeration unit was used to keep the cold freshwater
sump at 4 to 7 °C. The controlled temperature bath of 19 °C was
heated with a lOOO-watt immersion heater during the months when
seawater temperature was cooler.

Abalone Seed Growout off Point Loma. California

433

Figure 2. The red abalone. Haliotis nifescens. Approximate size is 3.8
cm. Photo by Jaelyn Chock.

Culture System: Space and Naval Warfare Systems Center. San Diego

The Biological Effects Program (BEP) bioassay facility in
Point Lonia is the current location for abalone seed growout ac-
tivities (Fig. 5). The BEP was established to test new experimental
ship hull coatings proposed for use in the U.S. Navy as well as to
evaluate sediment for contamination in San Diego Bay. The facil-
ity also houses the portable microcosms for environmental testing
(POMFRET); these microcosms can be deployed on-site to evalu-
ate the chronic effects of pollutants on resident marine organisms
(Henderson 1990). The core of the system consists of 12 outdoor
128-L tanks (Fig. 5). The tanks are designed for semi-continuous,
flow-through of untlltered seawater and are exposed to sunlight.
POMFRET flow control is accomplished with a "batch delivery"
system. A series of paired, adjustable- volume bins are periodically
filled with ambient water and are automatically emptied by acti-
vation of cap valves cycled by an adjustable-speed motor. Supply
water for the POMFRET system is pumped from San Diego Bay
near the entrance to the bay by a pair of 3 horsepower swimming
pool pumps. Only one pump is used during normal operation; the
other pump is on standby. Operation of pumps is normally alter-
nated biweekly to allow cleaning of the inactive pump's intake and
output plumbing (Henderson 1990). A gravity supply tank pro-
vides constant flow of ambient water to a feed tank (approximately
57-L volume), excess water overflows into a standpipe. Overflow
from each of the POMFRET' s 12 abalone tanks and four other
larger tanks is fed into the City of San Diego's sewer system. All
tanks are continuously aerated.

Culture System: Naval Ocean System Center, San Diego

Earlier investigations at the Naval Ocean System Center in
Point Loma (now known as SPAWAR Systems Center. San Di-
ego) from September 1976 through July 1977 were conducted to
determine the temperature that would promote the most rapid
growth in the red abalone. A culture system was developed that
permitted accurate temperature control. Several groups of 60-L
culture aquaria were used as temperature baths. Two 13-L poly-
ethylene containers in each aquarium confined the experimental

Figure 3. The green abalone. Haliotis fulgens. Approximate size is 3.8
cm. Photo by Jaelyn Chock.

groups. Concrete bricks in each container provided a substrate for
juveniles. Seawater was continuously aerated and changed twice a
week. One hundred twenty hatchery-reared red abalone, ranging
from 12 to 15 mm in shell length, were purchased from Ab Lab at
Port Hueneme. California. All were maintained on a diet of Mac-
rocystis pyrifera. These animals were cultured at a series of tem-
perature in the range 16.5-22 °C. Shell lengths and total weights of
each juvenile were recorded every 30-35 days.

In a larger study conducted at HSWRI. hatchery-reared juvenile
red abalone were cultured for 175 days at constant temperatures
of 13, 15, 17. and 19 °C. The juveniles, averaging 10-12 mm in
length, were purchased from California Marine Associates,
Cayucos. California. The abalones were divided into eight test
populations, two per temperature bath. Each group was fed ad
libitum with either the giant kelp. Macrucystis. or the feather-boa
kelp. Egregia. twice weekly throughout the entire study. Prelimi-
nary results on H. nifescens showed a seasonal response with
peaks in growth rates in late winter and spring, possibly caused by
the increased nutritive value of the giant kelp, M. pyrifera. There-
fore, to investigate dietary quality-temperature effects, hatchery-
reared juvenile red abalones were fed on a diet of either kelp at
constant temperatures. A parallel study examined growth of wild-
caught juvenile green abalone at constant and wanner tempera-
tures of 17. 19. 21. and 23 °C. The initial mean size of the green
abalones was approximately 30 mm. The feeding schedule was the
same as in the study of red abalone. Shell length (to 0.1 mm) and
blotted wet weight (to 0.1 gm) of each abalone were measured at
35-day intervals.

In the later study conducted at SPAWAR Systems Center. 35
hatchery-reared green abalones were grown for 1 5 mo at ambient
seawater temperatures. The mean starting size of the green abalo-
nes was 22 mm. Measurements were conducted sporadically ex-
cept for the fall-winter measurements (1997-1998) which were
made on 30—40 day increments.

RESULTS

First Data— Nutritional Observations (SPAWAR)

Data on juvenile H. nifescens shell growth were obtained for a
period of 317 days (Fig. 6). Distinct increases in shell growth were
observed at all culture temperatures from late January through

434

Lapota et al.

ABAIONES
|-B«ICK SUBSTRATE

AIR VALVES AND llNE
r-WATER lEVEl

• 3 lITiR BUCKET

1.27 CM. AIR IINE

B.08 CM. SEA WATER UNE -
3.S CM.STYROFOAM tID-,

nriiniiiiztn

1.9) CM. VAIVES

i™^ Bjr»i*»^.jm«i"iig'.»a«.-tgii»Mr«r.,MI^

%.

3

/

WOOD riATFORM

•5

TANK

■AIR STONE WITHIN
PERFORATED CAPSUlE

Figure 4. End view of tanks and schematic of water circulation througli abalone culture system used at the Hubbs-Sea World Research Institute,
Mission Bay, San Diego, California.

April 1977. Shell growth after April declined noticeably at all four
temperatures, and was reduced markedly from late September
through early January. Clearly, growth of juvenile H. ntfesceiis
was maximum in late winter and early spring at all temperatures
tested. However, the groups cultured at 16.5 and 17 °C grew faster
than those at 20 and 22 °C.

Constant Seawater Temperature Culture (HSWRI)

The mean starting size of the red abalones grown at constant
seawater temperatures (13, 15. 17, and 19 °C) ranged from 11.1 to

1 2. 1 mm and the mean starting total weight for each of the four
groups was 0.2 to 0.3 g. Following 175 days of culture, mean shell
growth was greatest at 19 °C and least at 13 °C (Fig. 7). The mean
final size for these temperatures was 29.4 and 24.4 mm; a mean net
increase of 17.9 and 12.3 mm, respectively. Mean weight gains
were also greatest in the group cultured at 19 °C and least in the
group cultured at 13 °C (Fig. 8) (Lapota 1982). The mean final
weights were 3.5 and 2.1 g. a mean net weight gain of 3.3 and 1 .8
g. respectively. Mean shell growth in the group cultured at 17 "C
was greater than the group cultured at 15 "C (Fig. 7), The mean

Figure 5. I'holDgraphs of the Biological KItVcIs Program test station along San Diego Bay. The right photograph shows the POMFRET used to
culture red and green abalones.

Abalone Seed Growout off Point Loma, California

435

Sep76 Oct Nov Dec Jan'77 Feb Mar Apr May Jun Jul

Survey Date (Month, Year)

Figure 6. Mean increase in shell length of four groups of 17 hatchery-
reared H. rufescens cultured from September 1976 through July 1977
at four constant temperatures at SPA WAR Systems Center. Measure-
ments were taken every 30-35 days.

final size was 28.6 and 26.5 mm; a mean net increase of 17.5 and
15.1 mm. respectively. Similarly, the mean final weights of these
groups were 3.0 g at 17 °C and 2.4 g at 15 °C; a mean net increase
of 2.8 and 2.2 g.

Shell growth rate maximums were observed between January
30 and March 6. 1978. for the abalones cultured at 13. 15. and
17 °C (Fig. 7). Maximum shell growth rates at 19 °C were evident
prior to January 30. Shell growth appears constant; however, the
observed maximum rates of shell deposition add some nonlinearity
to the observed growth. Shell growth rates at all temperatures
decreased by May 15. 1978.

Growth of H. rufescens on Macrocystis and Egregia Kelp
Diets (HSWRI)

Mean shell growth was fastest in both groups cultured at 19 °C
and slowest at 13 °C (Fig. 9). The mean increase in shell length at
19 ""C for the Macrocystis-fed and Egregici-ied groups were 3.2
and 3.5 mm/month respectively, and at 13 C were 2.2 and 2.6
mm/month, respectively. Growth of red abalone at 15 °C fed Egre-
gia was significantly greater than growth of those fed a diet of
Macrocystis: however, there were no significant differences in
rates of growth at the other culture temperatures.

There was a significant difference in the growth of red abalones

Jan 78 Mar 78 Apr 78

Survey Date (Month, Year)

May 78

.■Oz/-. •

ar ;-^-;7*C ■-&-- «

/ .' ,/ ~^ .A

/ a' / ^iiK— --

/ / /

/ / /

// X

1 1 1 i \

+ 13C

A 15C

O 17C

• 19C

Dec 77 Jan 78 Mar 78 Apr 78 May 78

Survey Date {Montti, Year)

Figure 8. Mean seasonal rates of weight gain in H. rufescens versus
seawater temperature. Growth period was 175 days.

fed Macrocystis at the four culture temperatures. Growth at the two
higher temperatures was significantly greater than growth at the
two lower temperatures of 13 and 15 °C. Also, there was a sig-
nificant difference in the growth of red abalones fed Egregia at
1 3 °C when compared with growth of red abalones at the higher
culture temperatures (Fig. 9).

Mean weight gains were greatest in the groups cultured at
19 °C and smallest in the groups cultured at 13 'C (Fig. 10). The
average monthly growth for the Macrocystis-fed and Egregia-fed
groups cultured at 19 °C were 0.6 and 0.7 g, while groups cultured
at 13 °C exhibited weight gains of 0.3 and 0.4 g. respectively.

Growth o/H. fulgens — Constant Seawater Temperatures (HSWRI)

The highest rate of growth was achieved at 21 °C. with a mean
increase in shell length of 5 mm/mo (Fig. 1 1 ). Slowest growth was
observed at 17 °C, with a mean increase in shell length of 1.7
mm/mo. Similarly, greatest mean weight gain (3.7 g/mo) was ex-
hibited by the group cultured at 2 1 °C; lowest mean weight gain
occurred at 17 °C (0.7 g/mo). There was a significant difference in
growth rates of green abalones cultured at the four temperatures (F
= 36.81). A series of t-tests showed that growth at 21 °C was
significantly greater than growth at all other test temperatures (p <
.05). The growth of green abalones at 21 °C was significantly
greater than the red abalones at its optimal growth temperature of
19 C.

I I Macrocystis pyritera

Egregia laevigata

15C 17C

Culture Temperature (C)

Figure 7. Mean seasonal rates of shell growth in H. rufescens versus
seawater temperature. Growth period was 175 days.

Figure 9. Mean increase and standard deviation in shell length of
hatchery-reared H. rufescens led two different diets versus constant
temperatures. Growth period was 175 days.

436

Lapota et al.

Mean Increase in Shell

Mean Increase per Month

O Macrocystis pyhfera
• Egregia laevigata

13C 15C 17C 19C

Culture Temperature (C)

Figure 10. Mean increase in wciglit of hatchery-reared H. rufescens
fed two different diets versus constant temperatures. Growth period
was for 175 days.

Growth ofH. fulgens — Ambient Seawater Temperatures (SPAWAR)

Abalones monitored over a 16-month period for growth in-
creased in size, from an initial mean of 21.58 mm to 50.57 mm.
Seawater temperatures throughout the year typically ranged from
14 °C in the winter months to greater than 25 °C in late summer.
Growth was greater (104 (jLOi/day) at this time (between August 10
and October 10. 1997) than during the cooler winter months (Janu-
ary 25-April 10, 1998) (6-25 [jim/day) (Fig. 12). Weight gain in
the green abalones was most pronounced from April 10 to No-
vember 4. 1998. Mean weights increased from 5 to 16 g in this
7-month period.

DISCUSSION

Shell growth and weight gain of juvenile red and green abalo-
nes appear to be temperature dependent. When constant, elevated
temperatures were maintained, growth rates of red and green aba-
lones increased substantially.

The results indicated that more rapid growth in juvenile red
abalone was obtained at 19 °C, with an average daily shell growth
of 0.1 mm/day. Maximum sustained growth in juvenile green aba-
lone displayed an average daily shell growth of 0.16 mm/day al a
constant culture temperature of 21 "C. similar to growth rates
observed in green abalone during the summer months of 1997-
1998 in Point Loma. Extrapolations of these laboratory growth
rates for an entire year predict growth of 36 mmly for red abalone
and 60 mni/y of growth for green abalone. However, since ocean
temperatures are not static along the coast and may have a range as
much as 10-12 °C during the year along .southern California,
growth predictions based on laboratory-cultured juveniles may
vary considerably from those observed in wild populations.

Nutritional aspects may be as important as temperature effects
on growth rates in wild populations. Previous studies have ob-
served that Egregia and mixed algal diets are a superior food
source for juvenile abalones (Leighton 1976). However, growth
rates of red abalones fed Egregia increased only minimally in this
study (Lapota 19781. Some nutritional components o( Macrocystis.
such as protein and carbohydrate levels, \ary considerably
throughout the year. Additionally, C:N ratios in Macrocystis vary
seasonally from 17:1 in the winter months to 40:1 in the summer
months (Jackson 1977). Nutrient concentrations in the vicinity of
kelp beds also \ary with season. Below 4-5 m, nilrale conceiitra-

17 19 21 23

Culture Temperature (C)

Figure 11. Mean sliell length (mm I and standard deviations of four
groups of 13 wild-caught H. fulgens cultured for 175 days at four
temperatures. Measurements were taken at 35-day intervals.

tions are greatest during winter-spring (upwelling) and lowest dur-
ing the summer.

Artificial diets containing crude protein levels (white fish meal)
in excess of 30% produced increased growth in H. discus when
compared to other artificial diets with lower crude protein levels
(20-309'f ) (Ogino and Kato 1964). Growth rates declined when the
diet contained less than 159^^ crude protein. Increased growth rates
exhibited by juvenile red abalones in our studies from January
through April 1977 may indicate changing nutrient ratios or pro-
tein levels in the kelp, which are otherwise limiting during non-
upwelling months. It is probable that the nutritional quality of the
kelp limits growth in the abalone even when optimal growth tem-
peratures are present.

Past Reseeding Efforts

The Experimental Abalone Enhancement Program in Califor-
nia began in 1978 with four approaches lo be tested for meeting the
goals of the program (Tegneret al. 1981 ). The Department of Fish

Shell Size (mm)

Growth Rate (um/Day)

60

55

-

,-«^

-

50

-

/

\

y*-

E

£ 45

s

to

% "0

.c
w

n 35
30
25

r

1

1
1

1

/

1 1

■

y^^ /-

^ /

1 1 1 1 1 1 1 ].. .-I — \ — 1 — I-

120
110

100

>.

90

g

RO

3

70

a.

60

Je

50

o

a>

40

^

r

30

»

>

20

10

jLil97Aug Sfp Ocl Now DecJan98Feb Mar Apr May Jun Jul Aug Sop Oc) Nov

Month, Year

Figure 12. Mean shell length (mml increase and growth rates of hatch-
ery-reared //. fulgens cultured at amhient seawater temperatures for
16 months at SPA WAR Systems Center, Point l.onia.

Abalone Seed Growout off Point Loma, California

437

:V

V San Diego County |^

Satt Diego Bay \

t

1 X

^ \

. • Coronado

} Pt. Loma

\^_/^ Ballast Pt.
^r Lighthouse

\

^

Pacific Ocean

\ *

\

'' ''^Ww^

'y/^ 4 Monument •

)

^000A ' P'- Loma
y^wyXh, Lighthouse ,

^^m

^•32=40'

uses 7.5 Pi. Loma

Offshore Boundary: 900' distance
Coastline Length: 1.2 mi

0 0.5

1 1 1

■•-

Figure 13. Proposed abalone seed planting site off Cabrillo National Monument, Point Loma, San Diego, California.

and Game closed the mainland coast between Palos Verdes and
Dana Point for 5 y to all sport and commercial diving activities.
This management technique was initiated to study the effects of
abalone hatchery seeding activities and recruitment into the closed
area. The other approaches used in the closed area were designed
to observe the effects of habitat improvements and modifications
on the settlement of endemic and seeded abalones. and to study the
effect of transplantation of adult abalones as brood stock into the
closed area.

From several large-scale seed plantings of red and green aba-
lones conducted in various coastal sites of southern California,
several important facts have emerged. Poor seed survival in trans-
plants may be attributed to stress from transport and handling.
Small abalone are cryptic and mobile, making survival assessment
difficult. Some of the earlier experimental seeding activities in
November 1979 in Santa Barbara County indicated that the habitat
chosen for these plantings is critical to the survival of the planted
seed. Approximately 9900 hatchery-raised red abalone seed, aver-
aging 31 mm in length, were planted in a boulder habitat. Four
months of surveys following the planting indicated that seeding
did not increase juvenile abundance in the study area (Tegner et al.
1981). Only two hatchery-raised abalone were found. Predation of
the seed by crabs, lobsters, and octopuses was observed within 2
days of the planting. Four months after another planting (mid May
1980) of 8900 red abalone seed in the same area, the experimental
site was found to contain significantly more juveniles. Juvenile
density changed from 0.33 per 10 m" to 3.88 per 10 m" (Tegner et
al. 1981 ). Of the 8900 planted seed, an absolute increase of juve-
nile abalones in the study after 4 months was estimated between
600 and 800 abalones. Apparently, only 9% of the seed could be

accounted for within the study area. Smaller hatchery seed (20 mm
in length) appeared to move out of the study area at significantly
higher rates than naturally occurring seed (Tegner et al. 1981).
Later field experiments conducted off Palos Verdes in 1981 indi-
cated poor recruitment irrespective of seed size (Tegner and Butler
1985). While the growth of seeded abalones was similar to labo-
ratory-grown abalones, survival of the seeded abalones was only
1%. These results contrast with reseeding efforts conducted in
Japan by the Abu Fishery Cooperative (Kojima 1995) and with the
increased recruitment from planting larger seed according to Inoue
( 1976). Recapture rates for seeded abalones in the initial size range
of 15^0 mm were from 12 to 51 9^ in the 1980-85 year classes
that had been fished (Kojima 1993). The survival rate of larger
seeded abalones approached 70'7r survival for seed 70 mm and
larger (Inoue 1976). Such different results from similar studies
raise questions that can be addressed in future studies in Point
Loma.

Present Activities

Based on these later observations, our group is currently grow-
ing green and red abalones seed to a size of approximately 70 mm
before attempting to reseed areas of Point Loma in Cabrillo Na-
tional Monument (Fig. 13). The National Park Service divided the
monument area into three zones, based on accessibility by park
visitors. Area III (southern tip of Point Loma) is relatively undis-
turbed by visitors to the park and is in close proximity to our
laboratory. All of these areas contained abundant populations of
abalones in the 1960s and 1970s, but have been completely deci-

438

Lapota et al.

mated by sport and commercial fisherman. Green abalone will be
planted in the low intertidal zone and red abalone will be planted
in the adjacent kelp beds at depths of approximately 8-12 m.
Approximately 1000 green and red abalones at a mean size of 70
mm will be distributed along a series of transects. Growth and

survival will be monitored at 6-mo intervals. The stocking density
will be varied within the area to assess future seed recruitment.
Hopefully, by eliminating or reducing human interference, we will
be able to observe increased abalone recruitment from our near-
term reseeding with larger abalone.

LITERATURE CITED

Cox. K. W. 1960. California abalones. family Haliotidae. Fish Bull. 118:
70.

Cicin-Sain, B.. J. E. Moore & A. J. Wyner 1977. Management approaches
for marine fisheries: The case of the California abalone. Sea Grant
Piiblicalitin 54:2. La Jolla. California.

Ebert. E. E. & J. L. Houk. 1984. Elements and innovations in the cultiva-
tion of red abalone, Huliotis riifescens. Aquaculutre 39:375-392.

Foster, G. R. 1967. The growth of Haliotis tuberculata: Results of tagging
experiments in Guernsey, 1963-1965. / Mar. Biol. Assoc. U.K. 47:
287-300.

Heiman. R. F. G. & J. G. Carlisle, Jr. 1970. The California marine fish
catch for 1968 and historical review. 1916-68. Fish Bull. 149:70.

Henderson. R. S. 1990. User data package for portable microcosms for
environmental testing system (POMFRET). Internal Report, Naval
Ocean Systems Center. Hawaii Laboratory. Kailua, Hawaii.

Inoue. M. 1976. Abalone seeding and its effectiveness, pp. 9-25. In: Ef-
fects of Release of Hatchery-Reared Juveniles. Japanese Society of
Scientific Fisheries. Kouseisya Kouseikaku, Tokyo.

Jackson, G. 1977. Nutrients and production of the giant kelp. Macrocyslis
pyrifera. off Southern California. Limnol. Oceanogr. 22(6):979-995.

Kikuchi. S. Y. Sakauai. M. Sasaki & T. Ito. 1967. Food values of certain
marine algae for the growth of the young abalone H. discus hannai.
Bull. Tohokit Reg. Fish. Uib. 27:93-100.

Kojima. H. 1995. Evaluation of abalone stock enhancement through the
release of hatchery-reared seeds. Mar. Freshw. Res. 46:689-695.

Lapota. D. 1978. Growth-temperature-survival relationships in abalone.
Hubbs/Sea World Research Institute Technical Report No. 79-1 16. San
Diego, California.

Lapota. D. 1982. Biological and regulatory feasibility of abalone aquacul-
ture in the California coastal /.one. Master's thesis. San Diego State
University. San Diego. California. 117 pp.

Leighton. D. & R. A. Boolootian. 1963. Diet and growth in the black
abalone, Haliotis cracherodii. Ecology 44(21:227-238.

Leighton, D. 1968. A comparative study of food selection and nutrition m
the abalone H. riifescens (Swainson) and the sea urchin Stronglocen-
irotus purpuratus (Stimpson). Ph.D. dissertation. University of Cali-
fornia, San Diego, California. 197 pp.

Leighton, D. 1972. Laboratory observations on the early growth of the
abalone Haliotis sorenseni and the effect of temperature on larval de-

velopment and settling success. U.S. Nat. Mar. Fish. Sen: Fish. Bull.
70(2):373-38l.

Leighton, D. 1974. The influence of temperature on larval and juvenile
growth in three species of southern California abalones. U.S. Nat. Mar
Fish. Sen: Fish. Bull. 72(41:1 137-1 145.

Leighton. D. 1976. An investigation of feeding, food conversion, and
growth in the abalone, with emphasis on utilization of the giant kelp.
Macrocyslis pyrifera. Final Report, Contract N6600I-76-M-S755,
Ocean Food and Energy Farm Project. U.S. Naval Undersea Center,
San Diego. California.

McBeth. J. W. 1972. The growth and survival of the California red abalone
in Japan. Vemis 31(3):l22&endash ;126.

Newman, G. G. 1968. Growth of the South African abalone H. midae. Div.
Sea. Fish. Union South Africa Invest. Report 67:1-24.

Ogino, C. & N. Kato. 1964. Studies on the nutrition of abalone. II. Protein
requirements for growth of abalone. H. discus. Bull. Jap. Soc. Sci. Fish.
30(6):52.3-526.

Sakai. S. 1962. Ecological studies of the abalone, Haliotis discus hannai
Ino. I. Experimental studies on the food habit. Bull. Jap. Soc. Sci. Fish.
28(81:766-779.

Schiel, D. R. 1993. Experimental evaluation of commercial-scale enhance-
ment of abalone Haliotis iris populations in New Zealand. Mar.Ecol.
Prog. Ser. 97:167-181.

Tegner, M. J.. J. H. Connell. R. W. Day. R. J. Schmitt. S. Schroeter & J.
B. Richards. 1981. Experimental abalone enhancement program. Cali-
fornia Sea Grant College Program: 1978-1980 Biennial Report. Sea
Grant Report no. R-CSGCP-004. La Jolla. California.

Tegner, M. J. & R. A. Butler. 1985. The .survival and mortality of seeded
and native red abalones, Haliotis rufescens. on the Palos Verdes pen-
insula. Calif Fish Game 71(3):150-I63.

Tomita. K. & K. Saito. 1966. The growth of the abalone Haliotis discus
hannai. at Rebun Island, Hokkaido. J. Hokkaido Fish. Sci. Inst. 23( 1 1 ):
555-560.

Tomita. K. 1972. Experiments on the food selectivity and digestion of
abalone seed, Haliotis discus hannai. J. Hokkaido Fish. Sci. Inst. 29(4):
17-23.

Tutschulte, T. 1976. The comparative ecology of three sympatric abalones.
Ph.D. dissertation. University of California. San Diego, Calilornia.

Journal of Shclljlsh Research. Vol. 19. No. I, 439^44. 2000.

THE DEMAND FOR OYSTER RELAYING ACTIVITIES IN LOUISIANA: 1976—1995

WALTER R. KEITHLY, JR.,' ASSANE DIAGNE," AND
RONALD J. DUGAS'

^Coastul Fisheries Institute

Wetland Resources Building

Louisiana State University

Baton Rouge. Louisiana 70803-7503
"Coastal Fisheries Institute

Wetland Resources Building

Louisiana State University

Baton Rouge. Louisiana 70803-7503
^Louisiana Department of Wildlife and Fisheries

1600 Canal Street

New Orleans. Louisiana 70890

ABSTRACT Louisiana usually leads the nation in the annual production of oyster meats. Production is derived from both leased
water-bottoms and the public seed grounds. A sizeable amount of the water-bottoms under lease have either conditional or restricted
status. To make the best economic use of leases under these two statuses, leaseholders will, at times, relay oysters from leases in
conditional or restricted areas to leases in approved areas. This paper examines relaying activities in Louisiana during the period
1976_1995. Specifically, an econometric model was developed and used to examine the demand for relaying activities in the state.
Results suggest that demand depends significantly on economic as well as environmental factors.

KEY WORDS: Louisiana, leases, oysters, public .seed grounds, relaying

INTRODUCTION

About 10% of the U.S. population lives within 50 miles of the
coast, and between 1950 and 1984 the population in coastal coun-
ties grew by more than 80'7r (USEPA 1989). This rapid rate of
growth, in conjunction with the absolute number of people living
along the coast (about 350 per square mile), has strained the fragile
ecosystems that support shellfish populations (particularly mollus-
can). As a resuU. many of the traditionally productive shellfish
nursery and habitat grounds have been destroyed and others have
become unsuitable for human activities. Some of the specific hu-
man-induced causes of deterioration of shellfish-growth areas, as
cited by the U.S. Environmental Protection Agency (USEPA
1990) include:

Industries-According to USEPA estimates. 1300 major industrial
facilities discharge directly into estuarine and near-coastal wa-
ters;
Sewage treatment plants-Almost 600 municipal treatment plants

discharge effluents into estuaries and near-coastal waters:
Nonpoint sources-More than one half of coastal pollution is at-
tributable to urban and agricultural nonpoint sources:
Combined sewer overtlow-Raw sewage and urban runoff are dis-
charged into estuaries after rainstorms in urban areas.
Although the Gulf of Mexico is considered "the most healthy of
our (i.e.. U.S.) coastal marine environments" (Lipka at al. 1990),
most, if not all, of the previously cited anthropogenically induced
causes of deterioration of shellfish-growing waters are present to a
greater or lesser extent in the Gulf of Mexico region and. specifi-
cally. Louisiana.

One readily available source for information on water quality in
the Gulf of Mexico is the compilation of classified shellfish-
growing waters published by the National Shellfish Register of
Classified Estuarine Waters. Information on Gulf of Mexico es-
tuarine water classifications for 1971 and 1995 is presented in

Table 1. Approved growing waters constituted about 63% of total
classified growing waters in 1971 but declined to less than 45% in
1995. Conditional growing waters increased from about 6% of the
total in 1971 to more than 159!- of the total in 1995. Restricted and
prohibited waters equaled about 40% of the total in 1995 compared
to only 32% in 1971.' With respect to Louisiana, 1.6 million acres
of estuarine waters (both leased and non-leased) were classified as
approved in 1995 while 400.000 acres were classified as condi-
tional and almost I million acres were classified as restricted.

The compilation of shellfish-growing waters provides one in-
dication of estuarine water quality: however, it is somewhat lim-
ited in scope (i.e., it tests only for elevated levels of indicator
organisms). Estuarine water quality in the Gulf of Mexico has also
changed over time as a result of physical changes in wetlands. In
Louisiana, for example, wetlands are being lost at a rapid rate due.
at least in part, to human action (see Turner and Cahoon 1988 for
details). Alteration of the wetlands has resulted in salinity changes
in many of the local ecosystems, impairing the productivity of
natural oyster reefs. As noted by Van Sickle et al. (1976). the
oyster industry in Barataria Basin. Louisiana, one of the most
productive basins in the state, "is steadily being squeezed between
encroaching salinity (and the accompanying predation and disease
problems) from the south and pollution from the north" (p. 17).
The encroaching salinity discussed by the authors is largely the
result of human activities. These authors concluded that further
coastal erosion will force production further inland, where higher
levels of coliform exist and. at some point, areas suitable for oyster
production will decline. In support of their conclusions, leased
water bottom acreage in Louisiana advanced from 32,000 acres in
1950 to almost 400,000 acres cuirently. Despite this sharp increase

'In 1971 the restricted classification was not used. Waters classified today
as restricted were classified as prohibited in 1971.

439

440

Keithly et al.

TABLE 1.

Gulf of Mexico shellfish estuarine waters classification trends, 1971
and 1995 (1000 acres)/

Year

Classification

1971

1995

% Change
1971 to 1995

Approved''
Conditional
Restricted
Prohibited

Total

3226 (62.9)'-
282 (5.5)
0(0.0)
1618(31.6)

5126(100.0)

2860(44.2)
997(15.4)
1597(24.7)
1015(15.7)

6469(100.0)

-11.4
254

-37.3
26.2

" Does not include classifications of offshore growing areas and/or unclas-
sified waters.

'' Definitions of classifications: Approved waters: Shellfish may be har-
vested for direct marketing; Conditional waters: Shellfish-growing waters
may be opened if they meet approved classification status under predict-
able conditions. Waters are opened when water quality standards are met
and closed at other times; Restricted waters: Shellfish-growing waters can
only be harvested if shellfish are relayed or depurated prior to marketing;
Prohibited waters: Shellfish may not be harvested for direct marketing.
^ Numbers in parentheses reflect percentage of the yearly total associated
with corresponding classification. Sources: U.S. Department of Commerce
(1997) and Bell (1978)

in leased acreage, annual oyster production in Louisiana has re-
mained quite stable in the long run. generally in the range 10-
million to 13-million pounds. This suggests that the productivity of
the "average" lease has fallen substantially during the past several
decades.

Louisiana usually leads the nation in the production of oyster
meats. Production is derived from both privately leased grounds
and public grounds, with production from the former historically
accounting for about 80% of the state's total annual landings; in
recent years, the share has fallen to approximately 50%. While
production from the public seed grounds has historically been
minor relative to production from the private leases, these grounds
serve two important purposes. They provide a source of market
oysters during the September-March period (the months during
which the public grounds are generally open to harvesting activi-
ties). Second, the public seed grounds provide a source of seed
oyster (i.e.. less than three-inch oysters), which leaseholders can
transplant to their private leases and harvest at a later time when
public seed grounds are closed.

Acreage leased in conditional or restricted waters can also
serve a purpose to the lease-based oyster operations, specifically
oysters can be moved from conditional waters to approved waters
and can be harvested when having met approved classification
status. The purpose is that of using the leases for relaying activi-
ties. The U.S. Food and Drug Administration (1995) definition of
relaying can be summari/cd as the transfer of shellfish from re-
stricted areas, conditionally restricted areas in the open status, or
conditionally approved areas in the closed status to approved or
conditionally approved areas in open status for the reduction of
pathogens as measured by the coliform indicator group or poison-
ous or deleterious substances that may be present, by using the
ambient environinent as a treatment status. Because all of the
closure statuses in Louisiana are based on fecal coliform level
criteria, the definition essentially refers to the transfer of moder-
ately polluted oysters to open areas for natural ilepuration.

The process of relaying can provide both the oyster harvester
and the environment with a resource restoration process. Many
oyster restoration processes are basically the introduction into the
environment of some sort of material for cultch attachment. The
oyster larvae attach and establishment of an oyster reef community
begins; in some areas of the country it takes several years for the
first generation inhabitants to establish themselves. The process of
relaying can be a shortcut to this process as relaying can provide
both the attachment material, which in this case is the oyster shell,
and a living community of oysters and fauna associated with an
oyster reef environment. The area from which the relaying oc-
curred will continue its existence and the area to which the relay-
ing takes place will obtain, for all practical purposes, a complete
restored oyster reef community.

Although potentially beneficial, the process of relaying is labor
and capital intensive. Hence, it is conducted only when economic
and environmental conditions indicate a favorable return on in-
vestment. The purpose of this paper is to examine the demand for
relaying activities in relation to economic and environmental con-
ditions faced by the industry during the period 1976-1995. While
the study is specific to Louisiana, the results should be applicable
to other states with lease-based oyster or other shellfish operations.

To achieve the purpose of this paper, a brief description of
relaying activities in Louisiana is presented in the next section of
the paper. Attention is then given to theoretical considerations and
the development of a conceptual model that is used to determine
the factors influencing the demand for relaying activities. Finally,
empirical results are presented, along with a brief discussion of
relevant findings.

DESCRIPTION OF LOUISIANA'S RELAYING ACTIVITIES

Relaying oysters in Louisiana requires a permit from the Loui-
siana Department of Health and Hospitals. The cost of the permit
is nominal, approximately $50. However, the applicant is also
required to post a S50()0 security bond, which is returned if no
violations are detected during the relaying process. In general, the
permits are valid for a 2-week period.

Two types of relaying activities are practiced in Louisiana. The
first is generally referred to as an experimental or controlled relay.
Relays of this type are conducted infrequently (Fig, 1 ). The state

Number of Permits

120

100

1976 1978 1980 1982 1984 1986 1988 1990 1992 1994
Eijiure 1, Annual number of experimental and regular permits issued
for relating: 1976-1995.

Louisiana Oyster Relaying

441

No. of Permits

Price ($/lb)

nni Inn

a

d

D I

1.5

1

0.5

1976 1978 1980 1982 1984 1986 1988 1990 1992 1994

Figure 2. Annual number of regular permits issued and deflated Loui-
siana dockside oyster price: 1976-1995.

permits "controlled" relaying for a limited duration from polluted
water bottoms in the public seed grounds or other areas not under
lease. In 1990. both the public .seed grounds in Vermillion Bay and
the southeast corner of Lake Pontchartrain (which is neither a
designated public seed ground area nor open for leasing activities)
were opened for controlled relaying. In 1985, the state-owned reef
in Bay Junop (that area of the reef in polluted waters) was opened
for a limited amount of time for controlled relaying, and in 1987
the state-owned reef in Vermillion Bay was opened.

The second type of relaying activity is simply referred to as the
regular relay. This entails the movement of oysters from leases in
conditional or restricted waters to leases in approved waters."
These regular relays are the primary focus of this paper. As indi-
cated in Figure 1. permits issued for regular relaying activities
were consistently less than 40 per year from 1976 until the mid
1980s. Then the number of permits issued advanced rapidly, peak-
ing at over 100 in 1987. Beginning in 1988. the number declined
sharply, though a moderate increase was observed again in 1990.""

While considerable detail is given in following sections of the
paper as to the reasons for the observed annual variation in number
of regular permits issued, some general observations are presented
here. First, the deflated price of the harvested product (i.e.. the
current price adjusted for inflationary effects) advanced rapidly
during the 1985-1990 period but declined sharply in 1991. and has
remained well below that observed during the mid 1980s to 1990
period (see Fig. 2). One would hypothesize, ceteris paribus, that
increases in the deflated price of the harvested product would
result in increased relaying activities.

Even though the deflated price increased through 1 990. a sharp
decline in the number of regular permits issued began in 1988. The
Louisiana Department of Health and Hospitals initiated a require-
ment in 1988 that a security agent be onboard any vessel relaying
oysters from polluted to approved waters. Given that this require-
ment would add expenses to relaying operations, one would expect

-Conceivably, this type of relaying could also entail the relaying of oysters
from leases in conditional or restricted waters to other leases in condi-
tional or restricted waters. One would expect minimal, if any. activities of
this nature because it would then require additional relaying activities
before the oysters could be marketed.

"In some instances, continuations for use of an issued perinit were re-
quested and approved. These continuations are not included in the analy-
sis.

1986-1995
Figure 3. Quarterly distribution of issued relaying permits: 1976-
1995.

that it would result in a reduced number of permit applications.
ceteris paribus. A cursory examination of the data suggests this is
the case.

The quarterly distribution of relaying permits is presented in
Figure 3 for two time periods: 1976-1985 and 1986-1995. As
indicated, the majority of relaying activities occurred during the
tli'st (January through March) and fouilh (October through Decein-
ber) quarters of the year. The amount of relaying activities in the
first quarter grew during the later time period. For the 1976-1985
period only about 28% of relaying activities occurred during the
first quarter, but by the 1986-1995 period the percentage had
increased to almost 50%.

THEORETICAL CONSIDERATIONS AND
CONCEPTUAL MODEL

Theoretical Considerations

Oyster leases are an asset used in the production of market
oysters. As such, an oyster lease is no different than agricultural
property where the land is an asset used in the production of a crop.
The implied goal of the farmer is to maximize the discounted
stream of returns generated from employment of his property
(owned or leased) over an infinite time horizon, or:

max(NPV) = Z(P, * Q,(q) - C,(Q))/( \ + r)' ( 1 )

where NPV = net present value of returns from oyster farming
activities: P, = the output price of the harvested lease-based oyster
product in time period t; Q, = the quantity of oyster output from
lease-based activities in time period t which in turn is a function of
the quality (q) of the asset: C, = cost of production in time period
t, which is a function of output Q,(q); r = discount factor.

The net present value of returns (the discounted profits) are, as
indicated, related to the output price (P,); the output quantity (Q,),
which is related to the quality of the asset (q): the costs of pro-
duction (C|): and the discount factor (r). As specified, an increase
in the output price or quantity will result in an increase in the net
present value of returns. Conversely, an increase in costs for a
given level of production (due to an increase in input costs) or an
increase in the discount factor will result in a decrease in the net
present value of returns."*

In general, demand for relaying activities can be considered as
a derived demand for a factor of production (see Layard and
Walters 1978 for details). As such, demand for relaying activities
will be directly related to the ability of these relaying activities to
enhance the net present value of returns from oyster farming ac-

■"An infinite time horizon is assumed for discussion purposes. The analysis
could be changed, without loss of generality, to allow for the sale ot

property after a fi.ved number of time periods.

442

Keithly et al.

tivities. The implications of this are muhifaceted. First, it implies
that the demand for relaying activities will increase (decrease) in
relation to an increase (decrease) in the output price (P,). ceteris
paribus. Second, it implies that the demand for relaying will be
positive only to the extent that it will achieve a short-run or pos-
sibly long-run increase in the output from lease-based activities
(Q,). A short-run increase in output may be achieved if the relayed
oysters are removed shortly after being placed on the approved
lease(s). To the extent, however, that relaying activities provide
attachment material (i.e., the oyster shell and the living community
of oysters and the associated fauna of an oyster reef environment),
these activities can enhance the long-run productivity of a given
lease.

The quantity of oyster output from lease-based activities (Q,),
as specified in Eq. (I), is a function of quality. Environmental
factors, such as the salinity regime, can affect annual or even
long-run quality of a lease. The derived demand for relaying ac-
tivities as a function of lease quality is, to a large extent, unknown.
For example, low potential production on approved acreage in a
given year due to an unsuitable salinity regime may also indicate
that potential productivity of leased acreage in conditional or re-
stricted waters is also low. In this scenario, there would be little
benefits from relaying as there would be little product to relay.

A third implication, based on the calculation of net present
values as presented in Eq. ( 1 ), is that an increase in relaying costs
will result in a reduction in the derived demand for relaying ac-
tivities, ceteris paribus. Specifically, an increase in relaying costs
results in an increase in overall harvesting cost, denoted as C,(Q).
This is intuitive in that as relaying costs increase, it becomes less
likely that the lease holder will achieve a positive return on his
investment from relaying activities. While one generally thinks of
costs in terms of inputs to the production process (e.g., fuel, crew,
repairs, etc.), there are other costs to be considered. One of the
most relevant is that of opportunity costs which, from an economic
perspective, refers to the lost value in not pursuing the next best
alternative. One alternative to relaying is transplanting from public
seed grounds. If the availability of oysters (seed and market) on the
public grounds is high, the need to relay from polluted to approved
leases is diminished. As such, the demand for relaying, in theory,
should be related inversely to availability on the public seed
grounds, ceteris paribus.

Finally, the issue of overall market supply needs to be consid-
ered. After controlling for price and environmental factors, high
volume sales may suggest readily available markets as opposed to
limited markets whereby quotas may be imposed by individual
dealers on the fishermen. The high volume sales associated with
available markets may necessitate the need for alternative supply
sources by the leaseholders. If the leaseholders are not able to
secure the needed supply from their leases in approved waters or
from ihc public seed grounds, they may turn to relaying as one
method to secure the additional supply. This is of particular rel-
evance when environmental conditions limit availability on leases
in approved waters or on ihc public seed grounds.

Conceptual Model

Based on the above discussion, demand for oyster relaying
activities in Louisiana is specified as follows:

pera, = h,, -i- h, * price, + b, * cost, -I- b, * acres, + b.,

* avpub, I + bs * avpri, + b„ * ppub, + b^ * ppri, + u,

(2)

where pera, = permits issued per 1.000 acres of leased water
bottom in year t; price, = deflated Louisiana dockside oyster price
in year t (in $/lb of meat); cost, = discrete variable indicating
whether security agent is required (equal to 0 before 1988 and 1
thereafter); acres, = acres of water bottoms leased by the state for
the cultivation of oysters in year t (in thousands); avpub,,, =
indicator of market oyster availability on public oyster grounds in
year t - I; avpri, = indicator of market oyster availability on
leased grounds in year t; ppub, = production of oyster meats from
public grounds in year t (million pounds); ppri, = production of
oyster meats from private grounds in year t (million pounds); b,,.
b|. . . . b7 = parameters to be estimated; u, = error term.

As indicated, the endogenous varible, permits issued in year t,
is specified on a per acre leased basis (permits issued divided by
acres of water-bottoms leased for the purpose of oyster cultiva-
tion). The rationale for this is that the amount of leased acreage has
increased significantly during the period of analysis and if all other
factors are held constant, relaying activities would increase simply
because of the increased acreage. Dividing permits issued by acre-
age is therefore an attempt to preserve homogeneity.

The model, as given in Eq. (2), includes seven exogenous vari-
ables. The rationale for including the deflated price (price,) has
been established. Given that the demand for relaying activities is
hypothesized to be positively (negatively) related to an increase
(decrease) in price, the sign associated with b, is anticipated to be
positive (c*(pera,)/r/(price,) > 0).

Costs, as discussed above, are also considered to influence the
demand for relaying activities. Although a time-series database
pertaining to harvesting and relaying costs does not exist, one cost
factor in particular is hypothesized to influence the demand for
relaying activities. Specifically, beginning in 1988 a requirement
was enacted that a security agent be onboard the vessel when
relaying activities are occurring. This requirement is thought to
increase overall relaying costs significantly. To account for this
increased cost, a discrete variable (cost,) equal to 0 before 1988
and 1 thereafter was included in the model. Given that the demand
for relaying activities is hypothesized to be related negatively to
increased costs, the sign associated with b, is anticipated to be
negative.

Acreage under lease (acres,) is included in the model in an
attempt to account for the long-run change in quality of the aver-
age oyster lease. As previously noted, while the amount of leased
water-bottoms has increased substantially during the period of
analysis, the overall long-run oyster production has remained
stable, indicating a substantial decline in the average production
per acre. This reduction in per acre produclivity is hypothesized to
be due. in part, to less desirable water bottoms being leased over
time. Specifically, one would expect the more suitable oysler
growing water bottoms to be leased initially. Remaining water
bottoms subsequently leased are therefore of lower quality on av-
erage.^ Relaying of oysters shell and the living community of
oysters and associated fauna of an oyster reef community from
leases in conditional or restricted waters to leases in approved
waters is one method to enhance the long-run productivity of the
leases in approved waters. Hence, one would anticipate that

^While this is the case im average, there are certainly exceptions. Due to
WL'tiands erosion and subsidence, for example, many historically produc-
(i\c areas arc now likely lo be of little value in terms of iheir ability lo
produce signiticanl quantities of oysters.

Louisiana Oyster Relaying

443

c)(pera|)/f)(acres,) > 0. suggesting that the sign associated with b, is
positive.

Indicators of oyster availability on pubhc grounds in year t - 1
(avpub,_|) and privately leased acreage (avpri,) in year t were
included as explanatory \'ehicles in the analysis. The indicator of
availability on the public seed grounds included seed and market
oysters and was based on square-meter samples collected by the
Louisiana Department of Wildlife and Fisheries in August each
year. It is stated in terms of total estimated sacks available on the
public seed grounds (in millions).'" As estimated availability of
seed and market oyster on public seed grounds increases, in theory,
there should be less need to relay from conditional or re.stricted
water-bottoms to leases in approved waters.^ Hence, it is hypoth-
esized that the sign associated with the coefficient bj is negative.
The indicator of oyster availability on leased grounds was the
estimated amount of wetland acreage throughout coastal Louisiana
that had a salinity regime a 10 ppt in April of each year; expressed
in millions of acres. *"

Finally, variables representing annual production from public
grounds (ppub,) and private grounds (ppri,) were included in the
analysis. As previously discussed, high levels of sales, after con-
trolling for price and environmental factors, may suggest increased
marketing opportunities. High volume sales, in turn, may indicate
a need to secure additional sources of product from nontraditional
sources. One of these sources is that of relaying oysters from
polluted to approved waters. Hence, the expected signs associated
with both bft and b, are hypothesized to be positive, that is.
cJ(pera,)/rMppub,) and d(pera,)/c)(ppri,) > 0.

Statistical Considerations

The model developed in the previous section can be expressed
in matrix form as follows:

ifX,b4-U,>0 (3)

TABLE 2.

Summary statistics for Tobit analysis of Louisiana oyster relaying
activities, 1977-1995.

: X,b + U,

0

ifX.b-nU, <0

t= 1.

., N

where y, = dependent variable. X, = vector of independent vari-
ables, U, = error term assumed iid N(0.a").

The model specified is referred to as the Tobit model and is
used often in economic studies, particularly those of a cross-
sectional nature (see Amemiya 1984 for details). Given the speci-
fication, an assumption is implicitly made that an underlying sto-
chastic index equal to X,b + U, is observed only when strictly
positive. In other words, y, will be positive given a value of X,b -i-
U, greater than zero. Otherwise, y, will equal zero. For example,
consider 2 years having identical attributes with the exception of
output price (price,). Furthermore, assume relaying activities were
observed in the year when output price was higher, and in the year
where output price was lower, no relaying activities were ob-
served. This would imply that price in the year with the higher

''Only data for the public seed grounds east of the Mississippi River (i.e..
Breton Sound seed grounds) were used in the analysis.

'The indicator of a\ailabilily on public seed grounds was lagged by one
period in the analysis. This retlects the fact that the survey taken to
estimate availability is conducted in August of each year and harvesting
activities for either seed or sack production is permitted from about Sep-
tember through the following March. Hence, it is the estimate of avail-
ability from time period t - I that will influence relaying activities in time
period t.

*lt is generally believed that an extended salinity regime <5 ppt results in
high mortality rates. The data required to construct a variable using the 5
ppt criteria was not available for the current study.

Parameter

Regression

estimates

Asymptotic

Coefficient

Elasticity of

Variable

Pi

t-ratio

aE(pera,)/dXi

E(pera,)

price,(P,)

4.971

4.149

0.1164

3.156

cost,(p,)

-14.098

-5.204

-0.3301

—

acres,(P3)

0.097

4.738

0.0023

8.235

avpub,_|{|34)

-0.814

-2.429

-0.0I9I

-0.194

avpri|(P,)

0.487

0.906

0.0114

0.215

ppub,(Pj

0.753

2.467

0.0176

0.613

ppri,((37l

0.657

3.134

0.0154

1.607

constantiPi,!

-36.725

-5.316

-0.8600

—

price had exceeded that threshold level required to relay oysters
(i.e.. X, -H U, > 0), while price in the other year was below the
threshold that would be required to relay oysters (i.e., X, -I- U, < 0).
Factors such as those specified in Eq. (2) (i.e., those in the matrix
X,) likely influence relaying and thus the Tobit model is appro-
priate for the current analysis.

As shown by Greene ( 1981 ), ordinary least squares (OLS) es-
timates of Eq. (3) are both biased and inconsistent due to nonnor-
mality of the error terms. Thus, some estimation procedure other
than that of OLS must be used if unbiased or consistent parameter
estimates are to be obtained. Because several different estimation
procedures have been developed and used and should in all cases
provide the same parameter estimates assuming a unique maxi-
mum, the different estimation approaches are not considered here.
For purposes of analysis, the software package SHAZAM version
7.0 (White 1993) was employed.

The unconditional expected value of the dependent variable in
Eq. (3) was shown by Tobin (1958) to equal:

E(yl = XPF(Z) -I- CTf(Z) (4)

where: Z = Xp/o, f(Z) = unit normal density function, F(Z) =
cumulative normal distribution function.

The unconditional expected value of the dependent variable,
E(y). represents the expected value of the dependent variable as-
sociated with all observations. The change in the unconditional
expected value of the dependent variable with respect to a change
in any exogenous variable can be expressed as:

f)E(y)/aX, = F(Z)Pi (5)

This expression is equivalent to the parameter estimates generally
associated with OLS estimation.

EMPIRICAL RESULTS AND DISCUSSION

Results of the Tobit analysis related to Louisiana relaying ac-
tivities during the 1977-1995 period are presented in Table 2.** The
first column lists the variables used in the analysis. The Tobit
parameters associated with each of the exogenous variables are
given in the second column. The asymptotic t-values associated
with the parameter estimates are presented in the third column of
the table. The unconditional or total expected change in the de-
pendent variable (pera,) due to a change in the specified exogenous
variables is given in the fourth column. Finally, the unconditional
elasticities, which measure the expected change in the dependent
variable (pera,) with respect to a 1% change in any of the inde-

''Due to the inclusion of a lagged variable (avpub,.,) in the regression
model, the first year (i.e., 1976) is lost from the analysis.

444

Keithly et al.

pendent variables (evaluated at the means), are presented in the last
column of the table.'"

The parameter estimates associated with relaying activities ap-
pear satisfactory and reasonable based on two criteria. First, all
estimates, with the exception of the indicator of oyster availability
on private leases (avpri,), conform to theoretical expectations with
respect to parameter sign. Second, the asymptotic t-values of all
parameters with the exception of avpri, are significant at the 95%
confidence level." Given the fact that no previous research has
been conducted in this area, comparison of the magnitudes of the
current parameter estimates cannot be compared with results from
previous research to ascertain conformity across studies.

The results indicate that a one dollar increase in the deflated
dockside price of the harvested product in year t will result in an
increase of 0.1 16 permits issued on a per acre basis. At the mean
value of leased acreage during the period 1976-1995 (285,000
acres), this translates into a demand for an additional 33 permits
for every one dollar increase in the deflated price, ceteris paribus.
Similarly, a 10% increase (decrease) in the deflated price was
estimated to result in excess of a 30% increase (decrease) in the
demand for permits per acre of water-bottoms leased. While deri-
vation of an elasticity with respect to cost, is not valid due to the
discrete nature of the variable, the results suggest that the require-
ment of the Louisiana Department of Health and Hospitals that a
security agent be onboard vessel when relaying reduced signifi-
cantly the demand for relaying activities.

Increases (decreases) in the estimated availability of seed and
sack oysters on the public grounds in year t - 1 were found to
reduce (increase) significantly the demand for relaying activities in
the current year. Overall, a 10% increase in avpub,., was found to
result in a decrease in pera, of approximately 2%. At the mean
number of acres leased (258.000). a 1 million sack increase in
available supply (seed or market oysters) was estimated to result in
an overall reduction of five permits issued for relaying activities.
ceteris paribus.

"'An elasticity with respect to the variable cost, was not derived because of
its di.screte nature.

"While possibly biased due to the limit observations in the analysis, or-
dinary least st|uare tests also suggested that the model performed ad-
equately. Specifically, the adjusted R" equaled 0.9,1 and. based on the
Durbin Walsin test statistic, there was no indication of serial correlation.

The indicator of availability on private leases (avpri,) was not
found to significantly influence the demand for relaying activities.
There are at lea.st two possible explanations for this finding. First,
the indicator (i.e.. coastal acreage with a salinity regime in excess
of 10 ppt) may not fully reflect annual variation in productivity of
leases due to annual environmental changes. Second, the environ-
mental factors that determine productivity on leases in approved
waters may. in similar fashion, determine productivity of leases in
polluted waters. Hence, if availability is low on leases in approved
waters, it will also be low on leases in polluted waters and costs of
relaying would, in turn, be prohibitive.

The results suggest that a 1 million pound increase in market
oyster production from public seed grounds was found to result in
an increase of 0.018 permits per acre leased. This translates to an
increase demand for permits equal to 4.5 when evaluated at the
mean number of acres leased during the period of analysis
(258.000). With respect to production from leased acreage, the
increase demand for permits at the mean amount of acreage leased
equaled 4.0. This suggests that increases in production from either
the public grounds or the private grounds have approximately the
same impact on the demand for permits to relay. When examined
on the basis of elasticities, the analysis suggests that a 10% in-
crease in the market production from public grounds can be ex-
pected to result in approximately a 6.1% increase in the demand
for permits while a 10% increase in production from the private
grounds results in a 16% increase in demand for relaying activities.

Overall, the results support the hypothesis that econoinic and
environmental factors largely determine the demand for relaying
activities. While controlling environmental factors that influence
relaying activities is outside the control of management agencies,
the development of options to control economic factors, particu-
larly costs, may be feasible. Controlling costs will, under optimal
conditions, encourage increased relaying activities. Because the
process of oyster relaying can be a shortcut to the establishment of
oyster reef communities when compared to other restoration pro-
cesses, further examination of the issue is wananted.

ACKNOWLEDGMENTS

The research reported herein resulted from the Coastal Econo-
my Strategy Development Project, conducted by Louisiana Sea
Grant and partially funded by the U.S. Economic De\elopment
Administration. May 1998.

LITERATURE CITED

Amemiya. T. 1984. Tobit Models: a survey. / Econometrics 24:3-61.

Bell, F. W. 1978. Food from the Sea. We.stview Press, Boulder, Colorado.

Greene, W. H. 1 98 1 . On the asymptotic bias of the Ordinary Least Squares
Estimator of the Tobil Model. Economelricii 49:505-.') 1.1.

Layard. P. R. G. & A. A. Walters. 1978. Microeconomic Theory. McGraw-
Hill Book Company. New York. New York.

Lipka. D. A.. R. E. Pull. I.. Wise. F. C. Kopller & W Wlntson 1990. The
GuH'of Mexico Program, pp. 209-217. In: W. W. Wise (ed.). Proceed-
ings of the Twelfth Intenialional Conference of Ihe Coastal Society.
Our Coastal Experience: Assessing the Pa.st. Conlronling the Future.
The Coastal Society. Gloucester. Massachusetts.

Tohln. J. 1958. Estimation of relationships for liniilcd depeiidcnl \arKihles.
Lconomelrictt 26:24- .16.

Turner. R. E. & D. Cahoon (cds.l. 1988. Causes ol wetland loss in the
ccnlral coastal Gulf of Mexico. OCS Sludy MMS S7-()12(). Minerals
Management Service. New Orleans. Louisiana.

U.S. Department of Commerce (USDOC). 1997. The 1995 national shell-
llsh register of classified growing waters. Office of Ocean Resources

Conservation and Assessment. National Ocean Service. National Oce-
anic and Atmospheric Administration. Silver Spring. Maryland.

U.S. Environmental Protection Agency (USEPA). 1989. Marine and es-
luarine protection: programs and aclivilies. EPA-50.V9-89-()02. U.S.
Fn\ ironmenlal Protection .Agency. Office of Water. Washington. D.C.

U.S. Environmental Protection .-Xgcncy (USEPA). 1990. Progress in the
National Estuary Program: report to Congress. EPA 50.1/9-90-005. U.S.
Environmental Protection Agency. Washington. D.C.

U.S. Food and Drug Adminisiralion. 1995. National Shellfish Sanitation
Program Manual of Operations: Part 1, Sanitation of Shellfish Growing
Areas. Center for Food Safety and Applied Nutrition. Office of Sea-
food. Program and Enforcement Branch. Washington. D.C.

Van Sickle. V. R.. B. B. Barren. L. J. BarreU, L. J. Gulik & T. B. Ford.
1976. Baralaria Basin: salinity changes and oyster distribution. Loui-
siana State University Sea Grant Puhhcation No. I.SU-T-76-()2. Loui-
siana Slate University. BaUm Rouge. Louisiana.

White. K.J. 199.1. SHAZAM User's Reference Manual Version 7.0.
McGraw-Hill Book Company. New York. New York.

Journal of Shclltlsh Kcsconh. Vol. 19, No. I. 44.'i-447. 2()()().

COMMUNITY INVOLVEMENT IN PROJECTS TO REDUCE NONPOINT SOURCE POLLUTION

ODETE DA SILVA PINHO

Coiiio.x Valley Project Watershed Society

Box 3007 Courtenay

British Columbia. V9N 5N3 Canada

ABSTRACT Baynes Sound, located on the east coast of Vancouver Island in western Canada, is one of the prime shellfish culture
areas of British Columbia. In 1994 the Baynes Sound Stewardship Action Group, a multistakeholder group, was formed to address and
to remediate nonpoint sources of bacteriological pollution threatening the economic and environmental health of the sound. Reme-
diation action projects for urban stormwater. malfunctioning on-site septic systems, and agricultural runoff have successfully involved
hundreds of community volunteers and dozens of financial supporters in simple and effective pollution reduction activities. The
communily-based "hot spots'" projects have engaged and educated citizens in actions to improve water quality, in ways government
authorities alone could not have done. This work demonstrates that partnerships among government, the shellfish industry, community
groups, and citizens can create a powerful means for improving water quality. The projects also show that citizens are capable of
creating positive environmental changes in their communities through monitoring, education, and remediation programs. Citizen
volunteers can be dedicated and passionate agents of change when provided with the opportunity to participate in creating healthier
communities that benefit themselves and a clean-vvater-dependent industry, such as shellfish culture.

A'£}' WORDS: nonpoint source pollution, citizen participation, bacteriological pollution, urban stormwater, agricultural runoff,
malfunctioning on-site septic systems

INTRODUCTION

Involving citizens in protecting water resources is recognized
as being a powerful tool for protecting water quality (Broadhurst
1996). Throughout Canada and the United States, funding agencies
have increasingly recognized the importance of community-based
projects and the value of environmental stewardship. This paper
outlines the actions that one western Canadian community has
taken to protect their local water resources from nonpoint source
pollution.

Baynes Sound, located on the east coast of Vancouver Island in
western Canada, is a prime shellfish culture area, producing AO'^/r
of all shell stock in the province of British Columbia (Comox
Valley Economic Development Society 1998). The shellfish in-
dustry of Baynes Sound comprises 520 ha of inteilidal area with
1 15 shellfish growing leases. This clean-water-dependent industry
has a current wholesale value of $6 million (CDN) primarily in
Pacific oysters. Crassostreci gigas. and Manila clams. Tapes phil-
ippinanim. The geoduck clam, Panope abrupta. harvest, which is
still in its infancy, generated $12 million revenue in 1997 for
license holders in the region (Comox Valley Economic Develop-
ment Society 1998).

Water quality deterioration of Baynes Sound was first identi-
fied in 1994 by Environment Canada during their Canadian Shell-
fish Sanitation Program (CSSP) survey. This decline in marine
water quality, attributed to nonpoint source pollution, resulted in
closure of 20'7r of the shellfish harvesting area in 1 994 and has had
a direct impact on the shellfish industry.

The Baynes Sound Round Table and the Baynes Sound Stew-
ardship Action Group (BSSAG) formed in 1994 to address and to
remediate nonpoint sources of bacteriological pollution threaten-
ing the economic and environmental health of the sound. BSSAG
is a multistakeholder group with representatives from four gov-
ernment agencies, the shellfish industry, and three community
groups. The group has collectively designed pollution reduction
projects for urban storinwater. malfunctioning on-site septic sys-
tems, and agricultural runoff, which have successfully involved

dozens of financial supporters and hundreds of community volun-
teers in simple and effective pollution control activities.

The partnerships formed among government, industry, commu-
nity groups, and citizens have created an action-focused group
capable of garnering sufficient financial support for the monitor-
ing, education, and remediation work. The representatives from the
multistakeholder group bring their knowledge and resources to
further their collective goal of clean water.

The initial inspiration and ideas for this community-based pol-
lution reduction approach came from activities undertaken by the
Washington Sea Grant Program and the Paget Sound Water Qual-
ity Authority in Puget Sound. Washington state. USA. The projects
undertaken in the Comox Valley were based on their work but
were redesigned to fit the needs and characteristics of the local
community (Pinho 1998b).

The community projects described in this paper summarize the
activities of approximately 200 local citizen volunteers involved in
pollution monitoring, education, and remediation activities. These
citizens have become informal teachers to their neighbors, cowork-
ers and families, encouraging people to see that Baynes Sound
belongs to all residents, who share a collective responsibility for
water quality. Each of the programs aimed at reducing nonpoint
source pollution included educating people about their personal
actions, their impacts on water quality, and the effects of shellfish
contamination. Citizens were given the "tools" to become better
stewards and to educate others that water quality in the Baynes
Sound is a community responsibility (Pinho 1998b).

The community-based projects were designed to address pol-
lution from stormwater discharges, failing on-site sewage systems,
and agricultural runoff Each of the programs involved citizens in
the field activities and also in decision-making for designing the
programs and their future directions.

URBAN STORMWATER MONITORING AND REPAIR OF
SEWAGE CROSS CONNECTIONS

The stormwater-monitoring project, managed by the Comox
Valley Project Watershed Society, involved citizens in identifying

445

446

PiNHO

pollution sources, prioritizing tliem. and reporting the results to the
two local governments. In 1996, forty community volunteers were
trained to monitor bacteriological water quality. They monitored
60 storm drains once per month for a 6-month period, collecting a
total of 381 fecal coliform samples (Pinho 1996). Of the 60 storm
drains monitored, 16 drains posed a high risk to shellfish or human
health (greater than 1000-5000 coliform fecal units/100 mL). As a
result of this intensive data collection and sharing, the City of
Courtenay and Town of Comox became aware that there were
sanitary sewer-storm drain cross connections.

A sanitary sewer-storm drain cross connection occurs when a
domestic sanitary sewer service is mistakenly connected to the
storm drain rather than to the sanitary sewer, thus carrying un-
treated household domestic sewage down the storm drain. This
plumbing error was completely unknown to the two municipalities
prior to stormwater testing by citizen volunteers. Over 70 sanitary
sewer-storm drain cross connections were repaired between 1996
and 1998, and repairs are continuing. The City of Courtenay alone
has spent $1 10,000 (CDN) on cross connection repairs from 1996
to 1998. In 1996, the two municipalities also purchased smoke-
testing equipment, an essential tool for locating the specific lines
in need of cross connection repairs (Pinho 1998c).

Figure I illustrates the dramatic difference between fecal
coliform outputs prior to and after cross connection repairs. Fecal
counts from this one storm drain, which discharges directly into
north Baynes Sound, now average 60 CFU/100 mL versus previ-
ous counts as high as 3 300 000 CFU/100 mL. Seven cross con-
nections were repaired at this site in 1996 and two repairs were
made in 1998 (Pinho 1996). These cross connection plumbing
errors were located in a residential complex constructed in the
1950s, and this source of fecal pollution continued unnoticed for
46 years. This pollution source was discovered and acted upon
only after community volunteers donated their time to water qual-
ity monitoring. During this 6-month monitoring period, citizen
volunteers collectively donated 720 hours to testing storm drains.

ON-SITE SEWAGE SYSTEM CARE AND MAINTENANCE
EDUCATION PROGRAMS

Sixty-five on-site septic system inspections were performed
randomly throughout the Baynes Sound area in 1996 (on systems
volunteered for inspection). In this crude study, undertaken with
the assistance of a septic company, sy.stems fell into one of four
categories: 17'/f were passing and functioning well; 42'7f were
displaying evidence of limitations; 23% were pre-failing, and 8'/f
were absolutely failing systems (Drake 1997). The study results
indicated a need for improved education, routine maintenance,
and. in some cases, complete replacements of on-site septic sys-
tems in the region.

Proper septic care and maintenance education campaign work-
shops, called septic socials, and community septic tank pump-outs
were conducted in 1996 and 1997. The Comox Valley Citizens
Action for Recycling and ihe linvironmenl (CAREl managed these
efforts, which included printing approximately 1000 information
kits for distribution (Drake 1997). LIghty community volunteers
were Involved in distributing the educational Information kits to
Iheir neighbors throughout the region, and ln\ lling ihem to a septic
social and oyster barbecue at a nearby resident's home

The septic socials have proven to be very popular and enter-
taining workshops, and have provided training to 1 13 rural resi-

dents on the proper functioning, ongoing maintenance, and trouble
shooting of septic systems. The workshop involves a slide presen-
tation, a video presentation, and an actual inspection of a local
septic system, by a septic pumping and maintenance company.
Neighbors living In rural areas often live great distances from one
another, thus making this popular event an attractive opportunity
for neighborhood socializing.

An Interactive full-size model of a septic system was con-
structed for the program to further educate and involve citizens in
understanding the proper functioning of septic systems. The sys-
tem is made largely of a PVC pipe frame with ripstop nylon covers
illustrating the general concept of a distribution box, septic tank,
and distribution pipes. The model is set up at community events
and parents are encouraged to "flush" their children down the play
toilet and have them crawl through the distribution box, into the
septic tank, and then "flow" out one of the three distribution pipes.
This has become a hugely popular and fun educational tool for
approaching rural residents on this unfamiliar subject.

ADDRESSING AGRICULTURAL RUNOFF THROUGH
STREAMSIDE FENCING AND VEGETATION

Between 1997 and 1998, an agricultural program aimed at pol-
lution prevention and fish habitat protection contacted 200 farm-
ers. This landowner contact program was managed by the Comox
Valley Project Watershed Society. In 1998, hobby and commercial
farmers were offered up to $700 to assist them with remediation
activities, resulting in the installation of 18,882 feet of streamside
fencing to limit livestock access to streams on 17 farms. In addi-
tion. 6000 trees and shrubs were planted on 26 farms, covering
over 7500 feet of riparian area (Pinho 1998a). The financial in-
centive in the program proved to be an essential element for the
success of this project; farmers were given an economic rationale
for envlronnienlal protection In this agricultural valley ribboned
with salmon streams that flow into shellfish-growing areas down-
stream.

The projects emphasized protecting streambanks and develop-
ing partnerships with farmers to assist them with stream protection
projects on their properties. The trust and partnerships de\eloped
among farmers, the 88 citizen volunteers, and the nonprofit soci-
ety, were also a significant accomplishment for Ihe long-Ierm pro-
tection of water quality and fish habitat In this agricultural \alley
In the Baynes Sound watershed.

The program Involved the farming community In the design
and delivery of the program. The project was guided by a volunteer
advisory committee and farmers were visited by the program co-
ordinator and a retired farmer. The Insight and credibility gained
from Involving Ihe agricultural conniiunlly In the program man-
agement was an essenllal component for farmer participation In
this project.

THE OVERALL IMPROVEMENTS IN WATER QUALITY

ll IS difiicull lo sunnnan/c quanlllalncly Ihe overall effective-
ness of these land-based vvaler quality projects on marine water
quality. Due to funding constraints, most of Ihe projects, with the
exception of the stormwater-monitoring program, lacked detailed
spatial and temporal monitoring to assist In any proper assessment.

Community Projects Reduce NPS Pollution

447

However, monitoring was conducted at five stations every two
weeks in north Baynes Sound. Comox Harbour. The marine water
quality monitoring for fecal cohform was conducted from 1993 to
1997 by trained Coast Guard Auxiliary volunteers. The results of
the marine monitoring effort indicate an improving trend in bac-
teriological water quality. From 1995 to 1997. the frequency and
magnitude of fecal coliform counts greater than 43 MPN/100 mL
decreased at all five stations in Comox Harbour. However, these
lower levels were also coincident with lower rainfall levels expe-
rienced in the second year of monitoring. Additional monitoring is
required to provide any definitive conclusions on marine water
quality improvements in the region (Pinho 1998d).

CONCLUSIONS

The clean-water-dependent shellfish industry can benefit from
forming alliances with government, community groups, and citi-
zens to reduce nonpoint sources of pollution. Community volun-
teers can be capable of creating tangible improvements in water
quality through monitoring, education, and restoration projects.
Citizens involved in these water quality projects often become
informal teachers to their neighbors, coworkers, and families, en-
couraging people to see that the community shares a collective
responsibility for water quality and for protecting clean-water-
dependent industries, like shellfish culture.

LITERATURE CITED

Broadhurst. G. 19%. Promoting stewardship of Puget Sound: more PIE
success stories. Prepared for the Puget Sound Water Quality Authority.
Olympia. Washington. USA.

Comox Valley Economic Development Society. 1998. Comox Valley com-
munity profile. Comox Valley Economic Development Society.
Counenay. British Columbia. Canada.

Drake. R. 1997. Baynes Sound "hot spots" remediation project; on-site
septic care and maintenance program final report. Prepared for Citizens
Action for Recycling and the Enxironment. Courtenay. British Colum-
bia. Canada.

Pinho. O. 1996. Baynes Sound "hot spots" remediation project: stormwater
monitoring results for Royston. Town of Comox. City of Courtenay
and Regional District of Comox-Strathcona. Prepared for Comox Val-
ley Project Watershed Society. Courtenay. British Columbia. Canada.

Pinho. O. 1998a. Comox Valley agricultural program 1998 final report.
Prepared for Comox Valley Project Watershed Society. Courtenay.
British Columbia. Canada.

Pinho. O. 1998b. Non-point source pollution: community based remedia-
tion project — the Comox Valley experience. Prepared for Comox Val-
ley Project Watershed Society. Courtenay. British Columbia. Canada.

Pinho, O. 1998c. Stormwater quality in the Comox Valley: results of
monitoring programs in 1996 and 1998. Prepared for Comox Valley
Project Watershed Society. Courtenay. British Columbia. Canada.

Pinho. O. 1998d. Water quality in the Comox Harbor: results of a moni-
toring program to investigate bacterial contamination. Prepared for
Comox Valley Project Watershed Society. Courtenay. British Colum-
bia, Canada.

J

Journal of Shellfish Rcsconh. Vol. 19. No. 1. 44'-)^.'i4. 2000.

MAINTAINING THE SHELLFISH NICHE IN THE NEW MILLENNIUM: THE PACIFIC COAST

SHELLFISH GROWERS APPROACH

WILLIAM F. DEWEY

Taylor Shellfish Company, Inc.
130 SE Lynch Road
Shelton. Washington 98584

ABSTRACT Intense population growth, associated nonpoint source pollution, competing uses of the estuaries (e.g.. pristine views,
recreation) and increased regulatory scrutiny associated with the Endangered Species Act and the Sustainable Fisheries Act are
jeopardizing the survival of West Coast shellfish farmers. The Pacific Coast Shellfish Growers Association (PCSGA) has recognized
that to maintain a niche for shellfish amongst all these competing interests, growers need to be proactive on several fronts. PCSGA
members are encouraged to get involved with local watershed planning committees, growth management planning. Soil Conservation
Districts, environmental groups, and other watershed stakeholder groups, and to educate others on the value of shellfish as part of a
healthy ecosystem. Shellfish farmers are also being encouraged to "walk the talk." It is no longer adequate to go to hearings, profess
shellfish as the "canaries in the mineshaft," and ask everyone upstream to fix the problems. Shellfish growers are being asked to review
and improve their overall environmental performance. PCSGA is embarking on an effort to develop a coastwide Environmental Code
of Practice (Best Management Practices) for shellfish farming. PCSGA is also exploring the development of the Environmental Code
of Practice into a programmatic Habitat Conservation Plan to provide regulatory stability under the Endangered Species Act and
impending salmonid listings. The Pacific Shellfish Institute (PSD, the research arm of PCSGA, is pursuing funding for shellfish
ecosystem research as a high priority. Being proactive and establishing the industry as the most responsible user group in the ecosystem
will assure that shellfish farming survives into the new millennium.

KEY WORDS: Environmental management system, environmental policy, environmental code of practice, ecosystem, eelgrass

INTRODUCTION

The Pacific Coast Shellfish Growers Association (PCSGA) is
the predominant shellfish industry organization of the United
States West Coast. PCSGA represents oyster, Manila clam, mus-
sel, and geoduck farmers. The Association, formed originally in
1930 for the annual procurement of Pacific oyster seed from Japan,
currently deals with a variety of environmental, water quality,
health, and regulatory issues coastwide.

The West Coast shellfish industry began in the late 1800s with
wild harvest of the delicate native Olympia oyster, Ostreota con-
chaphila, in Washington, Oregon, and California. Harvest pressure
and pollution forced a transition to hardier, more prolific Pacific
oysters, Crassoslrea gigas. beginning in 1921. While Pacific oys-
ters grew well on the West Coast, natural reproduction was lim-
ited, requiring growers to obtain seed annually from Japan. Piggy
backing in the cases of seed from Japan was the Manila clam.
Tapes philippinanim. It adapted well to the Pacific Northwest
climate, reproducing naturally in a number of bays and estuaries
throughout Washington and British Columbia. Canada. It has been
a focus of cultivation efforts since the 1960's. Blue mussels (Myii-
his trossulus and Mytilus galloprovincialis) have also been cul-
tured on a limited but increasing basis since the mid 1980's.
mainly in Washington. Although the Pacific oyster is the predomi-
nant species cultured, a variety of other oysters (Crassoslrea vir-
ginica, Crassoslrea sikamea, Ostrea ediilis. and Ostreola con-
chaphila). are cultured for the half-shell market.

Washington State is the dominant shellfish producer on the
West Coast and, consequently, home to PCSGA. The relative pro-
duction of cultured shellfish is represented in Figure 1. Production
estimates for the various species, compiled by the Pacific Shellfish
Institute (PSI). are presented in Table 1.

The past two decades have brought a number of significant
regulatory and environmental issues to bear on the shellfish indus-
try. In 1988, the PCSGA recognized it was loosing ground in a

variety of arenas. Shellfish growing waters were being down-
graded at an alarming rate. Population growth and shifting demo-
graphics were taking their toll both from a standpoint of pollution
and competing uses displacing shellfish farms. Growers made a
decision to expand from an office in Coast Oyster Company's
plant in Seattle, Washington, with a 1/3 time volunteer, to a ded-
icated office in Olympia. the Washington State capitol. with a
fulltime lobbyist-executive director. Since the expansion, the or-
ganization has been lobbying actively on environmental and regu-
latory issues, educating the government and the public about the
benefits of shellfish farming, partnering with the environmental
community, and promoting strong ci'edible working relationships
with regulatory agencies.

HUMAN IMPACTS

Socio-Political Changes

There has been tremendous population growth in the coastal
areas and as a result shifts in usage in many of the rural areas
traditionally farmed for shellfish. With a general decline in wild
fisheries and timber-related jobs, many of the coastal areas are
shifting from natural-resource-based economies to service-based
and high technology economies. Loggers and fishermen are mov-
ing out and retirees and service workers are moving in. These
population increases and shifts in the employment base bring a
variety of environmental and sociopolitical pressures. Newcomers
to the rural watersheds tend to be less accepting of the traditional
working waterfront than are their predecessors. These newcomers
are demanding that forests, riparian areas, beaches, and coastal and
wildlife habitats be preserved and enhanced (Huppert et al. 1998).
The increasing high technology, white collar work force places a
greater importance on protecting natural amenities and preserving
opportunities for outdoor recreational experiences. These newcom-
ers have a higher level of environmental awareness and tend to be
less accepting of unsightly aquaculture operations.

449

430

Dewey

Oregon
California

Hawaii

Mexico \_ \

Relative Production

High
Medium

Figure 1. Relative cultured shellflsh production on the West Coast of
North America.

The watert'roni properties adjoining shellfish beds are being
developed with affluent homes. These new homes are driving up
property values along the waterfront, resulting in higher property
taxes. These higher taxes are impacting aquaculture operations,
which are dependent on waterfront property for facilities, and are
also forcing longtime residents to sell and move inland.

Increased Pollution

The increased number of people means increasing nonpoini
pollution. Homes buill in these rural areas typically use on-site

TABLE 1.

Whole wet weight and value of production for shellfish produced on
the West Coast, ISA, in IWX |I'CS<;A|.

.Species

Production
(million Ihs.)

Production
(metric tons)

\alue
(million U.S. $)

Oysters (all forms)

88.6

4().2()()

55.0

Manila clams

7.0

3170

21.0

Mussels

1.5

680

2.5

sewage systems. If local governments do not have effective edu-
cation, operation, and maintenance programs in place, the on-site
systems are prone to failure, which results in shellfish growing
area downgrades. Increased development also brings with it a
higher percentage of impermeable surface and increased storm-
water runoff. With this storm-water runoff comes untreated do-
mestic animal waste, lawn fertilizers, pesticides, and polyaromatic
hydrocarbons (PAHs).

Competing Uses

More people mean more competition among users, not only for
pristine views but also for water skiing, windsurfing, personal
watercraft. fishing, and pleasure boating. The increased recre-
ational use comes with spatial conflicts and results in increased
pollution from boats without sewage holding tanks, inadequate
boater pump-outs for those that do. and insufficient shoreside toilet
facilities.

REGULATORY IMPACTS

Endangered Species Act

The Endangered Species Act (ESA) potentially may have a
very significant effect on the shellfish aquaculture industry. De-
clining wild fisheries, and in particular the declining salmon fish-
ery, are drawing significant attention to habitat concerns from
natural resource managers. In Washington State, both Puget Sound
Chinook salmon and Hood Canal summer chum salmon were
listed in March 1999 as threatened under the Endangered Species
Act. Other coastal salmon runs are already listed or have listings
pending. Section 9 of the ESA prohibits "take" of threatened or
endangered species. A "take" not only constitutes killing the spe-
cies but includes any activity that hurts or harms any aspect of the
species' lifecycle, including damaging critical habitat. Critical
habitat is not only habitat that is critical to the juvenile salmon, but
includes habitat that is critical to the salmonid prey species (e.g..
eelgrass is critical for herring to spawn on, herring is a critical
salmonid prey species, therefore eelgrass is a critical habitat).

The potential impact of the ESA is related to the anadromous
life cycle of salmon. Depending on the species, out-migrating ju-
veniles can spend several days to weeks utilizing the intertidal
areas. These young fish will migrate in and out with the tide,
staying in shallow water to minimize their exposure to predators
while feeding on abundant marine invertebrates. This same inter-
tidal area is used for shellfish culture and, generally speaking,
(here is a limited understanding regarding use o( the marine estu-
ary by juvenile salmonid.

Shellflsh growers conduct a variety of activities in the day-to-
day operations of their farms, which could impact salmonid critical
habitat during the out-migration of juveniles. Many of these ac-
tivities have been practiced for decades and. in some cases, are
"grandfathered" by state or federal agencies as acceptable. All
activities, even these grandfathered, are now falling under a new
level of regulatory scrutiny with listings of salmon as threatened
and endangered species. Individuals found guilty of a "take" under
ESA are subject to fines and/or prison.

Shellfish farming activities that may be coming under new or
more intense review include:

Inteniclai off-holtom rack and hat; or Umfiline culture for oys-
ters. The structures used in this type of culture have the potential
to impact eelgrass and other submerged aquatic vegetation (SAV)
through shading effects. Depending on the site and the orientation.

Shellfish in the New Millennium

451

the structures may cause suspended silt to settle out. modifying the
bottom substrate, and thus impacting salmonid prey species. The
structures, depending on their orientation, have the potential to
lead juveniles following the shoreline to deeper water and greater
predator exposure.

Traditional bottom culture for oysters. Bed preparation, crop
management, and harvest vary from farm to farm.

Clean-up dredging. Often thick beds are hand-harvested, and
dredged prior to planting to clean up the remaining oysters.

Burrowing shrimp control. Over the last 30 years, several West
Coast estuaries have experienced dramatic increases in populations
of burrowing shrimp (Upogehia pugetlensis and Callianassa cali-
forniensis). These shrimp make oyster beds unstable, causing
crops to sink and perish. Considerable effort has been expended by
the industry since the early 1960s to research methods to control
burrowing shrimp. In the early 1960s, the Washington Department
of Fisheries (WDF). now the Washington Department of Fish and
Wildlife (WDFW). working with other public agencies and the
growers, found carbaryl (Sevin®) to be an effective, safe control
for burrowing shrimp (Final Environmental Impact Statement -
FEIS. WDF 1985. and Supplemental Environmental Impact State-
ment - SEIS, WDF 1992.) Its use and effects have received con-
tinuing review and monitoring over the past 25 years, culminating
in the preparation of the WDF's FEIS and SEIS. Concurrently,
growers are in the process of developing an integrated pest man-
agement program for control of the burrowing shrimp. Control
involves spraying carbaryl on beds during a single summer ex-
treme low tide series. The application is strictly regulated and
limited to 600 acres in Willapa Bay and 200 acres in Grays Harbor
annually. Growers consolidate areas to be treated into large con-
tiguous blocks of land, minimizing recolonization from adjacent
infected beds. This method of control is only available to Wash-
ington growers. The marine application of carbaryl is not legal in
Oregon or California. In these states, due to lack of effective
controls, the burrowing shrimp have taken thousands of acres out
of oyster production.

Burrowing shrimp control is a complex and controversial issue.
While the shrimp population increase is generally acknowledged
by all, the magnitude has not been documented. Various theories
have been offered for the phenomenon, including fewer low sa-
linity events in the estuaries because of climate changes and/or
damming of the Columbia River. The shrimp are not tolerant of
low salinities. Another possible factor is that the populations of
salmon, which prey on larval stages of burrowing shrimp, and of
sturgeon, which are uniquely suited to prey on adult burrowing
shrimp, are severely depressed. Regardless of the cause, the bur-
rowing shrimp have effectively turned thousands of acres of di-
verse, productive oyster and eelgrass habitat into desolate, largely
monospecifc mudflats. The areas treated with carbaryl stabilize are
seeded with oysters, and often are recolonized by eelgrass within
the following year, providing a diverse habitat for a variety of
invertebrates {Dumbauld and Wyllie Echeverria in press). Carbaryl
treatments are generally required every 5 to 6 years to keep the
ground in production.

Rotolilling, mowing and harrowing. Some growers rototil beds
prior to re-seeding to control oyster drills and SAV. Some growers
also "mow" eelgrass growing on bottom culture oyster beds by
towing "V" bars behind workboats. Controlling SAV prior to
planting and during maturation improves growth and meat yield.
Eelgrass and other SAV reduce water circulation and consequently
the amount of food available to the oysters.

Seeding. Seeding involves substrate modification. "Mother"
shells, with young oysters attached, are spread on the bottom.

Harrowing. In areas where oysters are prone to sinking or
burying, growers tow spring harrows across beds to pull the oys-
ters back to the surface.

Mechanical drag and hydraulic dredge hatxesting of oysters.
Oysters up and down the West Coast have been harvested for years
with traditional drag dredges and to a lesser extent with (Hanks)
hydraulic harvesters. Both of these methods disturb the substrate
and eelgrass (or SAV) when present.

Clean-up dredging, rototilling. harrowing, and controlling SAV
all have implications for juvenile salmon and their prey species
depending on the time of year the activity is performed.

Substrate modification for clam culture. A number of Manila
clam farmers in Puget Sound, and increasingly in Willapa Bay, add
gravel to muddy substrate to improve clam productivity. Thorn et
al. (1992) determined there was an increase of certain salmonid
prey species associated with the gravel substrate. However WDFW
biologists are concerned the graveling activity can be disruptive to
juvenile salmon during their out-migration between mid March
and mid June. State biologists fear the graveling activity, which
occurs at high tide, could scare juvenile salmon to deeper water
where they are more susceptible to predation. or that important
salmonid copepod prey species would be buried. Growers cur-
rently curtail graveling during the salmonid out-migration period.

The Sustainable Fisheries Act

The Sustainable Fisheries Act. which was passed by Congress
in 1996 and amended the Magnusen-Stevens Fisheries Conserva-
tion and Management Act. requires the regional fishery manage-
ment councils to identify essential fish habitat (EFH) for the spe-
cies they manage in the exclusive economic zone (EEZ), typically
3 to 200 miles offshore. The councils were further directed by the
Act to develop conservation and enhancement recommendations to
protect EFH. The Pacific Fishery Management Council has devel-
oped EFH documents for salmonids, as well as for pelagic and
demersal fish. The original draft of the salmonid EFH document
was very controversial because of its misrepresentation of aqua-
culture impacts on salmon EFH, and proposed conservation and
enhancement measures that would have impacted shellfish culture
practices severely. Subsequent drafts have been modified to reflect
potential impacts more accurately.

The shellfish industry's concern with both the ESA and the
Sustainable Fisheries Act is that they trigger a federal consultation
with the NOAA National Marine Fisheries Service (NMFS) if the
activity requires a federal permit or if the company receives any
federal funding. Historically, the majority of shellfish farming ac-
tivities in the Pacific Northwest have fallen under a programmatic
U.S. Army Corp of Engineers (USCOE) Nationwide Permit 4.
Because it is a federal permit, the consultation process is triggered
under both Acts. Growers now find themselves burdened by a
whole new layer of regulatory bureaucracy by having to negotiate
with NMFS on farming practices.

U.S. Army Corp Of Engineers Nationwide Permit 4 (NWP 4)

Recent changes in the NWP 4 permit are impacting shellfish
culture operations in Oregon and California. Prior to 1991, the
USCOE showed little interest in regulating shellfish culture ac-
tivities; the NWP 4 permit covered oyster and clam digging. In
1991. new language was added to the NWP 4 that specified that
oyster seeding was covered by the permit, provided it did not occur

452

Dewey

in wetlands or vegetated shallows. In 1996, additional language
was added clarifying that oyster seeding was covered by the permit
so long as it did not occur in wetlands, in sites that support SAV,
or in sites that have historically supported SAV even though it may
not be present in that given year. Also, covered oyster trays and
clam racks were singled out as no longer being covered by the
NWP4.

As a consequence of these NWP 4 changes, a number of shell-
fish culture operations will be required to get individual USCOE
Section 10/404 permits. In Humbolt Bay, California, a large shell-
fish operator has been negotiating for an individual USCOE permit
for 2 years, with the result that substantial changes to their historic
farming practices are being required. Similarly, the Oregon De-
partment of Agriculture is no longer allowing oyster leases in areas
with SAV.

RESPONDING TO THE CHALLENGE

"Walking the Talk"

Recognizing the challenge posed by regulatory and environ-
mental pressures, the PCSGA Board of Trustees is challenging
growers to "walk the talk," The shellfish industry has long been
recognized for its water quality advocacy efforts. Growers are
regularly on the front line demanding clean water to grow their
shellfish. If growers are going to demand that of everyone else,
they should be willing to set a good example. Not only do growers
need to be aware of their own potential impacts to water quality,
they also need to understand where their farming operations are
having negative environmental impacts and, where practical, be
willing to mitigate those impacts. Conversely, where culture ac-
tivities are providing a beneficial effect on the ecosystem, those
activities should be recognized and promoted.

Gel Involved

In addition to having the Executive Director dedicated to the
issues, growers are encouraged to get involved locally in shoreline
and growth management planning and also with watershed com-
mittees. Soil Conservation districts, and local environmental orga-
nizations having similar goals. Growers are also encouraged to be
active in their communities. They are encouraged to sponsor spoils
teams, donate product to local fundraising events, adopt sections of
highway or beaches for litter patrols, sponsor portable toilets in
areas with high recreational use and the potential to impact grow-
ing waters, give farm tours, and give guest lectures at the local
high schools and community colleges.

Promotion and Education

Besides these community activities, the industry participates in
a variety of promotional and educational events over the course of
the year. PCSGA sets up a raw bar in the Capitol Rotunda in
Olympia for an afternoon during the legislative session lo ihank
legislators and lobbyists for their continued support of water qual-
ity initiatives. Growers served free oysters to passers by in a court-
yard adjacent to the Brooklyn Cafe and Oyster Bar in downtown
Seattle on Oyster Appreciation Day. Anthony's Restaurants" Oys-
ter Olympics and Elliot's Oyster House's Oyster New Years are
two large annual promotions that raise money for and the public's
awareness of clean water issues. Oysterfest, an annual festival in
Shelton, Washington, draws over 2(),()()() people over 2 days and
provides education on the benefits of shellfish and the need for

clean water. The festival includes the West Coast shucking cham-
pionship competition. The winner is sent to Maryland to compete
in the national championship.

Environmental Management System (EMS)

With increasing regulatory scrutiny bearing down on the in-
dustry, the PCSGA recognized it would need a tool to encourage
growers to "walk the talk." Patterned on the International Organi-
zation of Standards ISO 14.000 program, the PCSGA is develop-
ing an Environmental Management System. An Environmental
Management System includes an Environmental Policy and an
Environmental Code of Practice (ECOP) to implement the Envi-
ronmental Policy. The ECOP equates to Best Management Prac-
tices (BMPs).

PCSGA growers first explored the concept of BMPs in the
spring of 1997 with grant money from People for Puget Sound and
the help of three students from the University of Washington's
Environmental Management Program. Working with several
growers, the students drafted lifecycle BMPs for the shellfish in-
dustry. At the end of the student's 3-month effort they had devel-
oped a product the PCSGA Board adopted as a draft (Ahlers et al.
1997). The Board committed to refining the BMPs to a document
the whole West Coast industry could adopt and implement. An
important goal of the University of Washington's Environmental
Management Program, which encouraged industry support for the
process, was to make companies more profitable, while at the same
time, more environmentally responsible.

Since the completion of the draft by the students, PCSGA has
been educating member growers about the importance of the
ECOP. PCSGA has been coordinating with the British Columbia
Shellfish Growers who are also interested in developing an ECOP.
Paul Lupi, the executive director of the New Zealand Mussel In-
dustry Council, was invited to speak at a Sea Grant workshop
regarding their recently completed 3-year effort to develop the first
shellfish En\ironmental Management System in the world.

The PCSGA Board recognizes that for growers to embrace and
adopt an ECOP they needed to be involved in its development. The
Pacific Shellfish Institute (PSD, the research arm of the PCSGA.
has submitted proposals to the USDA Sustainable .Agricultural
Research Education and National Research Institute for funding to
facilitate development of the ECOP along the West Coast. The
goal is to include growers from all West Coast states, representing
all cultured shellfish species and all culture techniques, in a bot-
tom-up, nniltistakeholder process. Growers have approached
NMFS and the Sea Grant Marine Advisory Program seeking their
support in the effort. The Sea Grant Marine Advisory programs in
Washington, Oregon, California, and Alaska have indicated an
interest in assisting with facilitation of the process. A proposal has
also been submitted to the PEW Charitable Trust Foundation for
funding to support implementation of the ECOP.

Regulatory Stability

Natural resource industries lack regulatory stability in today's
environmental climate. The rules governing business operations
change frequently. This is true whether one is growing animals,
row crops, trees, or shellfish. One of the few tools available for
private landowners to achieve regulatory stability is the habitat
conservation plan (HCP) process under section 10 of the Endan-
gered Species Act. Growers have been discussing with NMFS the
possibility of developing the ECOP into an umbrella HCP. This

Shellfish in the New Millennium

453

could serve as a template for growers. Indixidual growers could
prepare customized farm plans using the umbrella HCP as a tem-
plate, and receive a "certificate of inclusion" under the umbrella
plan. HCPs are generally 50-year contracts with the NMFS. The
landowner agrees to land management that has protections for
threatened or endangered species that often go beyond what cur-
rent regulation might require. In return, the landowner gets an
incidental "take" permit and regulatory certainty. The incidental
"take" permit allows the owner to accidentally kill or harm the
threatened or endangered species as long as they are operating
v\ ithin the provisions of their agreed contract. Without an inciden-
tal take permit, indixiduals can be subject to fines and/or impris-
onment. This topic is of keen interest to Puget Sound shellfish
farmers because, as noted earlier, the Puget Sound Chinook
salmon were listed in March 1999 and the out-migrating juveniles
utilize grower's beds in the transition from fresh to salt water.

Identifying Research Needs

An important aspect of developing an effective ECOP for shell-
fish cultivation is identifying the negative impacts in order to
mitigate them effectively. Some impacts are understood, but many
are not. The Pacific Shellfish Institute is conducting a literature
review of existing research to identify needs for future research.

Goals 2010

At the PCSG.A annual meeting in October 1998. a process was
begun to set goals in eight research categories for the year 2010.
With the goals identified, growers were asked to identify the re-
search priorities and legislative initiatives necessary to achieve
those goals. Shellfish ecology is one of the categories, with iden-
tified research priorities that attempt to fill gaps in existing knowl-
edge and with the goal of minimizing negative impacts and en-
hancing positive impacts. The 2010 Goals, Research and Initiative
Priorities, are available on the PCSGA website at www.pcsga.org.
PSI is using the document to prioritize its research efforts as well
as circulating it to the various research institutions, granting enti-
ties, and resource management agencies.

Industry, Scientist, Resource Manager Disconnect

As West Coast growers have become more proactive in the
arena of shellfish ecology, it has become apparent that a segment
of the research and resource management community is at odds
with the industry. In the Chesapeake Bay on the East Coast of
North America, it has been recognized that oyster reefs are an
important part of the ecosystem, providing critical habitat and
filtration to the estuary. Millions of dollars are now being spent in
an attempt to rebuild oyster reefs in the Chesapeake Bay, not only
to revive an important fishery but. more importantly, to restore the
natural functions to the system.

On the West Coast, certain segments of the research and re-
source management communities have come to view eelgrass as
the ultimate indicator of the health of an estuary. While it is a vital
pan of any coastal estuary, eelgrass is not the only indicator of a
healthy ecosystem.

A consequence of the "eelgrass protectionist" philosophy on
the West Coast has been a series of studies, frequently cited by
resource managers, examining the effects of shellfish culture on
eelgrass. When the studies conclude a negative impact of a culture
activity on eelgrass, the resource managers have promoted regu-
lations to prohibit the activity. The recent changes to the USCOE

NWP 4 provide a good example. Unfortunately, the industry has
been ineffective at getting the West Coast scientific community or
resource managers to acknowledge the benefits of the shellfish to
the ecosystem. In the East, hundreds of thousands of dollars of
taxpayer's money tare being spent to restore oyster reefs; however,
in the West, the regulatory screws continue to be tightened such
that eventually farming shellfish will no longer be a viable eco-
nomic enterprise.

Working with the Western Regional Aquaculture Center,
housed at the University of Washington's School of Fisheries, the
shellfish industry has recently secured funding for research that
will investigate the oyster-eelgrass interaction to better understand
the overall ecological role of the oysters in relation to the eelgrass.
The Pacific Shellfish Institute is pursuing funding for similar stud-
ies related to suspended shellfish (oyster and mussel) culture.

The Ecological Benefits of Cultured Shellfish

Bivalves are efficient filter feeders. Oysters and other suspen-
sion-feeding bivalves play an important role in estuarine ecosys-
tems as biofilters, significantly enhancing water quality and clar-
ity, which have been the subject of studies in Chesapeake Bay
(Gottleib and Schweighofer 1996). In fact, now that the oysters are
virtually gone from the nutrient-rich Chesapeake ecosystem, algal
blooms go largely unconsumed. Light cannot penetrate the algal
blooms, and eelgrass and other submerged aquatic vegetation
(SAV) are declining, as are the fish species dependent on the SAV
for habitat and refuge.

Also, regarding the bivalve's filtering capacity, a recent (Oc-
tober 1997) report by the Environmental Defense Fund, "Murky
Waters: Environmental Effects of Aquaculture in the United
States" (Goldburg & Triplett 1997), noted that mollusk farming
"actually reduces nutrient pollution. Mollusk farmers do not feed
(their stock). Clams, oysters, mussels and scallops are filter feeders
that consume phytoplankton already in the water column. Mollusk
culture actually reduces the nutrients in marine systems, because
35—40% of the total organic matter ingested by the mollusk is used
for growth and permanently removed by harvest of the mollusk."

Oysters have been shown to have positive impacts on specific
components of epibenthic communities and share many of the
same attributes as eelgrass beds. Like other three-dimensional bio-
logical structures (including eelgrass beds and rubble reefs), oyster
shells modify tidal flow and sedimentary processes and serve as
important nursery and refuge habitats for juvenile fishes, shrimps,
crabs, and other invertebrates (Ambrose and Anderson 1990; Doty
et al. 1990; Breitburg 1991; Dumbauld et al. 1993; Williams 1994;
Eggleston and Armstrong 1995; Simenstad and Fresh 1995). In-
terestingly, in Grays Harbor, Washington, the USCOE uses oyster
shell to mitigate impacts to crab habitat from their dredging ac-
tivities.

Doty et al. (1990) compared the abundance of intertidal juve-
nile crab in oyster, eelgrass, and open habitats and found that the
oyster cover contained 4 to 6 times the crab abundance found in
eelgrass. Basically, the open areas contained no juvenile crabs, and
the eelgrass did not begin to play a protective role until mid June
when it grew out. There is another important distinction between
oysters and eelgrass relative to habitat: the oysters are there year-
round. While eelgrass grows year-round, in the winter the blades
are shorter, narrower, and fewer per shoot (Phillips 1984). Fur-
thermore, many shellfish growers note that winter storms fre-
quently eliminate almost all eelgrass in intertidal beds.

454

Dewey

Graveling of tidelands and the broadcast of oyster shell are
historic practices for enhancing shellfish production. Enhancement
of secondary productivity, with increased standing stocks of
epibenthic prey resources for juvenile salmon, as a result of inter-
tidal graveling has been documented (Thom et al. 1992). The
presence of oysters and gravel appear to have little, if any. adverse
impact on species diversity and overall density compared to unal-
tered habitats, although shifts in species abundance and dominance
may occur as a result of physical and biological modifications
(Simenstad et al. 1991; Thompson 1995). Some of these shifts in
species dominance may actually benefit outmigrating juvenile
salmon. For example. Simenstad et al. ( 1991 ) found that densities
of the harpacticoid copepod Tisbe spp.. an important prey item for
some juvenile salmonids (e.g.. chum salmon, Oncorhynchiis keta).
were enhanced in areas of oyster culture and shell compared to
bare mudtlat. Brooks (1995) found that Carophhim achenisiciim.
another critical prey resource for fish, was enhanced in actively
cultured oyster beds, and data from Armstrong et al. (1992) indi-
cate greater densities of gammarid amphipods and small tellinid
clams as prey for both salmonids and 0+ Dungeness crabs (Cancer
magister).

CONCLUSION

West Coast shellfish culture practices have recently come un-
der greater environmental and public scrutiny as natural resource
agencies and government officials direct more attention toward
protecting estuarine ecosystems for their biological productivity,
complex habitats, and diverse assemblages of aquatic species. The
Pacific Coast Shellfish Growers Association is challenging shell-
fish growers to respond to the pressure by proactive involvement
in their communities, through local planning, education, promo-
tion, and research. As important, the PCSGA is working with
growers and other stakeholders in the estuaries to develop an En-
vironmental Management System for the shellfish industry. The
EMS will establish an Environmental Policy and an Environmental
Code of Practice for shellfish farmers up and down the West Coast.
The industry is prioritizing research to better understand potential
impacts as they develop an effective code of practice. Establishing
shellfish growers as responsible users of the water surface, water
column, and tidelands will be crucial to .securing the future of the
industry in the new millennium.

LITERATURE CITED

Ahlers. D.. E. Linsay & T. Lynch. \997. Best Management Practices for
the Shellfish Industry Environmental Management Program. Univer-
sity of Washington. Seattle. Washington. 42 pp.

Ambrose. R. F. & T. W. Anderson. 1990. Intluence of an artificial reeion
the surrounding infaunal community. Mar. Biol. 107:41-52.

Armstrong. D. A.. O. Iribarne, P. A. Dinnel. K. A. McGraw. J. A. Schaffer.
R. Palacios. M. Fernandez. K, Feldman & G. Wilhams. 1992. Mitiga-
tion of Dungeness crab. Cancer mai'islcr. losses due to dredging in
Grays Harbor by developmcnl of intenidal shell hubitat: pilot studies
during 1991, FRI-UW-9205, Fisheries Research Insiiiute. IJniversily of
Washington. Seattle. Washington.

Brooks. K. 1995. Long-term respon.se of benlhic invertebrate communities
associated with the application of carbaryl (Sevin) to control burrowing
shrimp, and an a.s.sessment of the habitat value of cultivated Pacific
oyster (Cra.'i.wstrea x'X"'^) beds in Willapa Bay. Washington, lo fulfill
requirements of ihe FPA Carbaryl data call In. (i9 p.

Doty, D., D. Armstrong & B. Dumbauld. 1990. Comparison of carbaryl
pesticide impacts on Dungeness crab {Cancer maf>i.ster) versus benefits
of habitat derived from oyster culture in Willapa Bay. Washington.
FRI-UW-9020. Fisheries Research Institute. University of Washington.
SeaUle. Washington. 69 pp.

Dumbauld. B. R. 1997. A review of studies on the impact of oyster aqua-
culture to West Coast benthic invertebrate communities. J. Shellfish
Res. 16:312.

Dumbauld. B.. D, Armstrong & T. McDonald. 1993. Use of oyster shell to
enhance intertidal habital and mitigate loss of Dungeness crab (Cancer
magister) caused by dredging. Can. J. Fish. Aquat. Sci. 50:381-390.

Dumbauld, B. R. & S. Wyllie Echeverria. In press. Burrowing shrimp
control and eelgrass distribution in Washington State coastal estuaries.
Abstract. / Shellfish Res.

Eggleston. D. E. & D. A. Armstrong. 1995. Larval supply, active substrate
selection, and post-settlement survival as determinants of estuarine
Dungeness crab recruitment. Ecol. Monogr. 65:193-216.

Goldburg. R. & T. Triplett. 1997. Murky waters: Environmental Effects of
Aquaculture in the United States. Environmental Defense Fund Publi-
cations. I

Gotlleib. S. J. & M. E. Schweighofer. 1996. Oysters and the Chesapeake '
Bay ecosystem: A case for exotic species introduction to improve en- '
vironmental quality? Estuaries 19:639-650.

Huppert, D. D.. A. M Olson. M. J. Hershman. K. T. Wing & C. M.
Sweeney. 1998. Socioeconomic causes and consequences of coastal
ecosystems change. NOAA Coastal Ocean Program. Decision Analysis
Series No. II. NOAA. Washington. D.C.

Phillips. R. C. 1984. The ecology of eelgrass meadows in the Pacific
Northwest: A community profile. US Fish and Wildlife Service. US-
FWS/OBS-84/24. 85 pp.

Simenstad. C. and K. Fresh. 1995. Influence of intertidal aquaculture on
benthic communities in Pacific Northwest estuaries: scales of distur-
bance. Estuaries 18:43-70.

Simenstad. C. A.. J. R. Cordell & L. A. Weitcamp. 1991. Effects of sub-
strate modification on littoral flat meiofauna: assemblage structure
changes associated with adding gravel. FRI-UW-9I24. Fisheries Re-
search Institute. University of Washington, Seattle, Washington.

Thom. R. M.. T. L. Parkwell. D. K. Niyogi & D. K. Shreffler 1992. Effects \
of gravel placement on estuarine tidal flat primary productivity, respi- I
ration and nutrient flux. Prepared for Washington Department of Fish-
eries by Balelle/Marine Sciences Laboratory. Sequim. Washington. 42
pp.

Thompson. D. S. 1995. Substrate additive studies for the developnienl of
hardshell clam habitat in waters of Puget Sound in Washington State:
an analysis of effects on recruitment, growth, and survival of the Ma-
nila clam. Tapes phili/ypinarnm. and on the species diversity and abun-
dance of existing benthic organisms. Estuaries 18:91-107. ■

Washington Deparlmenl of Fisheries. Washington Department of Ecology I
1985. Final Environmental Impact Stalement: Use of Insecticide car- I
baryl to control ghost and mud shrimp in oyster beds of Willapa Bay
and Grays Harbor. Washington Department of Fisheries. Washington
Department of Ecology. Seattle. Washington.

Washington Department of Fisheries. Washington Department of Ecology. :
1992. Supplemental Environmental ImpacI Slalemeni: Use of insecti- I
cide Carbaryl to control ghost and mud shrimp in oyster beds of Wil- 1
lapa Bay and Grays Harbor. Washington Department of Fisheries.
Washington Department of Ecology.. SeaUle. Washington. 147 pp. J

Williams. G. D. 1994. Effccis of a large-scale estuarine intenidal habitat I
modification on disinhulion patterns and food habits of epibenthic fish 1
species in Grays Harbor. Washington. Master's Thesis. University of
Washington. Seattle. Washington.

Journal of Shellfish Reseorch. Vol. 19. No. 1. 453-464. 2000.

A RESOURCE-BASED METHODOLOGY TO ASSESS DOCK AND PIER IMPACTS ON

PLEASANT BAY, MASSACHUSETTS

S. L. MACFARLANE,' J. EARLY,^ T. HENSON,^ T. BALOG," AND

A. MCCLENNEN-^
'p. O. Box 1164

Orleans. Massachusetts 02653
'Island Foundation

589 Mill Street

Marion, Massachusetts 02738-1418
'Massachusetts Coastal Zone Management

3225 Main Street

Barnstable. Massachusetts 02630

64 Carol Avenue

Falmouth. Massachusetts 02536
' Bayplan Steering Committee

68 Evelyn 's Drive

E. Harwich, Massachusetts 02633

ABSTRACT The preparation of a four-town resource management plan for Pleasant Bay. Cape Cod, Massachusetts, required a
comprehensive assessment of the present number of private piers on the bay. the probability of numerous additional piers in the future,
and the potential impacts from piers on the resources of the bay. The planning group developed a methodology to account for various
components relative to piers and their use. The study area was segmented into 26 geographic subsections. Each subsection was
evaluated for nine factors representing biological, physical, and human use characteristics critical to the impacts of docks and piers.
The nine factors were semi-enclosed or open water bodies, water depth, shellfish habitat, eelgrass. fringe salt marsh, density of existing
structures, moorings, and navigational channels, and recreational activity. After assessment of the areas, each of the nine factors was
assigned a value of 0, 0.5, or 1 , w here 0 indicated the least significance and 1 represented the greatest significance. Results were
tabulated and mapped according to resource sensitivity. The results indicated that a significant portion of the bay's more secluded
shoreline is extremely resource sensitive. The environmental impacts from construction and use of docks and piers in these areas pose
a direct threat to the extensive and fragile resources, and these areas have been deemed inappropriate for new docks and piers. Less
sensitive areas may be more appropriate for construction of new docks and piers based on baywide criteria to be formulated once the
plan is implemented.

KEY WORDS: Docks, piers, resource assessment, boating impacts, management

INTRODUCTION to eelgrass, chemical leachates from treated wood, construction

impacts, fragmentation of beach habitats, sediment resuspension

To a shorefront property owner, one of the primary reasons for from boat propellers, boat paints, chemicals used in marine sani-

paying higher taxes for waterfront property is the immediate ac- tation devices, and petrochemicals.

cess to the water. A dock in front of a house represents easy access Arguments used by homeowners or their agents to gain a per-
to the water, status, a higher property value for re-sale, and a mit for a new dock suggest that docks can have a beneficial en-
property right. The structure is generally located in public waters vironmental effect. Carriker ( 1961 ) stated that a piling could create
in Massachusetts if it extends below the high water mark. To a a microhabitat. changing the overall circulation patterns such that
resource manager, a dock represents a potential conflict with re- a small gyre is created, allowing for better settlement of shellfish
source protection, actual negative effects on the marine environ- laiA'ae, How multiple docks in an area affect currents is not well
ment, and private intrusion in public waters that is not a private understood. Untreated pilings are known to attract settlement of
property right. These conflicting representations often result in certain marine organisms. Another argument we have heard is that
frustration to both the homeowner and manager because there is a dock is better than a boat on a mooring because less total habitat
generally no cohesive policy toward the siting of new docks. While may be affected. A boat at a dock is at a fixed location, while on
the structures and their use are cause for concern, the cumulative a mooring the mooring chain may scour the bottom as the boat
impacts have not been researched adequately. As a result, in de- swings (Walker et al. 1989. Short et al. 1993). Also, a dock cross-
veloping the Pleasant Bay Resource Management Plan (known as ing over a marsh is better than a path through a marsh because
the Bay Plan), we have taken a conservative approach toward the there is less total impact on the marsh vegetation,
siting of new docks based on our own observations as well as Negative effects of the structures can include physical displace-
information provided by other researchers. ment of habitat from pilings, pipes, or other upright structural

Although generally thought of as benign structures in the in- members. While not usually perceived as a great threat, the cu-

tertidal and subtidal zones, docks and piers (hereinafter referred to mulative effect of the number of pilings per dock multiplied by the

as docks) have been shown to be potentially problematic. Concern number of docks can be substantial. Docks can shade submerged

centers on the effects of docks in shallow embayments and in- aquatic vegetation (SAVs) (Wetzel and Penhale 1983. Shon et al.

eludes vegetation loss from shading, shellfish habitat loss, impacts 1993; Burdick and Short 1995). Seasonal docks that are removed

455

456

Macfarlane et al.

yearly can cause dead zones of anoxic sediment in some areas that
is up to twice the diameter of the pilings (Macfarlane personal
observations). However, permanent docks that remain in the water
during the winter can be lifted by ice, requiring reinstallation of the
pilings and reducing the amount of accessible shellfish habitat for
harvest. Use of pressure-treated wood can be toxic to marine or-
ganisms (Weis et al. 1991; Weis and Weis. 1992a.b, Weis et al.
1993). Lastly, there is an aesthetics issue regarding the value of
natural shorelines versus those having manmade structures.

Operation of the boats tied to docks can cause problems (Craw-
ford et al. 1994). Prop dredging can mechanically remove habitat
while also causing resuspension of sediment. Although turbidity is
a natural phenomenon in wind-driven resuspension episodes, tur-
bidity caused by boats has not been well studied and is poorly
understood (Yousef 1974: Yousef et al. 1980. Hilton and Phillips
1982). The effect of petrochemical spills and chronic addition of
petrochemicals to the water from boats operating at a fixed loca-
tion is also poorly understood as is potential damage from boat
paints, e.specially formulations of bottom paint.

CURRENT PERMITTING SYSTEM

Under the current permitting system in Massachusetts, a prop-
erty owner desiring a new dock must apply to three agencies for
three separate permits. First, the homeowner needs an Order of
Conditions from the local Con.servation Commission, the Select-
men-appointed body of volunteers responsible for administering
the state Wetlands Protection Act (MGLC. 131) and local wetlands
bylaw (if applicable). Second, a Chapter 91 (Waterways) license
from the Massachusetts Department of Environmental Protection
Waterways Program must be obtained. (In Massachusetts, the pub-
lic has additional rights of fishing, fowling, and navigation in the
intertidal zone. These latter rights are part of the public trust doc-
trine and were established in the Massachusetts Colonial Ordi-
nance of 1641-1647.) Third, a permit must be obtained from the
U.S. Army Corps of Engineers in accordance with Section 404 of
the Clean Water Act (Federal Water Pollution Control Act of
1948).

In accordance with Massachusetts" amended waterways regu-
lations (Chapter 91 ). no new dock could be perniilted in any area
designated by the State as an Area of Critical Environmental Con-
cern (ACEC) until and unless the town completed a state-approved
resource management plan. Pleasant Bay was designated as an
ACEC in 1987. As a result, there has been a moratorium on the
issuance of new dock permits since 1991. At the time the mora-
torium went into effect there were 165 docks in the Pleasant Bay
estuary, the majority of which were located in the more protected
areas of the bay.

The performance standard in the existing regulations for allow-
ing docks in an ACEC is "no adverse effect.' Prior to the mora-
torium, property owners applying for dock permits would institute
mitigating measures to counteract the potential problems outlined
above. Shading effects can be overcome by raising the structure to
sufficicnl height to allow for sunlight to reach the grass, adding
"holes" (through greater plank spacing) in the walkway for sun-
light penetration, or using plastic or metal grates. Permanent struc-
tures can be maintained by use of bubbler systems during the
winter months of ice. Shellfish can be seeded around the dock.
Pressure-treated wood can be replaced by plastic "wood." cedar,
locust, or other nonleaching iiiateriaK. To many conservation com-

missions, these measures, presented as mitigation by homeowner
representatives (engineers, consultants, and lawyers), were enough
to maintain the standard of no adverse impact. In addition, once
one person received a permit, it was difficult to identify an adja-
cent property as too fragile to permit a dock and the neighbor
generally received a permit as well. Moreover, each application
was judged on its own merits and although cumulative effects were
an interest protected by the state Wetlands Protection Act. these
cumulative effects were difficult to prove in potential court ap-
peals.

New Jersey approached the problem of docks proliferation
through development of a generic Environmental Impact State-
ment (Crawford et al. 1998). Because EIS provisions are lacking in
Massachusetts, the Pleasant Bay Technical Advisory Committee
(TAC) developed a methodology to look at the proliferation of
private docks in a different manner. The Bay Plan was based not
on individual lot-by-lot decisions or a generic basis, but rather by
a method for estimating cumulative impacts and determining
where docks could be built that would not have an adverse impact
on the system as a whole.

STUDY AREA

Pleasant Bay is located at the outer part of the elbow of Cape
Cod within the towns of Chatham, Harwich, Brewster, and Orleans
(Fig.l). Designated as an ACEC in 1987, over 9000 acres (3645
ha) are within the boundary. Chatham Harbor is not within the
boundary of the ACEC, but it is a major part of the estuary since
the inlet for the bay is in the harbor. The primary reason for the
harbor's exclusion from the ACEC designation was that Chatham
is home to the region's offshore fishing fleet and, in order to
maintain that industry, dredging is sometimes required, an activity
prohibited by the designation. However, the harbor was included
for the purposes of developing the management plan. Brewster has
a mere 40 ft ( 12 m) of shoreline, but it contains the largest portion
of the bay's watershed.

The bay is a shallow embayment. where over 509^ is less than
2 m deep at mean low water (MLWi. It has a migrating barrier
beach on its eastern flank that protects the bay from the Atlantic
Ocean. The shallow portions of the Little Bay have healthy eel-
grass (Zosteni marina (Linnaeus)) meadows and sometimes pro-
duce bay scallops (Argopecten irradians irradians Lamarck). The
habitat can support clams {Mya arciuiria Linnaeus) and quahogs
iMi'itt'naiia inenenaria Linnaeus 1758). Mussels (MyuIus cdiilis
Linnaeus) are frequently found near Chatham Harbor. Oysters
(Crassostrea virginka Gmelin) have not been a native species for
at least the last 50 years, although they have been found sporadi-
cally as a direct result of a privately leased oyster area in the bay.
The buy is ringed with fringe salt marsh, primarily salt marsh
cordgrass {Sparlina allerniflnra (Loisel)). There are numerous
pockets of larger salt marshes indenting the shoreline. Coastal
banks are the dominant land interface feature.

PLEASANT BAY RESOURCE MANAGEMENT PLAN

Although the state imposed a moratorium on the construction
of any new docks in Pleasant Bay, the towns were slow to get
together to develop a joint plan. Several attempts were made and
finally, in 1995, the four towns entered into an intermunicipal
Memorandum of Agreement to develop a joint resource manage-
ment plan lo protect the extensive resources of Pleasant Bay. The
resulting plan was appro\ed in Harwich in May 1998 and by

A Resource-based Dock Assessment Methodology

457

Pleasant Bay
Marine Features

Draft

ACH

CZM

ThoM iTw(a wva praducad trr the Cap« Cod

Co<TimlM(on lor th* f lasMnt B«v RoKxima

pattttllv fundad tTvTXjfh MawachutaD
CoBRalZon« Manafemanl bf ■ %rvil from
lh« Officx o'OcTBri vd Cat^ttl Kaourm
M<nafWT>an(, Nitlorul Ocaank end
ABrotfiharic MmlnJitntton, and tha Ui
Oapartmant of CommanM. Tha v(v*t
axpraaaad ara tSoaa of tha aulhoKal
and do not iiH imiiIIi rtfled tha viswi
of NOAA M inr o( lb tub - igenda. Thl)
Intamwflon It aval labia <n aRamai]v«
ftfftTiiti upon rnoueW.

Town Boundary
Major Road

2.500
nSuRPWir Fm
^ortfi Anifnfjn CUTuir 1*^7

Map Crorted oo laouwy 25. 1 996

Shoreline, ponds, and town boundaries from
towns' assessor maps of various dates with
modifications made by CCC CIS in 1997 based
on the Department of Environmental Protection
wetland mapping aenal photos (photos from
March 1993).

Ponds, streams, major roads - MassGiS;
drgitized from U5CS quadrangles, 1 :25000
scale, various dates.

Figure 1. Map of the Pleasant Bay Resource Management Plan study area.

458

Macfarlane et al.

Orleans and Chatham in November 1998. it was rejected by Brew-
ster in May 1998. However, even with Brewster's action, the plan
can still be implemented since many of the recommendations cen-
tered on use of the water and the directly adjacent waterfront.

One of the primary reasons for the towns to get together was
the dock issue. Each town had received numerous requests for
docks since the moratorium had been put into effect. Orleans alone
received at least 20 requests per year (Macfarlane personal obser-
vation), most of which emanated from new property owners. As
the economy improved during the 1990s, it became widespread
knowledge from realtors that a dock added anywhere from $50,000
to $100,000 to the value of a home on Pleasant Bay.

From 1995 to 1998. the towns worked together to develop the
plan. This was the first time such a large cooperative effort was
achieved in the history of the towns. The Selectmen in each town
appointed a steering committee representative. A technical advi-
sory committee was established, comprising shellfish constables,
conservation administrators, harbormasters, water quality special-
ists, and town planners from each town. A consultant was hired as
a coordinator. State and county governments and National Park
Service (from the Cape Cod National Seashore) personnel served
on the Technical Advisory Committee (TAC) as well.

The planning process revolved around five issues; structures
(docks and coastal engineered erosion control structures), shellfish
and aquaculture, boating, biodiversity, and public access. During
the winter of 1997. the Steering Committee sponsored workshops
on each issue (several hundred people participated) to define prob-
lems and make recommendations for the plan.

The Structures Workgroup reviewed the current statutes, by-
laws, and regulations of dock applications and permitting. Given
the history of permits approved prior to the moratorium, this group
of the TAC was frustrated by the lot-by-lot approach and the lack
of a generic EIS and agreed that there had to be another way to
approach the subject.

THE METHODOLOGY

The TAC compiled data to create maps on a GIS system with
the assistance of the Cape Cod Commission, a county planning and
regulatory agency. Base maps were created for land use. location
of shellfish resources by species, marshes and eelgrass, existing
docks and mooring fields, town landings, navigational channels,
scenic view points, access points, and all other resources. From the
base maps, overlays were made to determine conflicts. In Pleasant
Bay. shellfish are often located in a thin ribbon of land between the
edge of the fringe marsh and about 250 ft (76 ml offshore, in both
intertidal and subtidal lands. It is also the location of most of the
docks.

When ihc map of the docks was overlaiil onto the map of
shellfish, the upper part ol the Ri\er Complex in Orleans (Meet-
inghouse Pond to Namequoit Point) and the protected areas of
Crow's Pond and Ryder's Cove in Chatham exhibited the most
overlap. When moorings were added, it was obvious that boating
activity heavily impacted these upper areas. The question posed
was. Would these areas remain productive for shellfish if theic was
a dock every 150 ft (45 m), the average lot frontage?

The approach of the TAC was to look at the bay as a system.
Constructing a dock in a salt pond at the far end ol' the estuary was
far different in terms of impact than constructing a dock on the

open shoreline along either Little Bay or Big Bay. A method was
needed that could distinguish the differences between these two
areas and so a sensitivity index was developed. The process was
divided into ten steps, discussed below.

Step I: The bay was divided into 26 segments or subsections
that were different from one another in one or more ways. These
subsections included pond, river, and open bay shorelines (Fig. 2).

Step 2: The attributes of each subsection were described with
respect to biological, physical, and human use factors (Fig. 3). Did
the area support shellfish? Did the area have eelgrass and/or fringe
marsh? What was the depth of water 200 ft (60 m) from the edge
of the marsh or MLW. whichever applied? Was the area open
water, a river, or semi-enclosed? What was the ratio of docks to
parcels without docks? Was there a navigational channel within
500 ft (150 m) of shore?

We chose these questions because in total they would give us
a sense of the area and what the potential impacts of a dock would
probably be. Because aesthetics is a difficult and subjective con-
cept to quantify, we asked ourselves what was it about a dock that
produces a reaction when looking at them? The answer to us was
that docks make a natural shoreline look manmade. The greater the
number of docks within a visual path and the higher the docks
were, the less aesthetically pleasing the scene was. We judged
natural shorelines to be highly aesthetic and manmade structures to
be less aesthetic. Therefore, we were able to address the aesthetics
by using the ratio of parcels with docks to those parcels without
them.

Step 3: Nine criteria for the sensitivity index were established
and each criteria was evaluated according to one of the following;
high, medium, low; shallow, medium, deep; lots. some, none; yes.
some, no. A matrix was developed using these criteria for each of
the 26 areas (Table 1 ). The nine criteria were open or semi-
enclosed water body; ratio of parcels with docks to those without:
shellfish habitat; fringe marsh: eelgrass: water depth within 150 ft
(45 m) of shore; moorings within 500 ft (150 m) of shore; navi-
gational channel within 500 ft ( 150 m) of shore: recreational use.

Step 4: Terms were defined (Table 2).

1. Semi-enclosed or open water body: A semi-enclosed area,
such as a salt pond al the head of the estuary, would have
lower flushing capacity and would be the first area to show
signs of problems resulting from nutrient loading, drainage,
or toxic inputs. An open area would have greater exchange
with the incotning oceanic water.

2. Dock ratio: Number of lots with docks and number without.
.3. Shellfish habitat: The shoreline was evaluated with respect

to the historical, present, and town propagation projects to
determine an area's ability to support shellfish. It was also
evaluated with respect to populations of clams, quahogs, and
scallops; mussels and oysters were niit present in the waters
in the 20()-ft (60 m)-\\ide shellfish area adjacent to the
shore.
4. Fringe marsh: Presetu in m;in_\ areas, fringe marsh varies
considerabl) in u idth. Il protects coastal banks from erosion
and. while not as productive as extensive marshes, fringe
marsh has the capacity to filter nutrient-loaded groundwater,
contains invertebrates such as ribbed mus.sels {Modiolus de-
mises) that can aid in water clarity, and provides habitat for
fish and invertebrates. It has widths varying from minimal to
about 20 fi (6 111), with 10 to 20 ft (3-6 ni) being average. A

A Resource-based Dock Assessment Methodology

4?9

Pleasant Bay
Marine Features

Draft

BliCH

pvr^liS' hjndtd (rv^jvf^

CciMa' Zona MuUfamw b^ * gnM fm/^

Dafanmant al Ginvnarak. I
wprwiid ar* thaw a' C^a luttvylal
ml do not liar imir1f| rWWl S^a vtawt
^ FKVA 0( anr a"tt *">> ' afcmda*. TYil*
tnfom Son la ■ ■W labia I n a NamMl ■•
Ipfmtti upon noutA.

Town Boundary
Major Road

UaaaiiuKm S UK PWv (vd
M«p Cmud on January 16. 1 990

Shoreline, ponds, and town boundaries from
towns' assessor maps of various dates with
modifications made by CCC CIS in 1 997 based
on the Department of Environmental Protection
wetland mapping aerial photos (photos from
March 1993).

Ponds, streams, major roads - MassCIS;
digitized from USGS quadrangles, 1 :25000
scale, various dates.

Figure 2. Map of the Pleasant Bay Resource Management Plan subsections.

460

Macfarlane et al.

PLEASANT BAY: SUBSECTION AREA DESIGNATIONS AND DESCRIPTIONS

1. Meetinghouse Pond is an enclosed pond on the upper end of an estuary. If there is a problem with flushing in
the bay. it would be most noticeable here. There is a low dock to parcel ratio currently, with less than 25*7^ of the
parcels with docks, which means approximately 15'7c of the parcels could potentially each request a dock. It is a
known shellfishing area close to shore (approximately 200 feet from fringe marsh) and beyond, which would
coincide with the locations of potential dock placements. There is .some deep water shellfishing here, but not in the
fringe area. Fringe marsh may be found along the entire shoreline, while eelgrass is spotty, and the water depth is
shallow (shown in bathymetry results). The pond is heavily used for moorings, it has no navigational channel, but
is highly used by people coming and going for recreation. It is a busy place possibly due it's having both filling
and pump-out stations.

2. Kent's Point — Upper River, which as a river makes the designation of enclosed versus open a difficult one. It
is a narrow body of water, where over 50% of the parcels have docks. This is a known shellfish area with a
productive fringe marsh. There is spotty eelgrass, the area is shallow, moorings are medium density, there is
definitely a navigational channel and is a busy area for its size.

.^. Kent's Point — Namequoit is less narrow than the Upper River although it is very narrow around Mayflower
Point. This area has a medium density of docks to parcels, and is a known spot for shellfishing, although some
parts are not highly productive. There is fringe marsh in the majority of this area, although spotty in some
locations, and it has a medium density of moorings, definite navigational channel and heavy recreational use.

4. Lonnie's Pond is definitely enclosed with a naiTow channel leading to it. (This is a potential site for
eutrophication in the future.) There is a medium density of docks to parcels, known shellfish habitat in the fringe
and deep waters along the fringe, spotty eelgrass, deep water close to shore, heavy use of moorings, no
navigational channel and heavy navigational use.

5. Lonnie's Channel is narrow and shallow. There is one dock, some shellfishing in the river itself, fringe marsh
throughout the channel, no eelgrass, and heavy recreational use from people coming and going.

6. Arey's Pond is similar to Lonnie's pond and very active.

7. Namecpioit River may be described similarly to Lonnie's Channel except that the channel is wider which
provides more space for moorings.

Figure 3. Descriptive attributes of eacli Bay Plan subsection.

TABLE 1.
In\entory of resource and use issues relevant to docks and piers.

Number

Parcels

Water

Navigational

of

w/No

Shellfish

Fringe

Depth

Moorings

Channel

Recreational

Area

Docks

Dock

Habitat

Marsh

F.elgrass

w/in 15(1"

w/in \5{y

w/in 150'

Use

(1)

Meetinghouse Pond

11

32

Yes

Heavy

Liahl

.Shallow

Heavy

No

Light

(2A)

Kents Point-UppLT River

IS

10

Yes

Heavy

Light

.Shallow

Light

Yes

Heavy

(2B)

Kenls Point-Nanici|Uoit

(.^)

Lonnie's Pond

19

.^9

Yes

Heavy

Heavy

.Shallow

Light

Yes

Hea\y

8

8

Yes

Heavy

Light

Deep

Heaw

No

Light

(.^A)

Lonnie's Channel

1

6

No

Heavy

None

Shallow

None

Yes

Heavy

(4)

Arey's Pond
Nainequoil Riv

8

12

Yes

Heavy

Lighl

Sh^illow

Heavy

No

Light

16

32

Yes

Heavy

L.iihl

SIkiIIow

Heaw

Yes

Heavy

(fi)

Pah Wah Pond

6

7

Yes

Heavy

Light

Deep

Heavy

Yes

Light

A Resource-based Dock Assessment Methodology

461

TABLE 2.
Definition of terms.

Shellfisli Habitat: All traditionally state-regulated species (soft shell clams, quahaugs. mussels, scallops and oysters) that have historically,
currently or in the future could potentially support shellfish.
Shellfish: All species are of equal value.

A. Yes = evidence of being able to suppon shellfish

B. Slight = supports shellfish but not in abundance

C. No = no evidence of supporting or being able to support shellfish

Fringe Marsh: Bands of Spartina altemifiora with some S. patens in transition areas from the water to the upland.

A. Heavy = 10 feet or greater in width of marsh grass

B. Medium = 5-10 feet width

C. Light = <5 feet width

Eelgrass: Amount of eelgrass in a particular area

A. Heavy = sediment covered with eelgrass with few bare spots

B. Medium = eelgrass interspersed with bare sediment of equal proportions

C. Light = no eelgrass or a few sporadic individual plants

Ratio of docks: Number of lots with docks compared with total number of lots in a given area
Water Depth:

A. £4 feet at Mean Low Water (MLW) = low sediment disturbance

B. 3^ feet = medium disturbance

C. <3 feet = high disturbance

Moorings: Public mooring area where density of moorings exceeds three moorings within 500 feet from shore.

A. Heavy = more than 3 moorings or mapped public mooring field

B. Light = 0-3 moorings per land parcel

C. None = no moorings in area

Navigational Channel: Structures placed less than 500 feet from channel would cause impediments to navigation.

Yes = channel within 500 feet; No = channel greater than 500 feet
Recreational Activity: General public use of the area

A. High = heavy use usually from boating activity

B. Medium = some boating or other water use

C. Light = \ery little boating or other water use

marsh 5-10 ft (1.5-3.0 m) wide was considered to provide
some functions, but one that was less than 5 ft (1.5 m) wide
probably provides only minimal positive effects.

5. Eelgrass: The presence or absence and relative abundance of
eelgrass was assessed. The healthy meadows in the middle
of Little Bay were considered as representative of heavy
abundance, while a few sporadic plants were considered low
abundance.

6. Depth of water: A depth of 4 ft ( 1 .2 m) or more would have
relatively little resuspension of particles from most outboard
engines on most shallow-draft boats (Crawford et al. 1998).
A boat put in gear and docking on the return trip can have
an enormous impact and can even create a channel or hole
known as "prop dredging." A depth of 3^ ft (0.9-1.2 m) a
boat would cause medium disturbance and at a depth of less
than 3 ft (0.9 m) at MLW could cause resuspension.

7. Moorings: The harbormasters automatically give waterfront
property owners up to three moorings in front of their prop-
erty. A mooring field is a public mooring area where moor-
ings exceeded that density within 500 ft (150 m) of the
shore. Areas near town landings had such mooring fields
while other areas generally did not.

8. Navigational channel within 500 ft ( 150 m): Many areas are
highly used for both recreational and commercial activity. In
accordance with .state .statute, a private structure should not
be an impediment to navigation. Current channel locations
were located and it was determined that structures less than

500 ft ( 150 m) from the channel would cause an impediment
from wakes, and cross-channel operation.

9. Recreational activity: Although difficult to actually define,
the area was examined with respect to the general public use
of the area. A long, narrow river connecting to a pond with
a lot of boats would have a lot of recreational activity near
shore, as would most of the ponds because of the heavy
number of moorings. The open water where docks would be
located did not have much activity.

Step 5: We added a number to the descriptions as follows:

1. high. yes. lots, shallow = 1

2. medium, some, few = 0.5

3. low, no, deep = 0

Step 6: The words in our matrix were replaced with the num-
bers for each criterion at each location in a second matrix (Ta-
ble. 3).

Step 7: The numbers were analyzed with respect to the total
impact of docks and their use in the particular segments of the bay.
Areas with the highest sensitivity were deemed to be inappropriate
for new docks; areas with medium sensitivity may be able to
support new docks with additional criteria; areas of low sensitivity
could potentially be opened for new dock applications.

Step S: The data were mapped and analyzed and the cut-off
points determined (Fig. 4). Anything greater than 5.5 was deemed
to be highly sensitive.

Step 9: Additional criteria were developed for the medium and
low sensitivity areas for conservation commissions throughout the

462

Macfarlane et al.

TABLE 3.
An assessment of biological, physical, and human use impacts on the shoreline of Pleasant Bay (7/97)

Water

Navigat

#

Section

Area

Enclosed

Docks

Shellflsh

Fringe

Eelgrass

Depth

Moorings

Ch

Recreat.

TOTAL

Ranking

1

9

Bay/North Shore

0

0

0.5

0

0.5

1

0

0

0.5

2.5

Less

2

7

Little Bay

0

0

0.5

0.5

0.5

1

0.5

0

0,5

3.5

Sensitive

3

23

Old Field Pt

0

0

0.5

1

0.5

1

0

0

0.5

3.5

4

21

Chatham Harbor

0

0

0.5

0

0

1

0.5

1

4

5

11

Bay/Northwest

0

0.5

0.5

0

0.5

1

1

0

4.5

6

12

BayAVesl

0

0.5

0.5

0.5

0

1

1

0

4.5

7

22

North Beach

0

0

1

1

1

1

0

0

5

8

3A

Lonnie's Channel

0

0.5

1

0

1

0

1

5.5

9

14

Nickersons Neck

0

0

0.5

0.5

1

1

0.5

1

5.5

10

19

Frost Fish Creek

0.5

1

1

0.5

1

0.5

0

0

5.5

11

20

Ministers Pt

0

0

0.5

0.5

1

1

0.5

1

5.5

12

1

Meetinghouse

0

1

1

0.5

1

0.5

0

6

13

3

Lonnie's Pond

0.5

1

1

0.5

0

1

0

6

to

14

16

Crows Pond

0.5

1

0.5

1

0

1

0

6

15

8

Narrows. Sipsons

0.5

0.5

1

0.5

0.5

1

0.5

1

6.5

16

24

Pochet Inlet

0

1

1

0.5

1

0.5

1

0.5

6.5

17

4

Arey's Pond

0.5

1

1

0.5

1

1

0

7

18

6

Pah Wah Pond

0.5

1

1

0.5

0

1

1

7

19

10

Quanset Pond

0.5

1

1

0.5

0

1

1

7

20

13

Round Cove

0

1

1

0.5

0.5

1

1

7

21

18

Ryders Cove

0

1

1

0.5

1

1

0.5

7

22

15

Bassing Harbor

0

1

1

1

0.5

1

7.5

23

2A

Kent's Pl-Upper

1

1

1

(1-5

0.5

1

8

24

2B

Kent's Pl-Nam

0.5

1

1

1

0.5

1

8

Most

25

5

Namequoit Riv

0.5

1

1

0.5

1

1

8

Sensitive

26

17

Upper Ryders

0.5

1

1

0.5

1

1

8

Biol.. Phys.. Human Use Values = 0. .5, I

TOTAL (Sum) = 0 to 9

region to adopt in their local regulations, which will be done
through implementation.

Srep 10: As the final step, the method was presented to the
public,

CONCLUSIONS

The Bay Plan included the inethodology (described in its ap-
pendix). The public was made aware that the moratorium will
continue in the River Complex, Crow's Pond, Ryder's Cove.
Quanset Pond, Round Cove, Pau Wah Pond, the Narrows, and
Pochet. The plan was adopted in November I99S. and though
many seasonal residents were not available to comment on the plan
at that time, negative comments from them were minimal when
they returned. Additional criteria were developed by the Technical
Resources Committee (renamed after plan adoption) for areas
where docks may be permitted in the future. The Orleans Conser-
vation Commissions adopted the method and additional criteria in
December 1999 by formalizing them in their regulations; Harwich
and Chatham have not done so yet. The Bay Plan received a vote
of approval from state agencies as written, including the dock and
pier methodology.

As far as we have been able to ascertain, the methodology
described herein is the first such attempt to address the environ-
mental problems associated with private docks that was based on
a baywide approach. As resource managers, we often have to make

policy decisions based on incomplete information. We eagerly
anticipate results from research that further amplifies our knowl-
edge regarding the impacts of docks and their use on marine en-
vironments.

By eliminating the lot-by-lot procedures, we have also elimi-
nated a more subjective approach to the permitting procedure.
Using biological, physical, and human use parameters over wider
areas has resulted in a management plan that will be more difficult
to refute on appeal. However, we are willing to adjust the bottom-
line recommendations should research prove that our designations
have been overly conservative. Our charge in developing a re-
source management plan was to protect the bay. The following
statement in the introduction to the plan sums up our approach:

An i':>riuii'y left tiloiic will nurlttn' and care for itself with no
help of human hands. It is only when human activities in-
teifere with natural processes that the Bay responds by
slum-in^ signs of stress, damage and disease. This steward-
ship plan for Pleasant Bay is based on the premise that
hninan inler\ention in the natural processes of the Bay mast
be minimized. With this premise, the plan seeks to encour-
age a level (tf human u.w which does not upset the balance
of the Bay's ecosystem, endanger the productivity of its
wildlife, or invade the tranquility of those who seek its
shores. Accomplishing this will require residents, visitors
and commercial interests alike to place the long-term health
of the Bay above individual interests. It will require change
and sacrifice, and on-going ctmimitment to preser\-ing the
health, beaiitv. antl tratu/uilitv of Pleasant Bav for future
geiu'rations.

A Resource-based Dock Assessment Methodology

463

Pleasant Bay
Marine Features

Draft

UCH

CZM

Co*<t>l Zona h^jM^errmtt by ■ frvilfn>m
th* CXnc* al Ocwn nJ GiMt KoourcM
MmmiiMiC. NiOonal Ocaanlc ml
Aovo»c*>m^ MfnJntafriOon, and ffx U^,
Onptlnvrl of Convnarts. Thavtawt
»«liiMmf tra ChOH of lti« •u(ho4<tl
•rv] do rat imiiMiilf) n/Wl Ova <i4vnii
irf NOi^A w arrr a< ta (ub ' atendoa. Thlt
Infonnaflon h ivW labia In aRafnatli*

' ' Town Boundary
■^ Major Road

M^ CreaUd on la/^VY 16 ' ^^

Shoreline, ponds, and town boundaries from
towns' assessor maps of various dates with
modifications made by CCC CIS in 1997 based
on ^e Department of Environmental Protection
wetland mapping aerial photos (photos from
March 1993).

Ponds, streams, major roads - MassCiS;
digitized from USGS quadrangles, 1 :25000
scale, various dates.

Figure 4. Map of the Pleasant Bay Resource Management Plan subsections based on the numerical sensitivity index.

464

Macfarlane et al.

LITERATURE CITED

Burdick. D. M. & F. Short. 1995. The effects of boat docks on eelgrass
beds in Massachusetts coastal waters. Massachusetts Office of Coastal
Zone Management. Jackson Estuarine Laboratory. Durham. New
Hampshire.

Carriker, M. R. 1961. Interrelation of functional morphology, behavior and
autecology in early stages of the bivalve Mercenaria mercenaria. J.
Elislui Mitchell Sci. Soc. 77:168-241.

Crawford. R. E., C. J. Kamond & K. Blake. 1994. Recreational boating on
Waquoit Bay: Use and practices. Technical Report No. 102. Waquoit
Bay NERR. Waquoit. Massachusetts.

Crawford, R. E., N. E. Stoipe & M. J. Moore. 1998. The Environmental
Impacts of Boating. Proceedings of a workshop held at Woods Hole
Oceanographic Institution, Woods Hole. Massachusetts. December
7-9. 1994. Technical Report WHOI-98-03. Woods Hole Oceano-
graphic Institution. Woods Hole. Massachusetts.

Geist. M. A. (ed.). 1996. The ecology of the Waquoit Bay National Es-
tuarine Research Reserve. Massachusetts Department of Environmental
Management, Forest and Parks-Region 1 . Waquoit, Massachu.setts. 1 13
pp,

Hilton, J. & G. L. Phillips. 1982. The effect of boat activity on turbidity in
a shallow broadland river. J. Appl. Ecol. 19:143-150.

Short. F. T.. D. M. Burdick, J. S. Wolf & G. E. Jones. 1993. Eelgrass in
Estuarine Research Reserves along the East Coast. U.S.A.. Part I; De-
clines from pollution and disease; Part II: Management of eelgrass

meadows. NOAA Coastal Ocean Program. Durham. New Hamp.shire.

107 pp.
Walker. D. I.. R. J. Lukatelich. G. Bastyan & A. J. McComb. 1989. Effect

of boat moorings on seagrass beds near Perth. Western Australia.

Aciiiul. Bot. 36( 1 ):69-78.
Weis. J. S. & P. Weis. 1992a. Construction materials in estuaries; reduction

in the epibiotic community on chromated copper arsenate (CCA)

treated wood. Mar. Ecol. Prog. Ser. 83:45-53.
Weis. J. S. & P. Weis. 1992b. Transfer of contaminants from CCA-treated

lumber to aquatic biota. / Exp. Mar. Biol. Ecol. 161:189-199.
Weis. P.. J .S. Weis & L. M. Coohill. 1991. Toxicity to estuarine organisms

of leachates from chromated copper arsenate treated wood. Arch. En-
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Weis. P.. J. S. Weis & T. Proctor. 1993. Copper, chromium and arsenic in

estuarine sediments adjacent to wood treated with chromated-copper

arsenate (CCA). Est. Coastal Shelf Sci. 3,6:1 \-19.
Wetzel R. L. & P. A. Penhale. 1983. Production ecology of seagrass

communities in the lower Chesapeake Bay. Mar. Technol. Soc. J. 17:

22-31.
Yousef. Y. A. 1974. Assessing effects on water quality by boating activity.

U.S.E.P.A.. EPA Tech. Serv. No. EPA-670/2-74-072.
Yousef. Y. A., W. M. McClellon & H. H. Zebuth. 1980. Changes in

phosphorus concentrations due to mixing by motorboats in shallow

lakes. Water Re.';. 14:841-852.

Journal of Shellfish Research. Vol. 14. No. 1. 46.'i-;66. 2000.

POOR WATER QUALITY? NOT IN MY BACKYARD! THE EFFECTIVENESS OF

NEIGHBORHOOD POND ASSOCIATIONS IN THE PROTECTION AND IMPROVEMENT OF

SHELLFISH GROWING WATERS ON MARTHA'S VINEYARD

RICHARD C. KARNEY

Martha 's Vineyard Shellfish Group. Inc.

P.O. Box 1552

Oak Bhijfs. Ma.ssachusetts 02557

ABSTRACT Neighborhood pond associations have proven to be an especially effective force in the protection and enhancement of
local coastal ecosystems. Environinental managers who are wise enough to forge partnerships with these local organizations will be
rewarded with the energy, commitment, and passion reserved for issues that hit close to home. With the vigilance and dedication of
a Neighborhood Crime Watch, local pond associations are the eyes and ears that sound the first alerts of environmental pollution. With
the efficiency of local Minutemen, they rally to the cause as volunteer environmental advocates who offer financial commitment, a
wealth of expertise, and effective political organization.

The environmental accomplishments of neighborhood groups on Martha's Vineyard are impressive. The Edgartown Harbor Asso-
ciation funded a water quality study resulting in the establishment of a free sewage pump-out facility for boaters. The Friends of
Sengekontacket (Pond) provided the leadership to coordinate a local, state, and federal partnership to complete a major dredging project
that restored filled shellfish habitat and nourished an eroding barrier beach. The Lagoon Pond Association funded a court battle to limit
pier construction. The Tisbury Great Pond Think Tank and Tisbury Waterways, Inc. have addressed farm and roadway runoff with
fencing, buffer strips, and innovative catch basins. All have conducted successful public education programs, water quality monitoring
studies, and fundraising activities designed to protect shellfish habitats and water quality. Many have advanced protective zoning
initiatives.

KEY WORDS: Martha's Vineyard, water quality, neighborhood pond associations

Located abotit 7 miles off of Cape Cod in southeast Massachu-
setts, the 100 mi" island of Martha's Vineyard has historically had
an economy based on fishing and farming. Its rural seaside ambi-
ance has made it a popular tourist destination; so much so. that
now 95% of the island's economy is tied to tourism, seasonal
residents, and vacation home development. In the past 20 years
alone, the year-round and summer populations have doubled to
12,200 and 72,600, respectively. On a big holiday weekend, police
estimate that the population can soar to nearly 150.000.

This recent dramatic increase in human population and activity
threatens the shellfish resources. The increases in the number of
houses, boats, piers, and paved roads have taxed the island's natu-
ral systems. Ground and surface waters are at increased risk from
bacterial, chemical, and nutrient pollution, and the growth in boat-
ing and the associated construction of piers are destroying shellfish
habitat. Preserving and enhancing shellfish resources in the wake
of this explosive development is a monumental challenge.

The historical importance of the local shellfish industry has
been a key factor in its preservation. On Martha's Vineyard, shell-
fishing is a cultural icon on par with the proverbial motherhood
and apple pie. It should come as no surprise that when shellfish
beds began to close due to high fecal coliform counts, the populace
of Martha's Vineyard rallied. Neighborhood pond associations
were formed and/or strengthened in response to developments that
threatened the island's shellfish resources. Due in part to their
vigilance, a remarkable 98.8'7f of the Island's 176.261 acres of
shellfish waters are approved for harvest. Neighborhood pond as-
sociations are active participants in the protection and management
of all the significant shellfish growing waters on the island. These
associations have proven to be an especially effective force in the
protection and enhancement of local coastal ecosystems.

Although some associations existed as social organizations pre-
viously, the majority of the organizations formed in the late 1980s
and early 1990s in direct response to the environmental problems

that followed the building boom in the mid 1980s. Water closures
due to fecal coliform and proposed condominiums and subdivi-
sions were the impetus for the formation of the groups. The Chil-
mark Ponds and Lagoon Pond associations are primarily composed
of waterfront homeowner associations with 120 and 125 members,
respectively. The Edgartown Harbor Association, the Friends of
Sengekontacket (FOS). and Tisbury Waterways. Inc. (TWI) have
larger memberships (200-450) that include contributors without
pond frontage. Most of these organizations have tax exempt
501(c)(3) status, which encourages tax -deductible memberships.

The Tisbury Great Pond Think Tank. Edgartown Ponds District
Advisory Committee, and Squibnocket Pond District Advisory
Committee are more accurately described as agencies of local
government, but all have significant neighborhood representation.
Boldwater Association, a landowners group, is active in the Edgar-
town Ponds District, and the Riparian Landowners of Tisbury
Great Pond participate in the Think Tank. Local government man-
dates the pond district advisory committees; the Think Tank is an
ad hoc committee.

The regular annual budgets of these organizations are between
$4000 and $25,000, which is usually raised through memberships,
fundraising projects, and grants. Special projects have solicited
much greater funding. The Lagoon Pond Association raised
$75,000 to support the legal defense of its stand to limit construc-
tion of private docks. The Edgartown Harbor Association raised
$750,000 in private donations to fund a Woods Hole Oceano-
graphic Institution (WHOI) water quality study, which included a
floating laboratory and scientific equipment. The Friends of Sen-
gekontacket likewise has funded (over $90,000) studies by WHOI.

The effectiveness of these organizations is largely a function of
their dedication. En\'ironmental managers who have forged part-
nerships with these organizations have been rewarded with the
energy, commitment, and passion that are reserved for issues
"close to home." Waterfront land values are dependent on good

465

466

Karney

water quality, and local pond associations are the eyes and ears that
sound the first alerts of environmental pollution. With the effi-
ciency of local Minutemen. pond associations rally to the cause
with volunteer environmental advocates who provide financial
commitment, a wealth of expertise, and effective political organi-
zation. Most of the important shellfish ponds on Martha's Vine-
yard have shared jurisdiction between neighboring towns. The
pond groups have provided valuable forums to coordinate the ef-
forts of the boards of the adjacent towns, resulting in uniform
management policies for the ponds.

The environmental accomplishments of neighborhood groups
on Martha's Vineyard are impressive. All of the groups have been
effective in funding water quality studies in their respective water
bodies. Funding for these studies has been secured through private
donations, town appropriations, and state and foundation grants.
The Chappaquiddick Island Association, the Edgartown Harbor
Association, and the Friends of Sengekontacket have contracted
with WHOI for extensive investigations into sources of fecal
coliform contamination and baseline surveys of chemical param-
eters. The Friends of Sengekontacket has funded inquiries into the
impacts of dredging and proposed wastewater treatment, and
mapped the bathymetry of its pond. The Tisbury Great Pond Think
Tank has initiated land use studies for its watershed. Groundwater
studies in the vicinity of Edgartown Great Pond, Tisbury Great
Pond, and Chappaquiddick Island have been prompted by the pond
groups. The Lagoon Pond Association funds an annual water-
quality-monitoring program and conducts a survey yearly of the
number of boat moorings. The Massachusetts Division of Marine
Fisheries has conducted finfish surveys in Edgartown and Squib-
nocket ponds after urging by pond groups.

Armed with data from these studies, the pond associations have
implemented a multitude of corrective measures. They have pres-
sured the respective boards of Health to inspect and replace failed
septic systems. The Lagoon Pond Association has also encouraged
removal of underground oil tanks. Both the Edgartown Harbor
Association and Tisbury Waterways, Inc. have addressed boat-
related pollution. Edgartown presently provides free pump-out of
boat sewage at its wharf. Tisbury Waterways, Inc. was instrumen-
tal in launching the traveling barge PU. E - II, which pumped
10,000 gallons of sewage waste from boat head-tanks in its second
year of operation. Tisbury Waterways, Inc. also funds a summer-
time assistant to the harbormaster. This assistant patrols Lake
Tashmoo distributing pamphlets and educating boaters about
proper disposal of head tank waste.

The Lagoon Pond Association has proven itself to be an effec-
tive local political force. It was successful in its efforts to have the
pond and its near shoreline declared a District of Critical Planning
Concern and was instrumental in the establishment of pier guide-
lines to protect shellfish and eelgrass habitats.

Runoff from point sources and nonpoint sources has been ad-
dressed by a number of the organizations. The Think Tank mapped
all road drainages into the shellfishing pond, and designed and
implemented measures to redirect the road effluent into adjacent
wetlands for filtration. The Think Tank worked with waterside
farmers to fence livestock away from the shoreline, to adopt best
management practices for handling manure, and encouraged the
use of vegetative buffers to reduce agricultural runoff. Tisbury
Waterways, Inc. in a cooperative project with the Board of Health
secured a state grant for $50,000 to install and monitor innovative
limestone catch basins to remove oils and metals, and neutralize
acid-rain runoff.

The Chilmark Ponds Association, the Friends of Sengekon-
tacket, and Tisbury Waterways, Inc. have coordinated and expe-
dited dredging projects to remove sediments and improve circula-
tion. Both Chilmark Ponds and the Friends of Sengekontacket.
through its Barrier Beach Task Force, have developed manage-
ment programs for the beaches and dunes adjacent to their ponds.

Public education is a high priority for the local associations.
Through newsletters and annual reports, the membership are kept
informed of ongoing projects and are provided with steps that they
can take to ensure good water quality. The Edgartown Ponds Area
Advisory Committee newsletter has a regular feature called Pond
Reminders, which informs readers, among other things, that boats
with antifouling paint are not allowed and that by law, a 100 ft
no-cut buffer of natural vegetation must be maintained along the
shore. The Think Tank produced and distributed a brochure with
similar buffer recommendations, including a suggested list of na-
tive vegetation for landscaping. Both the Friends of Sengekon-
tacket and Tisbury Waterways, Inc. have provided support
for water quality education programs in the local schools. The
Friends of Sengekontacket sponsors an annual "Carry
In-Carry Out" anti-litter poster-contest for students, and the
winning designs are posted on the island's ferries and at beach
entrances.

In conclusion, neighborhood associations are established and
potent forces in the preservation of shellfish habitats on Martha's
Vineyard. They are models for effective stewardship of shellfish
growing waters and should be duplicated elsewhere.

Journal of Shellfish Research. Vol. 19, No. 1, 467-172, 2000.

MAKING A CASE FOR COMMUNITY-BASED OYSTER RESTORATION: AN EXAMPLE FROM

HAMPTON ROADS, VIRGINIA, U.S.A.

ROBERT D. BRUMBAUGH,' LAURIE A. SORABELLA,'
CARENE OLIVERAS GARCU,' WILLIAM J. GOLDSBOROUGH,^
AND JAMES A. WESSON'

Chesapeake Bay Foundation

142 W. York Street

Suite 318

Norfolk, Virginia 23510
'Chesapeake Bay Foundation

162 Prince George Street

Annapolis, Maryland 21401
' Virginia Marine Resources Commission

P.O. Box 756

2600 Washington Avenue

Newport News, Virginia 23607

ABSTRACT The eastern oyster [Crassostrea virainka) remains at historically low levels throughout the Chesapeake Bay. Recent
efforts to restore oysters in the bay have focused on establishing a series of sanctuaries, or no-take zones, to increase oyster broodstock
in selected tributaries. Oyster parasites continue to affect the rate of recovery in these tributaries; however, innovative management
strategies, advances in aquaculture technology, and the availability of disease-tolerant broodstock from the lower Chesapeake Bay are
providing ways to involve the public directly in restoration of this resource. A 1996 management decision to transplant large
wild-caught oysters onto an oyster broodstock sanctuary reef in the Great Wicomico River, Virginia, was followed by greatly increased
abundance of juvenile oysters throughout that river in 1997. Using that result as a model for strategic oyster reef restoration, citizens
and school students have been enlisted to grow large numbers of hatchery-produced native oysters for restocking other sanctuary reefs
throughout Chesapeake Bay. Efforts to supplement natural oyster populations in Hampton Roads, Virginia, began in May 1998, with
the transplanting of 65,000 hatchery-produced oysters grown by school students. The oysters were transplanted onto strategically
located sanctuary reefs constructed in the Lynnhaven and Elizabeth rivers. Surveys of these reefs following the oysters' spawning
season have revealed order-of-magnitude increases in the abundance of juvenile oysters on both reefs, and correspondingly high spat
settlement rates on oyster grounds surrounding the reefs. These results demonstrate that stocking strategically located broodstock reefs
with hatchery-produced oysters grown by citizens can be an effective strategy for oyster restoration in the Chesapeake Bay.

KEY WORDS: Crassostrea virginica. oyster, habitat, restoration, fisheries management

INTRODUCTION studies suggest that restoring oyster populations to the Chesapeake

Bay, particularly along the shallow margins, could significantly

The tremendous decline in the abundance of the eastern oyster, reduce concentrations of suspended particulates, improve light
Crassostrea virginica. in the Chesapeake Bay is attributed to sev- penetration, and increase dissolved oxygen concentrations in the
eral factors: excessive harvest pressure in the late 1880s and early bottom waters (Newell 1988: Ulanowicz and Tuttle 1992: Gerrit-
1900s, declining water quality and increasing sedimentation rates, sen et al. 1994; Kennedy 1996). Indeed, the concept of restoring
and more recently, the presence of two disease-causing oyster water quality through revitalized oyster stocks is increasingly un-
parasites commonly known as MSX and Dermo (Kennedy and derstood and accepted by the public, as evidenced by a recent
Brei&ch 1983: Hargis and Haven 1988, 1995: Ford and Tripp article in U.S. News & World Report (Zimmerman 1997) that
1996). As a result, oyster landings have plummeted in Virginia to described oyster restoration in the Chesapeake Bay as one of "Six-
less than 1% of the levels in the mid 1900s (Fig. 1). As recently as teen Smart Ideas to Save the Vv'orld."

the 1980s, the oyster fishery was the most valuable commercial Recent efforts to restore oysters to Virginia's tributaries have

fishery in the bay. At present, however, Virginia's oyster fishery two primary strategies: construction of oyster habitat using large

supports the equivalent of a mere seven full-time jobs, and the volumes of shell to recreate three-dimensional reefs on historic

region's oyster-processing industry relies on the importation of oyster grounds, and management of the remnant fishery to increase

oysters from outside state waters to support public demand for this the abundance and size of oyster broodstock on public oyster

resource (Kirkley 1997). grounds. A system of reconstructed reefs designated as oyster

In addition to the economic value that might be realized from broodstock sanctuaries (no-take zones) have been established in

a restored oyster fishery, a broad-based effort to restore the Chesa- Virginia tributaries (Fig. 2). The sanctuary reefs are intended to

peake's oyster populations could also yield profound ecological allow oysters to accumulate, mature, and reproduce, thereby en-

benefits. The role of oysters as a dominant suspension-feeder is hancing local oyster populations. An experiment in the Great

well documented. For example, Newell (1988) estimated that his- Wicomico River in 1996 used large adult oysters purchased from

toric (pre- 1870) oyster populations were capable of processing oyster fishermen to stock one such reef The experiment resulted in

significant fractions of the bay's water volume each day. Recent a significant increase in oyster settlement onto the sanctuary reef

467

468

Brumbaugh et al.

Year

{D Public Landings ■Private Landings I

Figure 1. Oyster landings in Virginia between 1957 and 1998. Land-
ings are divided between private and public oyster grounds.

and suiTounding oyster grounds the following year (Fig. 3A,B)
(Southworth and Mann 1998). Using this result as a model for
oyster restoration in other tributaries, a program involving private
citizens and school students in the grow-out of hatchery-produced
oysters for stocking sanctuary reefs was implemented in Hampton
Roads, Virginia, in the lower Chesapeake Bay. In the first year of
this effort, approximately 90,000 hatchery-produced oysters grown
by citizens and students were transplanted to reefs located in the
Lynnhaven and Elizabeth rivers.

Data collected by students participating in the Chesapeake Bay
Foundation's (CBF) Student Oyster Corps, along with surveys of
the sanctuary reefs after the transplanted oysters had spawned,
were used to evaluate the success of this project. The results dem-
onstrate that a citizen-based restoration effort using strategically
located sanctuary reefs and hatchery-produced oysters can lead to
significant increases in local oyster stocks.

METHODS

Large, presumably disease-tolerant oysters were collected from
the Lynnhaven River in spring of 1997 and transported to a com-
mercial hatchery for spawning (Middle Peninsula Aquaculture.
North, Virginia). Oyster larvae were settled onto shell grit to pro-
duce "cultch-less," or individual, juvenile oysters. The broodslock
oysters were spawned in the hatchery on June 5, 1997. and juvenile
oysters with a mean size of 26.2 mm were distributed to school
classes on October 4, 1997.

Teachers and students from 26 middle school and high school
classes grew the halchery-prodiiccd oysters in floaling cages se-
cured to docks in tidal waters near each school. Students from each
class constructed lloating cages ineasuring 8 x 2 x I ft (244 x 61
X 30 cm) using sewer-grade PVC pipe and vinyl-coated wire mesh
(14-gauge 2.'i-mm square mesh). Fach cage contained 2000 oysters
divided between iwii large plastic mesh bags (mesh si/e ."i mm).
The cages were placed in the water at a dock or marina convenient
to each class's school and were monitored monthly. When appro-
priate, oysters were transferred to bags with larger mesh sizes to
minimize restriction of water Oow.

Students monitored oyster growth anil sur\i\al and measured
surface water temperature, salinity, ani.1 (iirhjilil\; moiilliK data

sheets were submitted to CBF throughout the school year. Random
samples of 40 oysters were taken from each floating cage (20 from
each of the two mesh bags) and measured to the nearest millimeter.
Each class was provided with a refractometer (SPER Scientific
model A366ATC) and was instructed to calibrate the instrument
with distilled water prior to use each month. Secchi disks (20 cm
diameter) were provided for measuring water clarity.

At the end of the school year, the students transplanted their
oysters to sanctuary oyster reefs located in the Lynnhaven and
Elizabeth rivers in the lower Chesapeake Bay. The oysters were
transplanted to the reefs in high densities (approximately 200-300/
m") just below the MLW level in an effort to increase fertilization
success upon spawning. The reefs in the Lynnhaven and Elizabeth
rivers were constructed in May 1997 and May 1998, respectively,
using barge loads of clean oyster shells deposited on the river
bottom. Both reefs are approximately one-half acre in size and rise
1-2 m above the bottom, extending to approximately 0.25 m above
the MLW level.

Data submitted by the school classes was pooled across sites to
obtain monthly averages for oyster shell length, salinity, tempera-
ture, and water clarity. Monthly growth rates were computed and
correlated to water quality parameters. After the transplanted oys-
ters had spawned, recently settled juveniles (spat) were surveyed

Virginia Oyster Reef Resloration Sites

"?>3

\ "^

/J

^V^' Mi

I'iuiiru 2. I.tiialions of saniiiKir\ riel's In \ irnlnia's piirlitin iif Che.sa-
peake Hay. Inset shows loialions ol' sanctuary reefs In the Lynnhaven
and Klizabeth rivers in Hampton Roads, Virginia.

Oyster Restoration in Hampton Roads. Virginia

469

1996

1997

Year

B 1200

1994

1995

Year

.Shell Bar (0.5 nri) . .». . Haynie Bar (0.8 rrt)
-Cranes Creek (3 5 rri) — » —Reeton Bar (4 5 rri)

A— Sandy TOnl (1 .0 ni)

_ 0- - Whaley's Rat (6 0 rri)

Figure 3. (A) Mean abundance of juvenile oysters (spat/m^) on tlie
Great Wicomico River sanctuary reef. (B» Mean abundance of juvenile
oysters (spat/m-| on public oyster grounds in the Great Wicomico
River. Symbols represent different natural oyster bars near the reef.
Distance from the reef is in parentheses.

on both reefs in October 1998 using 0.25-m- quadrats. Twelve
replicate samples were obtained (using SCUBA) from each reef,
with samples divided evenly among high, middle, and low reel
elevations. Additional samples were taken from oyster grounds
throughout the Lynnhaven River that were exposed at low tide.
These sample were used to determine how far from the reef oyster
spat might have been transported prior to settlement.

RESULTS

Student-grown oysters increased from 26.2 mm in October
1997 to approximately 50 mm by June 1998, when the oysters
were transplanted to sanctuary reefs. A clear seasonal cycle of
growth was evident, with minimal growth observed between De-
cember 1997 and April 1998 (Fig. 4A). Juvenile mortality was less
than 1 % throughout the school year. One hundred oysters from the
same cohort were retained in floating cages and monitored for
growth and survival throughout 1998. By October 1998. these
oysters had increased to 77 mm. and mortality was only 6%.

Both temperature and salinity exhibited seasonal cycles within
documented ranges that favor oyster growth and survival (Fig. 4B)
(Galtsoff 1964: Loosanoff 1953. 1958). Temperature varied from

1 1 °C in the winter to 27 °C in July 1998. Mean salinity reached
a maximum of 25 ppt in October 1997, and a minimum of 10 ppt
in February 1998 following several winter "Nor'easter" storms
that brought heavy precipitation to the region between December
and February. Salinities remained low through the spring as a
result of higher than average precipitation rates in the region.
Water clarity (Secehi depth) showed two maxima in December and
March and ranged from less than 1 m to more than 2 m throughout
the year. The peak in December most likely reflects the decline in
phytoplankton biomass following the fall bloom, while the de-
crease through February was most likely associated with heavy
runoff and suspended solids from winter storm events.

A strong correlation was observed between salinity and growth
rate (r = 0.86. P < .003). However, there was no apparent corre-
lation between temperature and growth, or between water clarity
and oyster growth. Spat settlement increased dramatically on the
Lynnhaven River sanctuary reef between 1997 (prior to stocking)
and 1998 (post-stocking), from 8 to 181 spat/nr (Fig. 5A). While
similar pre-stocking data are not available for the Elizabeth River
reef, spat settlement there in 1998 was similar in magnitude to the
Lynnhaven reef In addition, dredge samples taken from shell bot-
tom close to the Elizabeth River reef suggest the observed settle-
ment rates represent a substantial increase over previous years.

Month

25

-■

1

1"

\

y

20

\

/

i

I

15

"N

\

"^

•
X

^

\

\
\

•

,'

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10

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.5'

***»^

♦

*

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5

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" N

0

1^

CO

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" «> s

§

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CD

<T>

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jb

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3 £
Q.
(U
O

C/5

Month

-TOf^O^TURE _ -

. SALNITY . -♦- - SBXH DBTH

Figure 4. (A) Mean size of hatchery-produced oysters grown by stu-
dents. (B) Mean monthly surface water temperature, salinity, and wa-
ter clarity.

470

Brumbaugh et al.

0)

E

to
a.

180
160
140
120
100

eo

60
40
20

0

Lynnhayen
Ri\er reef
stocked
5/19/98

Location and Year

Western
Brancli
(1.0 ni)

Location and distance from reef

Figure 5. (A) Mean abundance of juvenile oysters (spal/ni") on Hamp-
ton Roads sanctuary reefs. (B) Mean abundance of juvenile oysters
(spat/m") on intertidal oyster grounds in the Kynnhaven River in 1998.
Distance from the reef is in parentheses.

DISCUSSION

The results of this first year ot effort demonstrate that stoctcing
even modest inimbers of hatehery-produced oysters derived from
hardy broodstocl\ is useful for restoration of the Chesapeake Bay's
oyster populations. Spawning by oysters from the same cohort held
in floating cages through the summer was observed on July 10.
lyys (pers. obs.), which supports the notion that the transplanted
oysters are capable of spawning after being transplanted to the
reefs. Dredge samples taken near the Elizabeth River reef revealed
that spat were 10-20 times more abundant than juvenile and adult
oysters, suggesting poor recruitment prior to H)')8. Moreover,
there are few "boxes," or empty shells, which indicates that recent
oyster mortality is relatively low. Therefore, settlement appears lo
be a limiting factor for oyster populations in this river.

.Similarly, spat were frequently the only live oysters IihiikI in
quadrat samples from unreslored shell botlom in the l.vnnhaven
River. Spat abundance on natural shell botlom was highest near the
reef and decreased with distance from the Lynnhaven reef (Fig.
5B), similar to the trend observed in the Great Wicomico River in
1997 (Fig. 38).

While the absolute abundance of spat differs belwcen the Circal
Wicomico River and Lynnhaven River reefs (S.'id versus 181 spat/

nr), the order-of-magnitude increases in spat settlement observed
in both systems following initial stocking efforts are strikingly
similar. It is worth noting that in places like the Great Wicomico
River, where "background" spat settlement in recent years has
been on the order of 100 spat/nr. the impact of stocking efforts as
modest as those conducted in Hampton Roads in 1998 may not be
detectable. However, in places like the Lynnhaven and Elizabeth
rivers where spat settlement rates in recent years have been con-
siderably lower (e.g.. 8 spat/nr in the Lynnhaven in 1997). these
effects are more easily discerned.

The data collected in 1997 and 1998 by the student volunteers
demonstrate that, even in highly urbanized areas, restoration ef-
forts using hatchery-produced oysters and small-scale aquaculture
techniques can be effective. Juvenile mortality was very low
throughout the school year, and temperature and salinity ranges
were suitable for oy.sters to grow to maturity by mid-summer after
their first year of growth. Although the effect of suspended par-
ticulate matter on oyster feeding rates is well documented (e.g.,
Loosanoff 1962; Loosanoff and Tommers 1948). there did not
appear to be any relationship between water clarity and oyster
growth in this study. It is possible that suspended sediment levels
never exceeded threshold levels that decrease feeding rates, or that
the particulate material in the water column was not of an appro-
priate size fraction to affect the oysters" growth adversely. More
likely, growth was the result of synergistic effects between two or
more of the water quality parameters measured by the students
(Kennedy 1991). These relationships will be examined more
closely as more data are obtained through this program in subse-
quent years.

The Lynnhaven River is located in the high-salinity portion of
lower Chesapeake Bay where diseases have caused extensive oys-
ter mortality in recent decades (Buireson and Calvo 1996, and
references therein). Large, isolated oysters that survive in such
areas are thought to be more tolerant of the parasites MSX and
Dermo than smaller oysters (Gaffney and Bushek 1996) and thus,
are deemed more valuable as broodstock. While the ability of
transplanted oysters to pass on the trait of disease-tolerance is
poorly understood, concentrating large, presumably disease-
tolerant oysters on these reefs is thought to offer some hope of
overcoming the reproductive failure resulting from low densities
of broodstock. Surveys of the reefs and surrounding bottom in
future years will better illustrate the effect of broodstock selection
on restoration efforts.

Our strategy of using hatchery-produced shellfish as brood-
stock parallels efforts to restore shellfish stocks in other systems.
For example, in the Westport River, Massachusetts, a community-
based effort to restore bay scallops, Argopecten irnulians. has
been undertaken using hatchery-produced scallops and spat col-
lector bags deployed in arrays throughout that system (Tammi et
al. 1998; Turner and Soares 19981. In areas within that river having
historically high settlement of scallops, hatchery-produced scal-
lops were grown lo maturity by volunteers and held in floating
cages in an attempt to enhance scallop fertilization rates. Spat
collectors, consisting of monofilament fishing net material, were
manufactured by school students and cili/en \iiktnteers and used
lo collccl juxenilc scallops tor lurlhcr eiihaiicemcnt of natural
stocks.

One of the less tangible, but nevertheless important, benefits of
the Virginia oyster restoration effort is the increased public aware-
ness that has followed. Funding for reef restoration efforts typi-

Oyster Restoration in Hampton Roads, Virginia

471

cally has been through grants to the Virginia Marine Resources
Commission (VMRC) from outside sources, such as the U.S. En-
vironmental Piotection Agency (USEPA), National Oceanic and
Atmospheric Administration (NOAA), and Virginia's Chesapeake
Bay Restoration Fund (whose monies are derived from the sale of
"Friend of the Bay" license plates). As a result of these early
restoration successes additional private sources of funding are be-
ing made available for restoration work. For example, the Rotary
Club of Norfolk, Virginia, committed $28,000 toward construction
of two additional reefs in Hampton Roads, which were constructed
in the Lafayette River in \999.

In many parts of Hampton Roads, water quality conditions
prevent or restrict the taking of shellfish for human consumption.
Polluted waters are classified as either restricted, whereby shellfish
must be relayed according to specific state guidelines, or prohib-
ited, in which case no shellfish may be removed for consumption.
Despite these conditions, there is increasing public support for
restoring oyster reefs strictly for their ecological potential, such as
increased water clarity and the provision of fish habitat. The public
perception of oysters as a keystone species in the bay, combined
with existing support for the continuation of fishery enhancement
efforts, is especially encouraging. It demonstrates public commit-
ment to programs designed to restore the Chesapeake Bay, and an
increasing appreciation for the ecological, as much as economic,
importance of natural resources.

Since the initiation of this restoration project, CBF's Student
Oyster Corps has expanded to include more than 90 classes bay-
wide. A local citizens group called Restore the Oyster formed in
1997 to help advance oyster restoration efforts in Hampton Roads
by recruiting citizens to grow oysters for stocking local reefs. More

than 230,000 hatchery-produced oysters reared in floating cages by
citizens and students in CBF's Oyster Corps program were trans-
planted to the reefs in 1999. Additionally, selectively bred oysters
have been made available to commercial hatcheries and are now
being used to produce seed oysters for this effort. The first of those
oysters were transplanted to reefs in the spring of 1999. Continued
monitoring of the sanctuary reefs and surrounding oyster grounds
will better define the impacts that these stock-enhancement activi-
ties are having on local oyster populations.

ACKNOWLEDGMENTS

We greatly appreciate the dedication of the 26 teachers and
more than 1500 students who participated in the first year of
Chesapeake Bay Foundation's Student Oyster Corps project. They
collected much of the data used in this study and helped to shape
future restoration efforts. Ken Kurkowski of Middle Peninsula
Aquaculture and Peter Perina of Eastfield Farms contributed to the
success of this program through their participation in educational
workshops. Funding for the construction of the sanctuary reefs in
the Lynnhaven and Elizabeth rivers was provided by the USEPA
Chesapeake Bay Program, NOAA Chesapeake Bay Office/Office
of Habitat Conservation, and the Virginia Chesapeake Bay Resto-
ration Fund. Funding for the first year of Chesapeake Bay Foun-
dation's Student Oyster Corps project was provided by grants to
Chesapeake Bay Foundation by Toyota and Landmark, Inc./The
Virginian-Pilot. Carene Oliveras Garcia was supported as a Chesa-
peake Bay Foundation Summer Intern by the QEM/NASA Sharp
Plus Program through Norfolk State University. Lisa Drake pro-
vided valuable comments on an earlier draft of this manuscript.

LITERATURE CITED

Burreson. E. M. & L. M. Ragone Calvo. 1996. Epizootiology of Perken.sis
marinus of oysters in Chesapeake Bay. with emphiisis on data since
1985. J. Shellfish /?«.I5:17-34.

Ford, S. E. & M. R. Tripp. 1996. Diseases and defense mechanisms, pp.
581-660. In: A. F. Eble. V. S. Kennedy & R. I. E. Newell (eds.). The
Eastern Oyster, Crassostrea virginica. Maryland Sea Grant. College
Park. Maryland.

Gaffney, P. M. & D. Bushek. 1996. Genetic aspects of disease resistance
in oysters. J. Shellfish Res. 15:135-140.

Galtsoff, P. S. 1964. The American oyster, Crassostrea virginica Gmelin.
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Gerritsen. J., A. F. Holland & D. E. Irvine. 1994. Suspension-feeding
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Hargis, W. J. & D. S. Haven. 1988. The imperiled oyster industry of
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Hargis. W. J. & D. S. Haven. 1995. The precarious state of the Chesapeake
public oyster resource, contribution no. 1965. Virginia Institute of Ma-
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Kennedy, V. S. 1991. Eastern oyster, Crassostrea virginica. pp. 3.1-3.20.
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Habitat Requirements for Chesapeake Bay Living Resources, second
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Kennedy, V. S. 1996. The ecological role of the eastern oyster, Crassostrea
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Kennedy. V. S. & L. L. Breisch. 1983. Sixteen decades of political man-
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Kirkley. J. 1997. Virginia's Commercial fishing industry: its economic
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Gloucester, Virginia. 77 pp.

Loosanoff, V. L. 1953. Behavior of oysters in waters of low salinity. Proc.
Natl. Shellfish. As.mc. (1952):I35-151.

Loosanoff. V. L. 1958. Some aspects of behavior of oysters at different
temperatures. Biol. Bull. 114:57-70.

Loosanoff, V. L. 1962. Effects of turbidity on some larval and adult bi-
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Newell. R. I. E. 1988. Ecological changes in Chesapeake Bay: are they the
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Tammi, K. A.. S. J. Soares. W. Turner & M. A. Rice. 1998. Settlement and

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Journal of Slwllfisit Research. Vol. 19. No. 1, 473-476. 2000.

JUMP RUN CREEK SHELLFISH RESTORATION PROJECT

NANCY M. WHITE,' DANIEL E. LINE,^ J. D. POTTS,'

WILLIAM KIRBY-SMITH,^ BARBARA DOLL,' AND W. F. HUNT''

School of Design

North Carolina State University

Box 7701

Raleigh. North Carolina 27695
'North Carolina State University Water Quality Group

Box 7637

Raleigh. North Carolina 27695

North Carolina Department of Environment and Natural Resources

Shellfish Sanitation Section

P. O. Box 769

Morehead City. North Carolina 28557
^Dnke Marine Lab

111 Pivers Island Road

Beaufort. North Carolina 28516
'North Carolina Sea Grant

Box 8605

Raleigh. North Carolina 27695

Biological and Agricultural Engineering

North Carolina State University

Box 7625

Raleigh. North Carolina 27695

ABSTRACT The objective of this multi-agency project is to I ) quantify the effects of land use change on shellfish closures and 2)
assess techniques that can be used mitigate those impacts. This report is based on preliminary analyses conducted over the last 1 8
months. The project focuses on the 320-ha watershed in Carteret County. North Carolina, that is the drainage for Jump Run Creek.
Bacterial data from 1970 through 1998 indicate increased loading since 1974. which is when closure management began. Recent grab
sample data from the tributaries indicate high levels of bacteria during storm events and moderate levels during dry weather. The
majority of the loading is coming from the portion of the watershed draining an older, medium density neighborhood (single family
houses) and a trailer park. A door-to-door survey found two malfunctioning septic systems, more than 100 pets, and the presence of
wildlife. Change analysis of land use/land cover shows hydrologic modifications were instituted in the 1970s. Dye studies confirm that
water moves through the watershed in hours, indicating that time for bacterial mortality is insignificant. Future analyses include ground
water sampling, automated storm water monitoring, and DNA tracking of fecal sources. Planned mitigation practices will include
riparian buffer restoration, stormwater wetland, bio-retention, peat tllters, and education. Locations and sizing of practices will be
determined through GIS-based hydrologic analysis of the watershed in conjunction with a community-design/educational approach
involving neighborhood citizens.

INTRODUCTION agement is occurring in areas impacted by creeks draining water-
sheds witii extremely low development densities. This fact con-

This project focuses on restoration of a watershed draining to a «™s ^°'^^^ residents, fishing interests. Shellfish Sanitation staff,

shellfish resource at the mouth of Jumping Run Creek. The 320-ha ^"'^ "^^rine researchers. The objective of this project is to inves-

watershed contains mixed land use including single-family resi- "g^'<^ ^'^y this is happening and how it can be alleviated by 1)

dential. business, and industrial facilities. The natural land cover is quant'fymg the sources and loading rate of bacterial contamination

dense, coastal pocosin growing above sandy, relic dune ridges. In ^nd 2) by testing watershed-based restoration techniques for miti-

the upland portion of the creek, both banks are heavily forested gation.
with wide riparian buffers. The lower portion of the creek is bor-
dered by residential lawns with banks that are bulkheaded. METHODS

The creek empties into a significant shellfish resource for

which closure management began in 1974. The areal extent and Site Description
length of closure has increased steadily so that now the entire

resource is permanently closed. This is the trend for shellfish re- There is a variety of land uses within Jumping Run Creek

sources throughout North Carolina's coastal region, with a 12% watershed (Fig. 1 ). Twenty-four hectares are mobile (trailer) home

increase in closures in the last 5 years. and recreational vehicle (RV) housing. This area is characterized

Increased closures have occurred simultaneously with increases by gravel roads, grassed drainage swales, large trees, and lawns,

in population. However, like Jumping Run Creek, closure man- The trailer park uses traditional septic systems for waste manage-

473

474

White et al.

Medium Density
Residential

RV and MHP

Low Density
Residential

Industrial

Bogue Sound

Figure 1. United States Geological Survey 1994 aerial photograph with noted land use.

merit. The RV park has a new, low-pressure pipe septage treatment
(LPP) system. Low-density, single family residential (greater than
0.40-ha lots) encompasses 48 ha. Roads and some drives are
paved, but there are no sidewalks, and stormwater drainage is
conveyed in grassed swales. The lots are characterized by large
trees, expansive lawns, with wooded buffers between houses.
Waste management is traditional septic systems. Medium-density
residential (lots less than 0.4 ha) covers 109 ha. In the upper part
of the watershed, roads are paved, drives are gravel, and storm-
water conveyance is in grassed swales. Lots are covered primarily
by lawn and large trees and the creek area has a tree-based riparian
zone. In the lower portion of the watershed, drives and roads are

paved with both piped and grassed swale stormwater conveyances,
bulkheaded yards, and cleared creek edges. Waste management in
both areas is traditional septic. In the industrial zone, which covers
93 ha, there are paved roads and parking lots, sidewalks, gutters,
and combinations of grassed swales and piped stormwater convey-
ances. Buildings have small footprints and expansive lawn areas,
ornamental trees, and shrubbery beds. Waste management is in-
ground septic systems. Open space encompasses 48 ha of unde-
velopable steep slopes and pocosins along creek banks. These
landscape features are indicative of the White Oak River system,
which is composed of relic dunes ridges, steep banks, and heavy
veeetation.

Grab
Sample Sites

Grab Sites

BOGUE SOUND

Figure 2. United States Geological Survey 1994 aerial photograph on which sample locations are identified.

Jump Run Creek Shellfish Restoration Project

Bacterial Loading

475

16000-

14000-

12000-

10000-

MPN 8000-

6000

4000

2000

4^

La

1 r

1 2 3 4 5 6 7

i-^a^JJ.

1 \ r

■ Industriid

D Upper Watershed

■ Outlet
DMHP

■ RVP

9 10 11 12 13 14 15 16 17 If
Event
Figure 3. Results of bacterial grab samples by location.

Sampling

Grab samples were collected after storm events and analyzed
for fecal coliform, nutrients, and sediment. Fecal coliform analysis
was conducted by Shelltlsh Sanitation of the North Carolina De-
partment of Environment and Natural Resources (NC DENR) in
order for project work to be consistent with their data. The sample
sites were located to characterize the water draining from trailer
park, residential areas, industrial facilities. RV park, and at the
outlet of the creek before it is influenced by tidal waters (Fig. 2).
Samples from the creek outlet were collected at the lowest point of
the ebb tide.

Rhodomine dye studies were conducted to determine the time
needed for water flowing from each portion of the watershed to
reach the grab sample sites. The flow rates are collaborated with
data collected using a propeller-type, velocity flowmeter. Water
level relative to flow was noted using an in-stream gage plate. The
dye was dripped into the creek at a measured concentration and
rate using a peristaltic pump. Time was kept until the centroid of
the dye, as determined using a fluorometer. reached its destination.
This information and the flowmeter data were used to calculate
velocity in cubic meters per second (CMS).

The day after a 254-mm rainfall event, the research team con-
ducted a door-to-door survey in the watershed. Each survey team
included a certified Health Inspector as well as member of the

research team. At each location, septic leach fields were located
using a metal probe and checked for inundation as well as surfac-
ing septage. The imperviousness of the sites was measured. In
addition, residents were queried as to the number of pets in the
household.

RESULTS

The results of the bacterial sampling are summarized in Figure
3. After 1 1 sampling events, the majority of the loading was de-
termined to be coming from the upper reaches of the watershed
containing the trailer park and residential land uses. Additional
samples were collected at the tributary below the trailer park and
RV park to examine this in greater detail. These data indicated that
very little additional loading occurred as (he water flowed past the
RV park to the outlet. Contributions from the industrial area were
inconsequential and grab sampling at this location was suspended.
Data show levels from the trailer park, creek outlet, and below the
upper reaches of the watershed are similar, indicating that for these
events the trailer park area was contributing the majority of the
bacteria to the water column.

The results of the dye studies are summarized in Table I. It is
important to note that at relatively low flow levels, water moves
through the system in less a than a day. Furthermore, during winter
months when flow levels are high, bacterial life is extended (White

476

White et al.

TABLE 1.
Summary of travel time.

TABLE 2.
Summary of neighborhood survey.

Gauge

Other

Date (1998)

Hgt (m)

0.42

CMS

0.23

Travel Time

No Data

Location

Visits

SV

Dogs

Cats

Pets

Impcrviousness

January 21

MHP

47

0

23

6

3

3716 m=

January 22

0.35

0.16

MHP-Outlet. 5 hours

Medium Density

66

T

60

47

25

17.861 m-

February 4

0.91

0.50

MHP-Gauge, 2.5 hours

Low Density

14

0

7

0

0

8740 m-

February 23

0.51

0.30

No Data

RVP

1

0

0

0

0

460 m'

March 3

0.34

0.08

Headwaters-Gauge. 3 hours

Totals

128

2

90

53

28

29,900 m-

March 4

0.33
0.28
0.27

0.07
0.16
0.29

RVP to Outlet. 1.5 hours
No Data
No Data

2.9 ha

April 3
April 17

* Does not include roads

or ini.

ustrial

irea.

April 30

0.25

0.14

No Data

June 18

0.23

0.12

No Data

cover, time-char

ge analysis

to quantify where the

most significant

July 14

0.21

0.12

No Data

changes have occuired

These data

will be

analyzed spatially rela-

1996) due to low temperatures and reduced light levels, and the
contamination potential is higher.

Neighborhood survey results are summarized in Table 2. There
were only two septic system surface failures, less than 10% im-
perviousness, and more than 30 cats at one location — most pets
were located close to tributaries.

CONCLUSIONS

The surface septic system failures, found during the survey,
were not connected to the creek via surface runoff. Hence, these
did not appear to be a source of bacterial contamination. However,
the possibility that bacteria are transported via groundwater has not
been dismissed. The research team is concerned that septic leach
fields may be intersecting groundwater during wet weather, and, in
combination with porous, sandy soils, leaching bacteria into the
creek.

Lack of bacterial presence in grab samples eliminated the in-
dustrial area from consideration as a contributor. However, this
area is contributing large volumes of fresh stormwater, which is a
problem. NC DENR Wetland Restoration Program is participating
in the project, and they, based on project data, are planning to
convert some of the lawn areas, which are being donated by the
landowner, to wetlands. This will increase storage time and filtra-
tion as well as reduce the flow volume and velocities during storm
events of the water draining from more than one fourth of the
watershed.

Even counting the roads and industrial park areas, this water-
shed is less that '^'/( imper\ ious. which is well below the published
threshold for the initiation of water quality degradation (Schuler
1995). However, preliminary examination of aerial photography
indicates that extensive hydrologic modification of all land cover
has occurred. Photographs from the I9.^()s through the I99()s show
channelization, ditching, and bulkheading — modifications thai
cause stormwater runoff lo be delivered faster and in greater vol-
umes during storm events, allowing less lime lor bacterial stores in
the watershed, naturally occtnring or not. to be reduced. These
photos will be further analyzed using GIS-based. land use/land

live to 30 years of bacterial data for potential correlation.

Nevertheless, the impact of such alterations on bacterial load-
ing potential are not captured adequately by measuring impervious
surfaces. The hydrologic transport mechanisms as well as the lo-
cation of bacteria needs to be known in order to properly target and
design mitigation. To further this end, automatic monitoring sta-
tions have been installed in the watershed below the trailer park, at
the gage, and near the creek outlet. These will collect flow-
weighted storm samples, which will be composited for the rising,
peak, and falling portions of the storin hydrograph. These data will
clarify when the bacteria is loading, and in conjunction with base-
tlow-oriented grab samples, assist in differentiation between trans-
port pathways and the calculation of total loading by land use. If
necessary to determine source and transport path, dye studies will
be conducted on septic systems in the watershed and DNA analy-
ses on the fecal matter.

Once loading and transport pathways are known, the project
team plans to design and install innovative elevated bioretention
areas, peat and sand filters, and constructed wetlands to mitigate
the bacterial loading. In 1999, a neighborhood stakeholder group
participated in a design charette to assist in this effort. In this
manner, these facilities become neighborhood amenities not just
stormwater treatment devices.

ACKNOWLEDGMENTS

The authors wish to acknowledge and extend thanks to the
following folks and agencies without whom this project would not
be possible. Project Collaborators: North Carolina Cooperative
Extension Service, University of North Carolina Sea Grant; North
Carolina Department of Enviionmenl and Natural Resources —
Shellfish Sanitation and Coastal Management: Duke Marine Labo-
ratory. North Carolina State University School of Design, depart-
ments of Soil Science and Biological and Agricultural Engineer-
ing, and Carteret-Cra\en Electric Cooperative. Fundina Agencies:
North Carolina Department of Environment and Natural Re-
soiirces-DWQ 319; North Carolina Department of Environment
and Natural Resources DWQ- CWMTF; North Carolina Depart-
ment of En\ iroiimeni and Natural Resources DWQ WRP.

LITERATURE CITED

Schuler, T. 1995. F.nvironmenlal Land Planning Series: Site Plannini; for
Urban Stream Protection. Publicalioii # '15708. Center for Watershed
Proleclion. Fllicol City. Maryland. 232 pp.

White. N. M. 1996. Spatial analysis of fecal colif'orm bacteria fate and
transport. Doctoral Dissertation. North C;irolina Stale University, Ra-
Icieh. North Carolina.

Joiinwl of Shellfish Resecirch. Vol. 19. No. 1. 477-+Sn. 2000.

ONE SHINING MOMENT KNOWN AS CLAMELOT: THE CEDAR KEY STORY*

SUZANNE COLSON ' AND LESLIE N. STURMER"

^ Suwannee River Water Management District

P.O. Bo.x 376

Cedar Key. Florida 32625
'University of Florida Cooperative E.xtension Senice

Cedar Key Field Station

P.O. Box 89

Cedar Ke\. Florida 32625

ABSTRACT The heritage and culture of rural communities along Florida's Big Bend coastline in the Gulf of Mexico have been
linked intrinsically with commercial fisheries for generations. Over the past decade, closures of oyster harvesting grounds and a
state-imposed ban on gill nets triggered economic decline and depression in this area. A transition to shellfish aquaculture as an
alternative employmenuipportunity has been facilitated through the recent federally funded, job-retraining programs. Since 1993 when
the first program graduates were placed with leases, the industry has grown and now supports over 300 hard clam, Mercenaria
mercemirhi (Linnaeus. 1758). growout operations on 950 acres of state-owned submerged lands with sales (farm gate value) estimated
at $10 million in 1997. The promise of prosperity has created a new excitement and common bond among the individuals of these
communities. With a renewed sense of purpose and cohesion, people are working together to promote their livelihoods, and above all.
to protect the coastal waters so critical to the success of these ventures. This revitalization has also spun-ed a reaction and respon-
siveness to the emergent industry by local governments and by state and federal agencies. Citizens of Cedar Key have formed advisory
groups to work closely with elected officials and agency representatives in the planning and implementation of a wide range of water
quality activities. These include storm water and wastewater treatment, environmental education, and water quality monitoring
programs. Sustainable hard-clam aquaculture operations have proven to be an excellent opportunity to both protect and preserve the
region's environmental qualities as well as support economic activity.

KEY WORDS: Mercenaria mercenaria. hard clam, aquaculture. partnership, pollution abatement, shellfish restoration and reme-
diation, water quality monitoring, watershed management

IN THE BEGINNING

"A.sk even- person if he has heard the story and tell it strong
and clear if he has not. that once there was a fleeting wisp ofgloiy
called Clamelot. "*

The history, character, heritage, and economy of this modern-
day Clamelot are inextricably tied to Cedar Key's scenic, teeming
coastal waters. Tourism, annual seafood festivals, restaurants, and
commercial and recreational fishing all depend on good quality
water. Yet, during the past decade, broken septic and stormwater
systems have led to broken dreams, as a large number of the area's
commercial oystermen and fishermen were forced out of business
by contaminated Gulf of Mexico waters. With a renewed sense of
purpose and determination, the community is now working to-
gether to develop and promote new livelihoods and to protect the
coastal waters so vital to the success of these commercial ventures
and the survival of their beloved Clamelot.

CREATING NEW JOB OPPORTUNITIES

"It's true, it's true the climate must be peifect. all the year. In
short, there's .Hmply not a more congenial spot for happy eve raft-
ering than here in Clamelot."*

Florida's warm Gulf waters and high natural productivity levels
create a superb environment for marine life and, by extension, for
those who earn their living "on the water." The weather, market
trends, and a variety of other factors have always made life inter-

*With apologies to Alan Jay Lemer and Frederick Lowe, whose wonder-
fully appropriate lyrics to the musical Camelot. published in I960, were the
inspiration for this presentation.

esting, and at times uncertain, for local residents. Yet. the eco-
nomic picture in Clamelot has not always been "rosy" — it has
included the closure of oyster harvesting grounds due to water
pollution and a state-imposed ban on gill nets.

Economic survival in Clamelot requires a willingness to adapt,
and its residents have embraced a promising new industry with
gusto. Beginning in 1991. the Florida Department of Labor and
Employment Security introduced federally funded, job-retraining
programs in shellfish aquaculture for unemployed or underem-
ployed oyster harvesters and other seafood workers in a four-
county area. Trainees were prepared for their new businesses
through hands-on participation and a classroom curriculum. In-
struction was provided by Harbor Branch Oceanographic Institu-
tion and the University of Florida's Institute of Food and Agricul-
tural Sciences. The programs. Project OCEAN and Project
WA'VE, were headquartered in Cedar Key.

Through Project OCEAN, which incorporated both oyster and
hard clam culture technology, over 130 program graduates re-
ceived shellfish aquaculture leases in 1993 and the knowledge to
put the submerged lands into production. The success of this pro-
gram was the impetus for Project WAVE, which enabled displaced
net fishermen in the same region to be instructed in the business of
culturing hard clams. During 1995-1997, 69 fishermen were given
leases for the startup of individual- or family-operated farms. Most
of the trainees have made a successful transition to clam farming
and are operating productive and profitable leases. Currently, the
emergent industry now supports more than 300 hard clam growout
operations on 950 acres of state-owned submerged lands off the
coast of Dixie and Levy counties. Sales, (farm gate value) in 1997
were estimated at $10 million. Shellfish aquaculture is now a
primary source of income for many residents along the coast.

477

478

COLSON AND STURMER

Figure 1. Clam farmer harvesting his crop from lease areas located in
the productive waters of the Gulf of Mexico off Cedar Key, Florida.

COMMUNITY INVOLVEMENT

There may be only one road leading in and out of Cedar Key.
but there are many paths leading to the protection of the town's
water quality and. consequently, its lucrative shelltlsh industry.
When a statewide gill net ban was imposed in 1995. many com-
mercial net fishermen found themselves out of work. Looking to
reverse their misfortunes, they turned to clam farming, a newly
emerging industry in which success is directly tied to water quality
from the estuaries and from human activities in and around Cedar
Key.

When inadequate stormwater and sewage treatment systems
began posing threats to local water quality, citizens rallied to pro-
tect the natural resources on which their livelihoods were depen-
dent. They formed the Cedar Key Water Alliance to encourage
citizen participation in finding solutions to some of the towns most
pressing water resource concerns. The committee's advisory
groups worked closely with elected officials and agency represen-
tatives in planning and implementing a wide range of water quality
activities, including improved stormwater and wastewater treat-
ment systems and environmental education. The community re-
ceived substantial funding from the state's Surface Water Im-
provement and Management Program to conduct a master storm-
water system study and to develop a master stormwater plan. An
additional $500,000 has been appropriated for implementation of
stormwater projects, with funds provided through the Florida De-
pailment of Transportation's wetlands mitigation program. One ol
Cedar Key's top priorities has been to replace all existing septic
tanks with connections to the town's centralized sewer system. To
achieve this ambitious goal, volunteers surveyed existing homes,
as well as lots not yet on the system, and drafted a budget for both
short-term and long-term goals. Homes thai were within the ex-
isting collection area were targeted initially. With a $52,000 grant
from the Suwannee River Water Managemenl District lo purchase

the necessary supplies, committee members provided the labor to
connect over 42 homes. Next the group sought to expand the sewer
system to serve the more than 100 remaining homes still on septic
tanks. The city and its water and sewerage district garnered support
from their local legislative delegation, and in 1998 the Florida
Legislature appropriated $790,000 to eliminate every septic tank in
the community by the year 2000. The result will be ongoing pro-
tection of the town's water quality and preservation of a shellfish
industry vital to the community's economic survival. In addition,
the community is committed to promoting an extensive water con-
servation program. Activities in progress or already completed
include conducting a leak detection survey of all residences, ret-
rofitting commercial toilets, and implementing "xeriscape" land-
scaping.

WATER QUALITY MONITORING

The historic Suwannee River, immortalized by songwriter
Stephen Foster, begins in Georgia's Okefenokee Swamp, and emp-
ties into the Gulf of Mexico near Cedar Key. one of the few
remaining areas for shellfish harvesting in Florida. Designated an
Outstanding Florida Water, the Suwannee River has managed to
remain relatively free of the pollutants that have diminished the
health and tarnished the beauty of many of our nation's waterways.
Yet. even the Suwannee is in danger of becoming a casualty. In a
stretch of river known as the Middle Suwannee, nitrate levels are
at the highest level in 10 years. Animal waste and fertilizers from
this rural region's many dairy and poultry operations are thought to
be contributing factors, along with human waste from inadequate
or poorly functioning septic systems, and fertilizers from other
coinmercial and residential activities. To stem the tide of nutrient
loading and other pollutants into this Outstanding Florida Water,
state and federal agencies have joined in cooperative monitoring
efforts to track the quality and quantity of water flowing through
the river, its springs, and groundwater.

Recognizing that whatever Hows into the Suwannee will even-
tually wash into the Gulf, efforts also are being made to monitor
closely the condition of the state's coastal waters, vital to the
survival of the state's fishing, shellfish, and tourism industries.
Project COAST is one such monitoring project. Launched in 1997
by Florida's Suwannee Ri\er and Southwest Florida water man-
agement districts as a one-year water quality monitoring study.
Project COAST is now an ongoing program coordinated by the
University of Florida's Dcparlnicnt of Fisheries and Aquatic Sci-
ences in cooperation with the Florida Department of Environmen-
tal Protection and citizen volunteers. Using their own boats,
trained volunteers take water samples at fixed sites adjacent to fi\e
coastal communities along more than 100 miles of the west central
Florida coastline. They measure temperature, salinity, water clar-
ity, chlorophyll concentrations, nitrogen, and phosphorous, and
submit the data to the university for analysis. Results from this
cost-effective sampling program will be used to develop a long-
term data set. which will in turn he used to establish baseline water
quality conditions for coastal waters. The results also will provide
educational information concerning enxironmenlal issues to the
public.

CREATING QUALITY COMMUNITIES

The tiny coastal village of Suwannee suffered a severe eco-
nomic blow in 1991 when high bacterial contamination caused by
(lie town's poor septic systems prompted the federal government to

Cedar Key Clam Aquaculture

479

close Suwannee Sound to oyster harvesting, the community's main treatment needs. The District also helped city and county officials

industry. To help preserve and protect the area's water resources obtain $9.7 million in federal grants and loans to finance the

and revive the $1 million annual local shellfish industry, the Su- town's new wastewater treatment system, which is now up and

w annee River Water Management District allocated $25,000 for a running. The resounding success of that effort was the inspiration

detailed feasibility study that addressed the town's wastewater for what is now the Quality Communities Program.

Suwannee River Basin

g SRWMD Lands '.. Dekia/Keato(

H Suwannee River Basin '.SeachM
i^ SRWMD Boundary
• Quality Communites City ''-.^sieii

1:

-- )- -f ^'% '-

\ V Gilchrist \

C'0«spl<y .FanolBji r\)
DIXIfi \ ^'^ 4'- "^

HorseBhoe^ Chlefti

• Beach J? "• J-

t^ Cedar Key

MILES
Map Scale 1:823.680

Figure 2. Clamelot. in relationship to the Suwannee River Basin and Big Bend coastline of north central Florida. Locations of shellfish
aquaculture leases off Cedar Key, the boundaries of the Suwannee River Water Management District, land acquisitions of the Suwannee River
Water Management District, and cities targeted for the Suwannee River Water Management District's Quality Communities Program arc
shown.

480

COLSON AND StURMER

The goal of the Quality Communities Program is to help small
rural communities protect water resources and at the same time
improve their quality of life by offering technical expertise and
funding needed to complete critical water quality and community
infrastructure projects. Some of the state's poorest counties, in
terms of per-capita income, education, property values, and taxable
property levels, are located within the Suwannee River Water
Management District. These counties lack the resources to make
the necessary improvements to their drinking water supplies,
stormwater drainage systems, and wastewater treatment facilities.

The District has targeted 37 communities and some unincor-
porated areas for assistance so that by the year 2010 each one will
have the opportunity to become a Quality Community. The District
will set aside $500,000 per year for 10 years as "seed money" for
projects that will eliminate street and residential flooding, for pre-
engineering or feasibility studies for project cost estimates, and as
leverage for other available grants and funds. Additional funds
from the District's land acquisition and management program will
be used to purchase lands for stormwater storage and water supply
protection.

LAND ACQUISITION AND MANAGEMENT

Florida's land and water resources are forever linked. To pro-
tect the rivers, lakes, streams, and underground water supplies, the
lands around them must be managed properly. The Suwannee
River Water Management District cunently owns and manages
nearly 100,000 acres of riverfront and wetlands to provide natural
storage areas for flood waters, reduce loss of life and property due
to floods, protect ground and surface water resources of the region,
and protect natural systems associated with tloodplain ecosystems.
One of the District's key acquisitions in terms of coastal protection
was the purchase of Atsena Otie, a 60-acre barrier island located
near Cedar Key.

An island of great historical significance, Atsena Otie was the
original site of Cedar Key and in the IXOOs served as Army head-
quarters for General Zachary Taylor. It later grew into a prosper-
ous city with a school, hospital, post office, and several sawmills,
one of which belonged to the Eberhard Faber cedar pencil manu-
facturer. Around the turn of the century, residents gradually aban-
doned the island following a series of devastating hurricanes, and
the island has since remained uninhabited. Today the island and its
surrounding waters are home to a variety of animals, including
egrets, ospreys, ibises, turtles, squirrels, raccoons. Gulf sturgeon,
dolphins, and manatees. Cordgrasses fringe the island, and the
interior is alive with sand live oaks, red cedars, cabbage palms,
palmettos, and other vegetation.

The waters surrounding Atsena Otie may be harvested for
shellfish, and clam lease sites are located on each side ol' the
island. In the early I'WOs. private developers introduced plans to
build a residential community on the island. Concern over the
potential impacts of septic tanks and stormwater runoff on the
coastal environment and the local shellfish industry prompted the
District to purchase the land in 1997 for $.3.1 million, thereby
placing it under public ownership.

Today the U.S. Fish and Wildlife Service manages the island.
It is open to the public for swimming, hiking, fishing, and nature
observation but not for camping. Posted signs remind visitors to
carry out all of their trash, and a self-composling portable toilet has
been placed on the island to accommodate visitors' needs and to
reduce potential environmental impacts of human waste on the
nearby clam lease sites.

PRESERVING AGRICULTURE, PROTECTING
THE ENVIRONMENT

Residents of rural North Florida's Suwannee River Basin are
struggling to balance and preserve the two things most vital to their
economy and quality of life: clean and scenic natural resources and
agriculture. This watershed features the Suwannee River and one
of the largest concentrations of freshwater springs in the world. It
also contains a large percentage of the state's farms, dairies, cattle,
and poultry operations. When high nitrate levels were discovered
in the Suwannee River, its springs, and local groundwater, the
regional water managers sought the involvement of farmers, local
governments, environmental regulators, and all citizen stakehold-
ers in a collaborative effort to reduce nutrient loadings to the
watershed, the waters of which eventually empty into the Gulf.

The Suwannee River Basin Nutrient Management Working
Group, comprised of two dozen government agencies and inde-
pendent organizations, was formed to facilitate that effort. Three
technical committees, focusing on management of fertilizers, ani-
mal waste, and human waste, are gathering and coordinating in-
formation that will assist in the eventual design and implementa-
tion of a basinwide nutrient management plan. A program coor-
dinator conducts public meetings and workshops, and serves as a
liaison between agencies, agricultural interests, elected officials,
and the public. Approximately $6.3 million in state and federal
funds has been earmarked for voluntary, incentive-based, nonregu-
latory cost-share programs to initiate best management practices at
farms; 43 dairy farmers and 102 poultry producers in the two inost
highly impacted counties will be able to participate.

INTERSTATE COORDINATION

Florida and Georgia share the 10,000 square-mile Suwannee
River Basin, and the two states are working together to protect it.
In 1996 the Suwannee River Water Management District, Florida
Department of Environmental Protection, Georgia Department of
Natural Resources (DNR), and U.S. Fish and Wildlife Service
joined in an informal alliance to foster communication and coop-
eration between the two states and to develop a comprehensive
plan for safeguarding the water resources within the basin. Without
the use of interstate compacts or agreements, the agencies have
made significant strides in the areas of cooperative monitoring,
information exchange, and outreach. Alliance activities include
public meetings and workshops, a semi-annual newsletter, and a
satellite-image poster of the entire Suwannee River Basin.

Perhaps the most important accomplishment yet is the coordi-
nation of monitoring activities throughout the entire Suwannee
River Basin. The Suwannee River Water Management District and
the Georgia DNR Environmental Protection Division are perform-
ing monitoring on a parallel schedule, using the same parameters
and methods at 72 sites in Florida and 73 in Georgia. Following
completion of the testing for ammonia, nitrates, total phosphorous,
fecal coliform, and trace metals, the agencies will publish a joint
report under the auspices of the Alliance.

"Dim'! lei it he foifiol thai once there was a s/nil Jor one brief
shining moment llial was known us Clamehil."*

ACKNOWLEDGMENTS

The poster presentation at the l99iS International Conference on
Shellfish Restoration was crafted with production assistance by
Cynthia Johnson and Berry Shafii, Suwannee River Water Man-
agement District.

Journal of Shellfish Rescunh. Vol. 19. No. 1. 481-485. 20U0.

INCREASED DENSITY OF LARGE RANGIA CLAMS IN LAKE PONTCHARTRAIN AFTER THE

CESSATION OF SHELL DREDGING

S. W. ABADIE AND M. A. POIRRIER

Department of Biological Sciences
University of New Orleans, Lakefront
New Orleans, Louisiana 70148

ABSTRACT Raiigia cuneata is a relatively large clam found in the oligohaline areas of Atlantic and Gulf of Mexico estuaries. Rangia
is common in Ldke Pontchartrain. Louisiana, and accumulated shells supported a mining industry from 1933 to 1990. Shells were u.sed
primarily for construction of roadways, parking lots, and levees, and in the production of cement. Based on mean densities, a 1954
study found that large clams (> 20 mm) were abundant (95/m-); however, large clams were found to be less abundant in 1973 (39/m-),
1982 (2/ni"). and 1984 (41/m") studies. Because baseline and comprehensive time sequence studies were not done, it is unclear whether
shell dredging caused the differences in abundance. Fifty-five sites were sampled in 1996 and 1997 from lakes Maurepas, Pontchar-
train, and Borgne to determine the abundance and distribution of Rangia. Lake Maurepas and Lake Borgne were included to provide
information about Rangia in adjacent estuaries that have salinities lower and higher than Lake Pontchartrain. Data from the Lake
Pontchartrain sites were used to determine whether the number of large clams had increased after shell dredging was stopped in 1990.
Large clams were abundant at most sites (Lake Pontchartrain mean density 126/m"), but absent in a 350-km" area affected by saltwater
intrusion and hypoxic conditions from the Inner Harbor Navigation Canal (IHNC). Although large clams were absent from the eastern
part of the lake in earlier studies, the highest density (602/m-) was found in this area. Based on the current distribution and density
of large clams, shell dredging had a significant impact on density and recovery has occurted since cessation of dredging.

KEY WORDS: Rangia cuneata. shell dredging, size classes. Lake Pontchartrain, density, distribution

INTRODUCTION

Lake Pontchartrain is an embayment in a large estuarine system
in southeastern Louisiana, It has a mean salinity of 4 ppt. a mean
depth of 3,7 m, and a surface area of 1,630 km" (Fig, 1), Saline
water enters from adjacent estuaries through natural tidal passes
and a navigation canal. Flow through the Inner Harbor Navigation
Canal (IHNC) causes salinity stratification and episodic bottom
hypoxia (Poirrier 1978, Junoi et al, 1983. Schurtz and St, Pe 1984).
Fresh water sources are streams and New Orleans area outfall
canals. Fresh water also enters from the Mississippi River through
the Bonnet Can'e Spillway as leakage when the river is high, and
when the spillway is opened to prevent possible Hooding of New
Orleans and other downstream communities (Barbe and Poirrier
1991). During this study, the spillway was open from March 17 to
April 18, 1997,

Lake Pontchartrain is located north of New Orleans, and more
than 1.5 million people live in its basin. With increasing urban-
ization in the New Orleans area over the last century, concerns
about possible declines in water quality, fisheries, and recreational
use of the estuary have been raised (Houck et al. 1989). A major
environmental concern was adverse impacts from dredging for the
shells of the common rangia. Riinf>ia cuneata (Gray), from depos-
its in the estuary.

Shell dredging began in 1933 and was stopped in 1990. The
volume of shells harvested increased until the mid 1970s and then
declined (USACOE 1987). Shells were used for foundations, road-
ways, as an ingredient in many industrial products, and for oyster
cultch. The shells harvested, according to estimates from the
1980s, had an annual gross value of $34 million. Dredging was
allowed in 449'r of the lake; it was prohibited near shorelines,
bridges, and oil and gas wells and pipelines. Dredging was regu-
lated so that only \9c of the bottom was disturbed at a time.
Suction dredges drew up bottom sediment, and shells were sepa-
rated from the sediment by washing on screens. The discharge of
the sediment slurry directly into the lake increased turbidity near
the dredge. Turbidity increases also were probably caused indi-

rectly by shell dredging, which results in the formation of uncon-
solidated bottom sediments that are easily resuspended by wind.
Besides increased turbidity, introduction of to,\ic substances from
the sediment into the water column and disruption of the bottom
invertebrate community were additional environmental concerns
(USACOE 1987).

Early studies (GSRI 1974) on the effects of shell dredging did
not show any significant environmental effects; however, studies
by Sikora and Sikora (1982) found an average density of only l/rtr
of Rangia > 20 mm. They attributed the low densities of large
clams to the adverse effects of shell dredging. R, Darnell obtained
information on the distribution and abundance of large Rangia (>
20 mm) during studies of Lake Pontchartrain in 1953-1954 (Sut-
tkus et al. 1954), These data were unpublished but cited by Sikora
and Sikora (1982) and later presented in an environmental impact
statement prepared by the U.S. Army Corps of Engineers (USA-
COE 1987). Darnell found large Rangia clams were present at an
average density of 95/m- (USACOE 1987), Other studies that
included information on the abundance and distribution of Rangia
in Lake Pontchartrain were Tarver and Dugas (1973) and Poirrier
et al. ( 1984), who found large clam average densities of 39/m- and
41/m-, respectively. These studies also found that few clams oc-
curred in the areas of the lake that were dredged. It was not clear
whether the high numbers of large clams reported by Darnell
(USACOE 1987) were indicative of the natural condition of Lake
Pontchartrain in the 1950s, because shell dredging began in 1933
and management practices were thought to be sufficient to avoid a
severe impact on Lake Pontchartrain.

Rangia cuneata is generally found in estuaries from the upper
Chesapeake to Vera Cruz. Mexico (LaSalle and de la Cruz 1985).
It can make up 95% of the benlhic bioinass in low salinity estuaries
(Cain 1975). It is a nonselective filter feeder that is important in
converting energy from suspended organic material to clam bio-
mass (Gaston et al, 1996). Juvenile and adult Rangia provide food
for fish, duck, and invertebrate predators (Darnell 1961, LaSalle
and de la Cruz 1985). Rangia clams, which are dioecious, shed
gametes directly into the water. A rapid change in salinity of 5 ppt

481

482

Abadie and Poirrier

can trigger spawning (Cain 1975). In Lake Pontchartrain. clams
become sexually mature at about 24 mm (about 2-years-old), and
spawning peaks occur from March to May and from late summer
to November (Fairbanks 1963). Adult Rangia can withstand a
salinity range of 0 to 25 ppt (Hopkins et al. 1973). However.
permanent populations do not occur at salinities above 15 ppt
(Hopkins et al. 1973). Filtration rates are highest at 5 ppt and
decrease with increasing salinity (Holley and Foltz 1987).

This study was conducted to determine whether there has been
an increase in the density of large Rangia after shell dredging was
banned in the summer of 1990. A more detailed description of this
study is presented in Abadie (1998). If the density of large clams
had increased significantly, it would indicate that shell dredging
had a significant effect, and that recovery from the adverse impact
had occurred. Francis and Poirrier (1998) showed an increase in
Secchi disc transparency after shell dredging was stopped. This
water quality improvement may be related to an increase in the
density of large clams.

MATERIALS AND METHODS

Lakes Maurepas. Pontchartrain, and Borgne were sampled to
determine the abundance and distribution of Rangia (Fig.l ). Lake
Maurepas, which has a mean salinity of 0.5 ppt, and Lake Borgne.

which has a mean salinity of 12 ppt (Perret et al. 1971), were
included to provide information about Rangia in adjacent estuaries
with salinities lower and higher than Lake Pontchartrain. Six sites
in Lake Maurepas, five sites in Lake Borgne, and 44 sites in Lake
Pontchartrain were sampled from November 1995 to October 1997
(Fig.l). These sites were sampled once to obtain information on
the spatial distribution of large Rangia. Temporal (seasonal) in-
formation was obtained by quarterly sampling of five sites on an
east-west transect that extended through the middle of the Lake
Pontchartrain (Fig.l). Additional samples from this transect were
taken to study the effects of the March 17, 1997, Bonnet Carre
Spillway opening. As a result, it was sampled seven times: No-
vember II, 1996, March 12, 1997, April 30. 1997, June 6, 1997,
July 28. 1997, September 17, 1997 and October 29, 1997.

Three replicate samples were collected from each site with a
(15 X 15 cm) petite Ponar dredge. Each sample was emptied into
a 0.6-mm sieve bucket to separate organisms from fine materials in
the .sediment. All material retained on the sieve was transferred
from the bucket into 500-niL specimen jars and fixed in a 10%
buffered formalin solution with rose bengal. In the laboratory, the
sample was washed on a 0.5-mm sieve to remove formalin and any
remaining sediment that could pass through the sieve. Each sample
was examined with a Wild M-5 dissecting microscope to remove
Rangia from remaining sediment. The Rcnigia specimens were
measured to the nearest millimeter and the lengths were grouped

Fi);ure 1. M:i|) of lakes Miiiin'pus, l'(intcluir(ruin. ;ind IS(ii'};iu', l.oiiisiiiiui. Mean (Icnsit) oi'lar^u l> 21 niml Riiiii;i(i ciinctilu arc indiciiti'd at each
of the 55 sites sampled I'roni Novemher l'W5 to October 1997. The five sites alonji the east-west transect, sampled seasonally, are marked with
an asterisk. Mean densities that are circled signify sites that are significantly different from the four sites on the east-west transect not affected
by hypoxia.

Recovery of Large Rangia in Lake Pontchartrain

483

into 5-mm increment size classes (0-5. 6-10. 1 i-15. 16-20. 21-
25. 26-30. 31-35. 36^0. and 41-45 mm).

Surface and bottom water temperatures, salinity, and dissolved
oxvgen were measured concurrent with clam sampling using a YSI
model 85 meter. A weighted 20-cm Secchi disc was used to mea-
sure transparency and depth.

All historic data (Tarver and Dugas 1973. Sikora and Sikora
1982. Poirrier et al. 1984, USACOE 1987) were converted to
N/ni". The designation of large clams, equaling 20 mm or more in
length, was made in the baseline study by Suttkus et al. ( 1954). and
was used to make comparisons among studies. A one-way
ANOVA was used to test the following null hypotheses: ( i ) there
has been no increase in large clam density since shell dredging was
stopped; (2) there has been no change in the density of clams in the
0-10 mm and 1 1-20 mm size classes in the studies by Sikora and
Sikora (1982), Poirrier et al. (1984). and this study; and (3) there
were no temporal or spatial differences in the distribution of large
clams.

RESULTS

Water depth at the study sites ranged from 1.8 m at the near-
shore stations to 6 m near the Inner Harbor Navigation Canal, and
averaged 4 m. Surface salinity ranged from 0.1 ppt in Lake Mau-
repas to 8.9 ppt in Lake Borgne. The salinity of Lake Ponchartrain
decreased to freshwater conditions after the March 1997 Bonnet
Carre Spillway opening, and returned gradually to pre-opening
conditions by October 1997. Secchi disc transparency ranged from
8 cm after the Bonnet Carre Spillway was opened to 290 cm in the
fall near the north shore. Water quality measurements followed
known seasonal trends (Francis et al. 1994) and known responses
from past Bonnet Carre Spillway openings (Poirrier and Mulino
1977). Specific effects of the 1997 spillway opening on Raiifiia
and other benthic invertebrates will be included in reports from
other ongoing studies.

The number of Rangia greater than 2 1 mm/m~ from each site is
presented in Figure 1. The density of large clams in Lake Pon-
chartrain ranged from zero in the southeastern region, near the
IHNC, which is subjected to salinity stratification and episodic
hypoxia, to 602/m" in the eastern lobe, and averaged 1 26/m" for all
areas. The average density of large Rangia from the three lakes
was 137/m-. A comparison of average densities (N/m") found by
this study with densities found by previous .studies (USACOE
1987. Tarver and Dugas 1973. Sikora and Sikora 1982. Poirrier et
al. 1984) indicates high densities in the 1954 study, low densities
in the 1973. 1982. and 1984 studies, and recovery in this study
(Abadie 1998) (Fig. 2). Results of the one-way ANOVA showed
that there was a significant difference in the number of large clams
among the studies (P < .0001 ). The average densities for the 1954
study and the 1998 study were significantly different from the
1973. 1982. and 1984 studies, but there were no significant dif-
ferences between the 1954 and 1998 studies (Fig. 2).

An analysis of variance showed that there were statistically
significant differences in the density of clams >21 mm among the
Lake Pontchartrain sites. A means comparison for each of the 55
sites was performed and analyzed against four of the five sites on
the east-west transect. Sites on the east-west transect were used in
this comparison, because data from them was based on a total of
21 replicate samples, contained seasonal information, and ranged
over a salinity gradient. One site on the east-west transect was
excluded from the comparison; this site was north of the IHNC and
was known to experience by episodic hypoxia (Schurtz and St. Pe
1984). Sixteen sites, located near the Inner Harbor Navigation

CO

c

<U

00

(U

s

3
C

<a

00
cd

u

>

<

150

100-

1954 1973 1982 1984 1998

Year

Figure 2. Historic average densities and standard error of large Ran-
gia cuneata in Lake Pontchartrain. Historic averages were complied
from USACOE (1987), Tarver and Dugas (1973). Sikora and Sikora
(1982). Poirrier et al. (1984). and this study. A one-way ANOVA and
means comparison were used to assess the effect of year on the average
density of large Rangia in Lake Pontchartrain. Means with different
letters are significantly different at the .05 level.

Canal, were significantly different from the remaining four east-
west transect sites (Fig 1).

A one-way ANOVA was used to compare the densities of 0-10
and 1 1-20 mm size classes (Abadie 1998) with two earlier studies
(Sikora and Sikora 1982. Poirrier et al. 1984). There were signifi-
cant differences in the average densities of 0-10 mm clams among
the 1982 (3.164/m-). 1984 (266/m-j, and 1998 (278/m-) studies,
but no differences were found between the 1984 and 1998 studies
{P < .0001). No significant differences (P = 0.7620) were found
in the densities of 1 1-20 mm size class among the three studies:
1982 (31/nr). 1984 (22/m-). and 1998 (57/nr).

The distribution of size class frequencies for all clams collected
from lakes Maurepas. Pontchartrain. and Borgne was bimodal
(Fig. 3). The clams ranged from 0 to 45 mm and were divided into
5-mm size classes. Peaks occurred in the 0-5 mm and the 26-30
mm size classes. The 0-5 mm size class made up 41% and the
26-30 mm size class 21% of the total clams collected. The per-
centages in the other sizes classes were 6-10 mm size class 16%,
1 1-15 mm size class 4%. 16-20 mm size class 1%. 21-25 mm size
class 10%. 31-35 mm size class 6%. and the 36^0 and 41^5 mm
size classes 1% each.

The analysis of the seven, seasonal east-west transect samples
(Fig. 1) showed significant spatial and temporal differences were
in the 0-5. 6-10. and 1 1-15 mm size classes; significant spatial
differences were present only in the 21-25. 26-30. 31-35 and
36-40 mm size classes among the five sites. There were no clear
trends related to seasons or differences among sites for clams < 21
mm. The introduction of Mississippi River water through the Bon-
net Carre Spillway may have affected trends. The density of large
clams (> 21 mm) was lowest at site four, where Rangia may have
been affected by hypoxia, and greatest at site five.

484

Abadie and Poirrier

Figure 3. Rangia cuneata size class frequencies from 55 sample sites in
lakes Maurepas, Pontchartrain, and Borgne, Louisiana.

DISCUSSION

A comparison of the density of large Rangia from prior studies
(Tarver and Dugas 1973. Sikora and Sikora 1982, Poirrier et al.
1984. Darnell in USACOE 1987) with current data (Abadie 1998)
shows there was a significant decrease from the early 1950s
through the 1980s in the density of large Rangia. The comparison
also revealed that current densities are not significantly different
from those found in DarnelFs 1954 study (USACOE 1987) (Fig.
2). The decrease in the density of large Rangia appears to be
related to the intensity of shell dredging, and the best explanation
for the increase in the density of large clams is the cessation of
shell dredging since the summer of 1990. When shell dredging
began in 1933. about 0.25 million cubic yards of shells were har-
vested per year. Until 1956, production was less than 2 million
cubic yards per year. Production increased after 1956, peaked at
7.5 million cubic yards in 1976, and then declined (USACOE
1987). Many live Rangia clams were harvested in the early years
of production, but by the early 197()s. the number of live clams
decreased (Tarver 1972). Although restrictions were in place, a
comparison of current data with historic data confirms that shell
dredging had a significant effect on the density of large clams.

The severity of the impact of shell dredging on Lake Pontchar-
train was unclear (GSRI 1974, USACOE 1987) because no studies
were conducted before 1933, when dredging started. The only
early quantitative data on benthic invertebrates was (he density of
large clams from Darnell's studies during 1953 and 1954 (USA-
COE 1987). These limited data may not have been adequate lo
characterize past conditions, and the contribution of other pertur-
bations such as urban runoff and saltwater intrusion to any de-
crease in clam density was unknown. Direct removal of large
clams by dredging was not thought to produce an effect because,
with the restrictions in place, it was supposed to take up to 4 years
to disturb an area equivalent to the area permitted for dredging.
However, some areas were disturbed more frequently than others
(USACOE 1987). Sikora and Sikora (1982) suggested that dredg-
ing decreased sedimeni bulk density and large clams sank into the
less dense sedimeni. However, experiments by Taylor Biological
Co. Indicated that clams could maintain their position in Lake
Pontchartrain's sedimeni bulk densities (USACOE 1987).

The absence or low densities of large clams from silcs iiorlli of
the Inner Harbor Navigation Canal (IIINC) indicates ihal episodic
hypoxia from salinity stratification due to sall-waler intrusion
(Poirrier 1978. Junot et al. 1983. Schurt/ and St. Pe 1984) affects

the establishment of older, larger Rangia. Based on the sites that
had a statistically significant difference in the density of large
clams, a 350-km- area is affected (Fig 1). The IHNC was com-
pleted in 1963, and changes in the salinity regime and episodic
hypoxia occurred along with the effects of shell dredging. The
combined effects of shell dredging and salt-water intrusion may
have contributed to the lakewide reduction of large clams. Shell
dredging produced trenches that were about 2 m wide and 1 m
deep. These trenches were generally backfilled by sediment dis-
charge. However, when they were not completely filled, they may
have distributed saline, hypoxic waters from the area near the
IHNC to other areas of the lake.

Many large Rangia were found in the eastern lobe of Lake
Pontchartrain, and relatively high densities were found in the more
saline waters of Lake Borgne (Fig. 1). No large Rangia were
reported from the eastern lobe of Lake Pontchartrain in past stud-
ies, including Darnell's 1954 study (USACOE 1987). This is the
first quantitative report of Rangia densities from Lake Borgne.

Sikora and Sikora (1982) reported average lakewide densities
of 3,164/m" for 0-lOmm Rangia. which were much higher than
the average of 266/m" reported by Poirrier et al. (1984), or the
average of 278/m" found in this study (Abadie 1998). Sikora and
Sikora' s (1982) mean densities were higher than the highest site
densities of 902/ni" and l,568/m~ reported by Poimeret al. (1984)
and this study (Abadie 1998). More small clams may have been
present in the past due to lack of competition from large clams, the
numbers of which were reduced from dredging activities. How-
ever, this is not supported by the comparable values reported by
Poirrier et al. (1984).

The cuiTcnt Rangia population shows a bimodal distribution
(Fig. 3). indicating heavy recruitment may occur continually (Fair-
banks 1963). Intermediate size classes are found in lower frequen-
cies due to intense predation pressure (Darnell 1961). As clams
increase in size, growth rate slows, predation pressure drops, and
a stable peak of larger clams is evident.

In the analysis of the large size classes over time along the
east-west transect, two trends arc clear. The number of large clams
at each site remains stable throughout the year. Thus, the 1997
spillway opening probably did not have a deleterious effect on the
density of large clams. The second apparent trend is the low num-
ber of large clams through the year al the site north of the IHNC.
Low densities of large Rangia were also found at 15 other sites in
the vicinity due to episodic hypoxia (Fig. I ).

The increase in Secchi disc transparency after shell dredging
was stopped (Francis and Poiirier 1998) may be related to the
increase in density of large clams. It is apparent that large clams
have returned to north shore and mid lake sites. However, densities
remain al low at south shore sites due lo the effects of the IHNC.
Francis and Poirrier (1998) found water clarity at both a north
shore and a mid lake site was better than at a south shore site.
Therefore, the high filtration rate associated with high densities of
large Rangia may help improve general water quality.

ACKNOWLEDGMENTS

We v\(uild like lo acknowledge the generous linancial suppi)rt
of this work by Frecporl-McMoRan. Inc. We would also like to
thank Dr. Pamela O'Neil for her suggestions on statistical analy.ses
and review of the manuscript. Dr. John Francis for his comments,
and Chris Schichle for his help wilh the benthic collections.

Recovery of Large Rang/a in Lake Pontchartrain

485

LITERATURE CITED

Abadie. S. W. 1998. Distribution and abundance of the bivalve RaiigUi
cimeala in lakes Maurepas, Pontclianrain and Borgne. Master's Thesis,
University of New Orleans, New Orleans, Louisiana. 142 pp.

Barbe. D. E. & M. A. Poirrier. 1991 . The urbanization of the watershed in
metropolitan New Orleans, pp. 147-154. In D. Dhanuith;iran. H. C.
McWreath, and A. I. Johnson (eds.). Proceedings of the 27"' annnual
conference Water Maiuigement of River Systems and symposium Re-
source Development of the Lower Mississippi River. American Water
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Cain, T. E. 1975. Reproduction and recruitment of the bracki.sh water clam
Rangia cimeata in the James River, Virginia. U S Natl. Mar. Fish. Serv.
Fish. Bull. 73:412-430,

Darnell. R. M. 1961. Trophic spectrum of an estuarine community, based
on studies of Lake Pontchartrain, Louisiana. Ecology 42:553-568.

Fairbanks, L. D. 1963. Biodemographic studies of the clam /?(»i^?/a cuneata
Gray. Tulane Stud. Zool. 10:3-17.

Francis, J. C. M. A. Poirrier, D. E. Barbe. V. Wijesundera & M. M.
Mulino. 1994. Historical trends in the Secchi disk transparency in Lake
Pontchartrain. Gulf Res.Rep. 9:1-16.

Francis, J. C. and M. A. Poirrier. 1998. Recent trends in water clarity of
Lake Pontchartrain. Gulf Res. Rep. 11:1-9.

Gaston. G. R.. C. M. Cleveland. S. S. Brown & C. F. Rakocinski. 1996.
Benthic-pelagic coupling in northern Gulf of Mexico estuaries: Do
benthos feed directly on phytoplankton? Gulf Res. Rep. 9:231-237.

Gulf South Research Institute (GSRl). 1974. Environmental impact of shell
dredging in Lake Pontchartrain. GSRI Project No. 414-665-41. Gulf
South Research Institute. New Iberia. Louisiana. 275 pp.

Holley. M. E. & D. W. Foltz. 1987. Effects of multiple-locus heterozy-
gosity and salinity on clearance rate in a brackish water clam. Rangia
cuneata. J. Exp. Mar. Biol. Ecol. 3:121-131.

Hopkins. S. H., J. W. Anderson & K. Horvath. 1973. The brackish water
clam Rangia cuneata as indicator of ecological effects of salinity
changes in coastal waters. U.S. Army Engineer Waterways E.xperiment
Station. Vicksburg. Mississippi. Contract Report No. DACW39-71-C-
0007. 257 pp.

Houck. O. A.. F. Wagner & J. B. Elstrott. 1989. To restore Lake Pont-
chartrain. A Report to the Greater New Orleans Expressway Commis-
sion. New Orleans. Louisiana. 269 pp.

Junot. J. A.. M. A. Poirrier & T. M. Soniat. 1983. Effects of saltwater
intrusion from the Inner Harbor Navigation Canal on the benthos of
Lake Pontchartrain. Gulf Res. Rep. 7:247-254.

LaSalle. M. W. & A. A. de la Cruz. 1985. Species profiles: Life histories
and environmental requirements of coastal fishes and invertebrates
(Gulf of Mexico) — Common rangia. U.S. Fish Wildl. Serv. Biol. Rep.
82 (11.31). U.S. Army Corps of Engineers, TR EL-82-4. 16 pp.

Perret, W. S.. B. B. Barney, L. R. Walter, J. F. Pollard, W. R. Mock. G. B.
Adkins. W. J. Gaidry & C. J. White. 1 97 1 . Cooperative Gulf of Mexico
estuarine inventory and study. Louisiana. Phase I. area description and
phase IV. biology. Louisiana Wildlife and Fisheries Commission. New
Orleans. Louisiana. 175 pp.

Poirrier. M. A. 1978. Studies of salinity stratification in southern Lake
Pontchartrain near the Inner Harbor Navigation Canal. Proc. La. Acad.
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Poirrier. M. A. & M. M. Mulino. 1977. The impact of the 1975 Bonnet
Carre Spillway opening on epifaunal invertebrates in southern Lake
Pontchartrain. J. Elisha Mitchell Sci. Soc. 93:11-18.

Poirrier. M. A.. T. Soniat. Y. King & L. Smith. 1984. An evaluation of the
southern Lake Pontchartrain benthos community. Final Report to the
Louisiana Department of Environmental Quality. New Orleans. Loui-
siana. 79 pp.

Schurtz, M. H. and K. M. St. Pe. 1984. Water quaUty investigation of
environmental conditions in Lake Pontchartrain. Report on Interim
Findings to Louisiana Department of Environmental Quality. Water
Pollution Division. Baton Rouge. Louisiana. 85 pp.

Sikora. W. B. & J. P. Sikora. 1982. Ecological characterization of the
benthic community of Lake Pontchartrain. Louisiana. Publication No.
LSU-CEL-81-18. Baton Rouge. Louisiana. 214 pp.

Suttkus. R. D.. R. M. Darnell & J. H. Darnell. 1954. Biological study of
Lake Pontchartrain. Annual Report, 1953-1954. Zoology Dept.. Tulane
University. New Orleans. Louisiana. 59 pp.

Tarver. J. W. 1972. Occurrence, distribution and density o( Rangia cuneata
in lakes Pontchartrain and Maurepas, Louisiana. La. Wildl. Fish.
Comm. Tech. Bull. No.l. 8 pp.

Tarver, J.W. and R. J. Dugas. 1973. A study of the clam Rangia cuneata
in Lake Pontchartrain and Lake Maurepas, Louisiana. La. Wildl. Fish.
Comm. Tech. Bull. No.5. 97 pp.

U.S. Army Corps of Engineers (USACOE). 1987. Clam shell dredging in
lakes Pontchartrain and Maurepas, Louisiana — Draft Environmental
Impact Statement and Appendixes. United States Army Corps of En-
gineers. New Orleans District. New Orleans, Louisiana.

Jotmmt oj Shellfish Research. Vol. 19, No. 1, 487-491, JdUO.

EVALUATION OF A NEW TAGGING TECHNIQUE FOR MONITORING

RESTORATION SUCCESS

M. J. STEWART AND R. G. CREESE

Leigh Marine Laboratory
University of Auckland
P.O. Box 349
Warkworth. New Zealand

ABSTRACT Venerid clams, Aiistrovenus srurehbuiyi. were tagged with small aluminum discs, enabling relocation using a metal
detector. Tag loss varied between treatment types, being highest for small, densely packed clams. Over three sites the mean tag loss
across all treatments was 10% (± 2.87) after 7 months. This is likely to be an overestimate as only a subsample of individuals was
recaptured. Laboratory studies showed no significant difference in survival, growth, or condition between tagged and untagged clams.
Ability to rebury was not affected by tags; all tagged and untagged individuals burrowed within 24 h of being placed in tanks. The
technique was also found effective for a deeper burrowing tellinid bivalve. The extensive movement of four whelk species made
relocation difficult, but the technique still holds potential for the tag and recapture of these gastropods. Studies previously considered
difficult are feasible with this technique.

KEY WORDS: Aiistrovenus stiiulibitryi. clam, restoration, metal detector, monitoring, tag and recapture. New Zealand.

INTRODUCTION

Austrovemis stutchburyi (Wood 1828) is a shallow-burrowing,
filter-feeding clam found in sheltered, soft-shore, intertidal habi-
tats around New Zealand. Adult clams have an average shell length
of 30—10 mm. Populations of A. stutchburyi are vulnerable to
increased sedimentation from coastal development and overhar-
vesting. Consequently, this popular resource has declined at many
locations throughout New Zealand, although the extent of this
decline has only recently been recognized. Our research investi-
gates the potential for restoration of infaunal clams through studies
of the ecology of A, stutchbuiyi: the study included manipulative
field experiments to assess movement patterns, predation rates.
and responses to translocations. Despite being used increasingly
overseas, restoration is a novel technique for New Zealand.

Restoration requires monitoring of biological parameters to de-
termine success (Pratt 1994). In the long term, reproductive output
and the ability to establish self-maintaining or sustaining popula-
tions are the most critical considerations, but in the initial stages of
shellfish enhancement the important parameters are survival and
growth. Estimates of these can be obtained at the population level
by using cohort analysis or through the analysis of growth rings
(Lutz and Rhodes 1980). More direct estimates can be obtained by
tag and recapture techniques (Brousseau 1978, 1979; Craig 1994).
In high latitude marine environments, bivalves often lay down
annual rings that correspond to seasonal growth spurts (usually in
summer). This phenomenon has been reported for some A. stittch-
biiiyi populations in southern New Zealand (Coutts 1974. Marsden
and Pilkington 1995). but many other studies have shown that shell
rings are too variable to be relied upon for calculations of age or
growth (Larcombe 1971. Coutts 1974. Blackwell 1984. Martin
1984), Tag and recapture procedures are considered more likely to
provide accurate estimates of survival and growth for this species.

There are several methods of externally marking shells for later
recapture of known individuals; for example, paint (Dobinson et
al. 1989). alizarin red. a calcium stain (Peterson et al. 1995). or
numbered tags. New Zealand bivalves commonly been tagged with
numbered plastic tags glued to the shell (e.g., A. stutchbuiyi. Mar-
tin 1984, Paphies subtriangulata. Grant 1994. P. austral is. Hooker

1995). The tag and recapture technique requires a reasonable re-
capture rate, which can be difficult to achieve for infaunal bi-
valves. Conventionally tagged animals often migrate out of the
area where they were released and it takes considerable time and
effort to sieve through large amounts of sediment to find them.
This also makes estimates of mortality difficult, as there is no way
of knowing how many tagged animals were missed.

One way to avoid this is to cage bivalves on the shore, which
is a common experimental technique (Hurlberg and Oliver 1980;
Virnstein 1980; Martin 1984). However, this procedure is difficult
to implement on beaches visited by large numbers of people and in
areas impacted by harvesting. In soft-sediment habitats, cages can
influence water flow and sedimentation in experimental plots and
these effects must be assessed using appropriate controls (Hurlberg
and Oliver 1980), A new technique with the potential to avoid
these problems tags bivalves with small aluminum tags glued to
the shell, and relocates them using a highly sensitive metal detector
run over the surface of the sediment. This technique was pioneered
in South Africa for the highly mobile surf clam, Donax serra
(Dugan and McLachlan, 1999). Tag loss in their study was
around 4% and tagging was found to have no significant effect on
condition or behavior. Such a tagging method would enable A.
stutchbuiyi to be relocated over a wide area, without caging. Prior
to this. Neves et al. (1989) tested techniques of telemetry on the
freshwater mussel Actinonaias ligainentia. Using epoxy resin,
magnets were secured to the valve, the mussels were placed at
known locations, and a systematic search conducted with a mag-
nometer. The degree of successful relocation of tagged mussels in
their study was not given.

Estimation of growth and mortality depends on the assumptions
that tagging does not affect behavior, increase the probability of
predation or disease, or negatively effect growth or longevity
(Southwood 1966). Many infaunal bivalves, including venerids,
are well suited to external tagging because they are hardy, have a
heavy shell (a tag therefore adds little weight), are reasonably large
(tags can therefore be positioned so as not to interfere with opening
or closing of the valves or protrusion of siphons), and their bur-
rowing behavior means tags are not visible to predators.

This paper evaluates the potential use of aluminum tags and a

487

488

Stewart and Creese

metal detector in tag and recapture studies of A. stiirclibiiryi. Field
and laboratory studies were used to test assumptions about tag loss
(as recommended by Trebbie et ai. 1993) and the effects of tags on
clam condition and behavior (as recommended by Martin 1984).
Preliminary trials were also carried out on co-occurring bivalves
and whelks.

Where volume
al. 1991)

tt/6 (shell height x length x breadth) (Savari et

METHODS AND MATERIALS

Tagging

Aiisiroveniis stittchbuiyi were collected at low tide as this is
when the clams are accessible in the field and least active
(Beentjes and Williams 1986: Williams et al. 1993). After blotting
with a paper towel, the clams were air-dried to provide a clean dry
surface for attachment of tags. Aluminum tags were attached to the
valve away from the apex and shell margin, using a clear, two-part
epoxy resin (Araldite). Tags were 1x5x5 mm and weighed 69
mg (± 1.7 mg). All clams were also given a second tag consisting
of a dot of enamel paint, which had been found previously to
remain on the shell for at least 10 months. This allowed later
estimates of the rate of loss of the aluminum tags. Enamel paint
rather than plastic tags, was used because of the large number of
shellfish in the experiment. After the glue had hardened and set,
tagged clams were returned to the tank, prior to use in various
experiments. The large clams were removed from the water for
approximately 1.5 h in total and smaller clams for a shorter time.

Assessing Tag Effect

Tagged (treatment) and untagged (control) A. suilchhuiyi were
kept in aquaria (320 x 250 x 150 mm) and monitored to determine
the effect of tagging on mortality and growth. A 3-cm layer of
sediment was placed in each aquarium prior to adding clams in
order to mimic the natural environment. Sediment had been sieved
through 2-mm mesh sieve to remove large macrofauna. Each
aquarium held 10 small clams (10- 1 8mm) and 10 large clams
(25-32mm). Clams in control aquaria were subject to the same
drying process as those tagged. Before being placed in the aquaria,
all clams were measured (to the nearest 0.1 mm) and weighed
before and after the addition of tags. Three randomly positioned
replicate treatment and control aquaria were used.

The burrowing behavior of tagged and untagged clams was
observed over the first 48 h. Aquaria were checked for mortality
weekly, and clams were re-measured monthly. After 5 months, the
physiological status of the tagged and untagged clams was com-
pared using condition indices. To ensure that the polenlial effects
on different components of the condition analyses were detected,
three separate indices were used. These were dry weight condition
index (Cl-dry), gravimetric condition index (Cl-grav), and bod>
condition index (BCD.

Cl-dry

dry tissue weight (g) x 100

shell weight (g)
(Crosby and Gale 1990, Marsden and Pilking 1995)

Ci-grav = :

dry tissue weight (g) x 100

internal shell cavity capacity (g)

Where internal shell cavity capacity = total whole live weight
dry shell weight (Crosby and Gale 1990)

dry tissue weight (g) x 100

Tag Loss and Tag Relocation

Loss of aluminum tags was assessed both in the laboratory
experiment on tag effect and in the field as part of a transplant
experiment. Field studies were carried out at two intertidal sites.
Point Wells and Lews Bay in the Whangateau Harbour near Leigh,
in northeastern New Zealand (Fig. 1 ). A total of 4500 cockles were
double-tagged and returned to the Whangateau Harbour (see Fig.
I ), where they were transplanted to three separate sites (two at
Point Wells and one at Lews Bay). Each group of 1,500 clams
consisted of equal numbers of small (10-18 mm) and large (27-35
mm) individuals. Within each size category, the clams were trans-
planted into either packed, high-density plots (200 clams/0.25 m")
or spaced out, low-density plots (50 clams/0.25 m~). There were
three replicates of each combination of treatments (clam size and
density), giving an orthogonal multifactorial design. Tag loss was
assessed during the experiment from marked cluckers (empty
valves still attached at the hinge) retrieved in visual searches at the
transplant sites (conducted weekly where possible). The assump-
tion was made that tag loss from cluckers was representative of tag
loss from live cockles.

The transplant experiment enabled the effectiveness of tag re-
location to be assessed. A metal detector (Minelab sovereign XS)
was moved across the sediment surface and when a tag was de-
tected (signalled by an increased tone), the area was marked and
the sediment was carefully excavated to expose the tagged clam.

Applications to Other Species

Preliminary trials were conducted to test the effectiveness of
the aluminum tagging methodology for Maconwna liliaiui (Iredale
1915) and several species of whelks. M. liliaiui was chosen for
comparison with A. stiitchhiiiyi as it is found in similar habitats,
but it is deep-burrowing, living approximately 20 cm below the
surface. Whelks were chosen to test the method on co-occurring
species that are highly motile.

Macomoiui liliana were collected from the field and then
tagged using the same method as for A. snitrhhiiryi. They were

BCI =

shell cavitv volume

I'iSiirc 1. Transphint ev|Hriim'nts iisinn the uluminum tans were con-
ducti'd ut l.i'ws lSa> and Point Wi'lls in the Whangateau Harbour {=
36°26'S, 174 4ft'El in northeastern New Zealand.

New Tagging Technique for Monitoring Restoration

489

held in salt-water, tlow-through tanks (nemighl and then returned
to the field on the next low tide. Three replicate plots were set up.
each containing 20 adult M. liliana. These plots were checked
regularly using the metal detector.

Four species of whelks were collected from the field. These
were tagged using the same method as for A. stutchhidyi and
released in the same area they were collected from. The release site
had a permanent marker from which movement by whelks could
be calculated. This tag and release process was repeated twice.
On the first, 15 each of Coiiiinella imicidosa ( Marty n 1784), C.
adspersa (Brugiere 1789), C. virgata (Adams 1863). and C glan-
diformis (Reeve 1847) were tagged. On the second occasion, 30 C
maculosa. 50 C. glandiformis, 20 C. virgatci. and 1 1 Lepsiella
scohina (Quoy and Gaimard 1833) were tagged. As with the
clams, the metal detector was swept over the sediment surface to
locate the whelks. When a whelk was detected the location was
marked with a plastic straw. When no more whelks were detected,
each whelk marked by a straw was identified and recorded.

RESULTS

Tag Effect

Ability to rebury was not affected by the tags. All tagged and
untagged A. stutchbiii-yi burrowed within 24 h of being placed in
laboratory tanks, and they remained burrowed for the entire ex-
periment.

Mortality was not significantly different between tagged and
untagged A. sliitchbiiiyi, for both the small and large clams (Fig.
2). However, there is an apparent difference in mortality between
small, tagged clams and control clams. But the fact that the trend
is toward higher mortality for control clams (36.7% versus 16.7%
for tagged clams), certainly does not indicate an effect of the tag
and is most likely due to problems with water supply. A two-way
ANOVA (data pooled across tanks) showed no significant differ-
ence in mortality between treatments {P = .3336) or sizes (P =
.0736) and no treatment * size interaction {P = .3336).

Although there was no obvious affect of the tagging procedure
on mortality, there may have been a more subtle, sublethal impact.
This was investigated by examining three indices of physiological
condition. Three-way ANOVAs (treatment, size of cockle, tank)
for each index revealed there were no significant interaction terms
(P < .05 for all three indices). There was no significant tank affect

DU -

^45-

o

E 30-

D. 15 -

T

T

n _

1

1

1

x^*

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c<S^''

c<!^'

.^^

o!.<i^

A*

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^°'

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Figure
months

Size and tag treatment

2. Percent mortality of tagged and untagged clams after 5
. Error bars are standard error, (n = 3).

o
O

•

BCI

*

bU -

■

Cl-grav

1

50 -

▲

Cl-dry

■

40-

■

30 -

■

•

20 -

•

•

10 ^

0 ^

1

1

1

1 1

.\^*

#

A^°^

ci^"

cj^*

\»

i~<i^

^o

s#

.<^^°^

\*

i.<^^

Size and tag treatment

Figure 3. Condition indices for tagged and untagged clams after 5
months in the laboratory. Error bars are standard errors, (n = 14
small clams, n = 26 large clams). Replicates have been pooled.

and therefore data were pooled across the three replicate tanks for
graphical representation (Fig. 3). There was no significant effect of
treatment {P < .05 for all three indices), for tagged clams versus
control clams, which was the comparison of interest. There was. as
expected, a significant effect of size for all three indices, an artifact
of the indices used.

There was no difference in growth between tagged and un-
tagged clams, principally due to the fact that the clams did not
grow significantly over the study period (Table 1 ). Dobbinson et
al. ( 1989) also found a lack of growth for A. stulchbiiryi within the
time frame of their experiment. Tagged clams that had been in the
field for nearly a year were just beginning their summer growth
spurt, with small clams having grown 2 mm or more over the
months of September to October. This suggests that the tag had
little, if any, effect on growth, even for small clams.

Tag Loss and Tag Relocation

No tag loss occurred during the 5 months that A. stiitchhinyi
were held in the laboratory. Mean tag loss in the field over three
sites, across all treatments, was 10% (± 2.87). Because of the low

TABLE L

Initial and final mean size of tagged and control clams, used in
laboratory experiments investigating tag effect.

Month 1

Month 5

Size

Treatment

Mean

SE

Mean

SE

Small

Tag

15.4

0.65

15.2

0.65

Small

Tag

14.8

0.66

14.5

0.58

Small

Tag

15.3

0.54

15.3

0.65

Small

Control

15.3

0.73

1 5. 1

O.I I

Small

Control

15.3

0.65

15.8

0.70

Small

Control

15.0

0.67

15.5

0.76

Large

Tag

29.6

0.56

29.3

0.72

Large

Tag

29.3

0.59

29.1

0.64

Large

Tag

28.9

0.80

28.8

0.81

Large

Control

29.8

0.63

29.9

0.70

Large

Control

29.7

0.62

29.2

0.57

Large

Control

29.1

0.48

29.3

0.59

490

Stewart and Creese

TABLE 2.
Relocation rate for tagged whelks in the field.

Total Relocation

All Species

C. virgata

c.

maculosa

C. glandiformis

L. scobina

Day

n = 111

n = 20

n = 30

n = 50

n = 11

1

10.8%

2U9r

3%

10%

18%

6

1.8%

30%

0%

2%

18%

10

0%

0%

0%

0%

0%

mortality of transplants (and therefore low numbers of cluckers
retrieved), tag loss among treatments could not be compared sta-
tistically using this method. However, observational data suggest
tag loss was greatest for small, densely packed clams. Although
experimental plots were never permanently marked, it was pos-
sible to relocate plots on every sampling occasion. Relocation was
accurate enough to avoid disturbing large areas of sediment.

Applications to Other Species

Macomona tiliana were successfully relocated in the field after
2 months and there was no evidence of mortality for tagged indi-
viduals in the field. There has been a low return rate for the
whelks, which is attributed to them moving away from the area,
between tides, too quickly to be tracked. On the first sampling.
only 16% of all whelks were relocated 1 day after release. All of
these were either C. adspersa or C. maculosa. Three days later
only one or two whelks were relocated. On the second sampling.
there was a 4.5% incidence of tag loss before release. After 2 days.
10.8% of all whelks were relocated. Relocation rates were highest
for C. virgata (20%) and L. scobina (\^%) (Table 2). After 6 days,
total relocation was only 1.8%, but relocation for C virgata in-
creased to 30% and remained at 18% for L. scobina (Table 2).
After 10 days no whelks were detected. During this experiment,
the whelks that were relocated were invariably solitary individuals,
illustrating that the metal detector was sensitive enough to pinpoint
a single tag approximately 3 cm under the sediment surface.

DISCUSSION

The aluminum tags had no detectable effect on growth, mor-
tality, or behavior of A. stiitchbiiryi. In addition, no effect on
condition was found for three separate condition indices, including
the gravimetric condition index (Cl-grav), which is the recom-
mended condition index to assess whether animals have been un-
der stressful conditions (Crosby and Gale 1990). However, (he

time of year when tags are attached may affect growth and con-
dition. Growth for shellfish is often seasonal, and attaching the
tags during a period of high growth may have more effect than
attaching them at another time of year. A long-term study of tag
effect is required to investigate this, but the laboratory studies
conducted here (in summer) suggest that any effect is likely to be
small, irtespective of season. The advantage of using aluminum for
the tag is that it is light enough not to affect behavior and is
rust-resistant in saline conditions.

The fact that no clams held in the laboratory lost their tags may
have been due to an absence of abrasive forces such as currents
and the movement of abutting shellfish (which would be experi-
enced in nature). These laboratory trials do confirm that the glue
and tag are able to remain bonded to the shellfish in salt water for
at least 5 months. Tag loss in the field was relatively low and did
not reduce the effectiveness of the method. Because A. stiitchbiiryi
were in clusters, only a few tags were required to locate the plots.
The metal detector is sensitive enough to locate a single tagged
clam, but as no individuals moved away from the experimental
plot this was unnecessary. In tenns of evaluating the performance
of restoration, this technique worked well for the ongoing moni-
toring of experimental transplants. With minimal effort it was
possible to relocate experimental plots without the necessity of
permanently marking them. For a full-scale restoration project, the
time and effort required to tag all individuals would obviously be
prohibitive. However, the technique would still work well if even
a small proportion of the seed shellfish for enhanceinent were
tagged for ongoing monitoring and treated as representative of the
population. Also, this technique holds potential for ecological
studies that seek to provide more information on which to base
decisions about restoration alternatives.

The technique pioneered by Dugan and McLachlan (1999).
and further developed in this paper, has allowed the recapture and
tracking of bivalves. Dugan and McLachlan ( 1999) were able to

TABLE 3.
Burial depths of some common intertidal New Zealand .soft-sediment bivalves.

Common

Species

Name

Macomiina liliciiui

wedge she!

Aitstravcnus sttttchhtiryi

Cdckie

Niictilu luirlvifiiaini

mil shell

Pophies ventricosa

liiheroa

Pcipliies iiuslrulis
Pnphies siihtritmKulaUi

pipi

Maximum

Size

Burial Depth

Reference

.■iD-WInim

=2()cni

Morton & Miller (1973)

30-l()nini

Top few cm

Morton tV Miller (1973)

<l()nim

Top few cm

Morton & Miller (1973)

>l.^()iiim

Top few em

Hooker (unpubl. data)

-.tOem

Stace (1991)

s.S0mm

Top S-IOcm

Morion & Miller (1973)
Hooker (1995)

=90mm

Top S-IOcm

Hooker (I99.'ii

New Tagging Technique for Monitoring Restoration

491

track the longshore movement of individuals. In this paper, we
were able to use a metal detector to successfully monitor clams
transplanted for small-scale experimental restoration. The metal
detector used (Minelab Sovereign XS). has a detection range of
approximately 20-30 cm below the surface for a 1 mm x 5 mm x
5 mm aluminum tag. The detection range is a function of tag size
and burial depth of the target organism. The detection range can be
improved by increasing the size of the tag. but this will ultimately
be limited by the size and shape of the bivalve. Reported burial
depths of some common New Zealand soft sediment bivalves sug-
gest that aluminum tags may possibly be used for all these species
(Table 3). Further experiments are needed to test for tag effects and
the relocation efficiency of these other species, many of which live
deeper in the sediment than A. stutchburyi. However, as reported
here, the burial depth of M. liliana (= 20 cm) did not hamper the
relocation of this species.

There was a low return rate for the whelks, as they rapidly
move away from an area between tides, too quickly to be tracked.

However the whelks that were relocated illustrated that the metal
detector was sensitive enough to pinpoint a single tag. The recap-
ture rate varied between the species tagged, being greatest for C.
virgata. Therefore, while it may not be possible to follow move-
ments of whelks over the long-term, the technique may still yield
important infomiation on movement patterns of other species.

Overall we believe that the simplicity, reliability, and versatil-
ity of this metal detection technique opens many new avenues for
researchers in the area of soft-sediment ecology and restoration
monitoring.

ACKNOWLEDGMENTS

This work was funded in part by a grant in aid of research from
the Auckland Regional Council. Thank you to Jenny Dugan for her
advice when purchasing the detector and during this project. The
tagging of so many shellfish was only possible with the help of
many students at the Leigh Marine Laboratory.

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Jounwl of Shellfish Research. Vol. 19, No. 1. 493-541, 2000.

ABSTRACTS OF PAPERS

Presented at the 4th International Abalone Symposium

Cape Town, South Africa
February. 2000

493

4lh International Abalonc Symposium, Cape Town. South Africa Abstracts, February 2000 495

CONTENTS
Vol. 19, No. 1 JUNE 2000

V. J. Allen, I. D. Marsden, and N. L. C. Ragg

The use of stimulants as an aid to wean fishery-caught biacivfoot abalone {Haliotis iris) to artificial food 301

S. Apisawetakan, M. Chanpoo, C. Wanichanon, V. Linthong, M. Kruatrachue, S. E. Upatham, T. Pumlhong, and
P. Sobhon

Characterization of trabecular cells in the gonads of Haliotis asinina 50 1

C. Anguiano Beltrdn, R. Searcy Bernal. and A. Esparza Hernandez

The effect of irradiance on the survival and growth of abalone postlarvae Haliotis fiilgens fed with Navicula incerta .. 501
M. N. Bautista Teriiel and O. M. Millamena

Diet development and evaluation for juvenile donkey's ear abalone, Haliotis asinina Linn.: lipid levels 501

S. J. Boarder and M. Shpigel

Comparative growth performance of juvenile Haliotis roei fed on enriched Ulva rigida and various artificial diets 502

L. Botes, G. C. Pitcher and P. A. Cook

The potential risk of harmful algae to abalone farming on the south coast of South Africa 502

J. Cdceres Martinez, C. Alvarez Tinajero, and Y. Guerrero Renten'a

Rikettsiales-like prokaryotes in cultured and natural populations of the red abalone Haliotis rufescens. blue abalone

Haliotis fiilgens. and the yellow abalone Haliotis corrugata from Baja California, Mexico 503

J. Cdceres Martinez and G. D. Tinoco Orca

S>'mbionts of red abalone Haliotis niffscens from Baja California, Mexico 503

L. Carreon Patau, S. A. Guzman del Proo, J. Belmar P., J. Carillo L., R. Herrera F. and A. Villa B.

Microhabitat. distribution and abundance of juveniles of Haliotis fiilgens and H. corrugata in Bahia Tortugas. Mexico 503

M. Chanpoo, S. Apisawetakan, A. Thongkiikiatkul, C. Wanichanon, V. Linthong, M. Kruatrachue, S. E. Upatham,
T. Pumthong, P. J. Hanna, and P. Sobhon

Localization of the egg-laying hormone (ELH) in the gonads of a tropical abalone, Haliotis asinina Linnaeus 504

Y. P. Chitramvong, M. Kruatrachue, E. S. Upatham, S. Singhakaew, and K. Parkpoomkamol

The pallial organs of Haliotis asinina Linnaeus, 1 758 (Gastropoda: Haliotidae) in Thailand 504

J. G. Correa Reyes, M. del Pilar Sanchez Saavedra, and N. Flares Acevedo

Isolation and growth of seven strains of benthic diatoms, cultured under two different light conditions 505

J. G. Correa Reyes, M. del Pilar Sanchez Saavedra, J. Arturo Simental Trinidad, and N. Flores Acevedo

Chemical composition of eight strains of benthic diatoms, cultured under two different light conditions 505

S. Cummins and P. J. Hanna

Location of egg-laying hormone in reproductive structures and neurons of Haliotis using antibodies raised against

recombinant fusion proteins 506

C. Cuthbert and G. Burnell

Preliminary trials with "green" artificial diets for juvenile abalone and sea urchins 506

S. Daume and A. Krsinich

Growth and survival of Haliotis rubra post-larvae feeding on different algal species 506

S. Daume, A. Krsinich, S. Farrell and M. Gervis

Settlement and early growth of the abalone Haliotis rubra in response to different algal species 507

R. W. Day. G. P. Hawkes. and V. Gomelyuck

Are abalone shell layers deposited annually? Validation using manganese vital staining 507

R. Day, C. Culver, A. Kuris, A. Belcher and D. Morse

The parasite Terehrasabella heterouncinata (Polychaeta) manipulates shell synthesis in Haliotis rufescens 507

S. De Waal and P. Cook

Quantifying the physical and biological attributes of successful ocean seeding sites for farm reared juvenile abalone

(Haliotis midae) 508

M. A. del Rio Portilla

Population genetics of the yellow abalone, Haliotis corrugata. in Cedros and San Benito Islands 508

S. De Waal and P. Cook

Use of a spreadsheet model to investigate the dynamics and economics of a seeded abalone population 508

496 Ahstracis. February 2000

4th International Abalone Symposium. Cape Town. South Africa

F. Diaz, M. A. del Rio Portilla, M. Aguilar, E. Sierra, and A. D. Re Araiijo

Preferred temperature and critical thermal maxima of red abalone Haliotls riifescens 509

K. Doeschate, B. M. Macey and V. E. Coyne

Characterisation of the enteric bacteria of the abalone Haliotis midae, and their role in the digestion of

ingested seaweed 509

B. Drew, Dean Miller, T. Toop and P. Hanna

Identification of expressed HSPs in blacklip abalone {Haliotis rubra) during heat and salinity stresses 509

S. Du and K. Mai

Ontogenetic changes in the activity of main digestive enzymes during the larval and juvenile stages of abalone.

Haliotis discus hanni Ino 510

S. Edwards, C. Burke, S. Hindruin and D. Johns

Recovery and growth effects of anaesthetic and mechanical removal on greenlip (Haliotis laevigata) and blacklip

(Haliotis rubra) abalone 510

N. G. Elliott, B. Evans, N. Gonad, J. Bartlett, R. Officer and N. Sweijd

Application of molecular genetics to the understanding of abalone population structure — Australian and South African

case studies 510

A. Enriquez, A. Shimada, C. Vdsquez and M. T. Viana

hi vitro digestion of cellulose with stomach extracts from abalone (Haliotis fulgens) 511

B. Evans, R. W. G. White and N. G. Elliott

The use of microsatellite markers for parentage analysis in Australian Blacklip and hybrid abalone 511

A. C. Fermin and S. Mae Buen

Compensatory growth after intermittent food deprivation and refeeding in the donkey's ear abalone. Haliotis asiiiiua
(Linnaeus 1 758) 511

A. C. Fermin, M. Bautista Teruel and S. M. Buen

Effects of sequential feeding with seaweed and artificial diets on growth dynamics and survival during on-growing of
abalone. Haliotis asiiiiiia {Linnaeus 1758) 512

C. A. Finley and C. S. Friedman

Examination of the geographic distribution of a Rickcttsia-Uke prokaryote in red abalone. Haliotis rufrsceiis. in

northern California 512

C. A. Finley, C. S. Friedman and T. J. Mulligan

Life history of an exotic sabellid polychaete. Terehrasabella heterouncinata: influence of temperature and

fertilization strategy 513

C S. Friedman, K. B. Andree, T. T. Bobbins, J. D. Shields, J. D. Moore, K. Beauchamp and R. P. Hedrick

"Candidatus Xenohaliotis californieiisis. " a newly described bacterial pathogen and etiological agent of Withering

Syndrome found in abalone. Haliotis spp.. along the west coast of North America 513

C. S. Friedman, T. Robbins, J. L. Jacobsen and J. D. Shields

The cellular immune response of black abalone. Haliotis cnuiierodii Leach, with and without Withering Syndrome ... 514

K. J. Friedman, G. Maguire and K. O. Halm

Evaluation of on-shore and sea-based culture systems for Roe's abalone (Haliotis roei) in Western Australia 514

O. J. G. Gonzalez Aviles

Seeding competent cultured larvae of the blue abalone Haliotis fulgens into some wild stocks off the Island of Cedros

Baja Califiirnia. Mexico 514

H. Roy Gordon, N. Qing, N. Uki, R. Fields, R. Flares, A. Ziomi, M. Tokley, R. Roberts, P. Cook, A. du Plessis and

G. Burnell

World .ibalonc supply, markets & pricing 514

H. R. Gordon

World abalone supply, markets and pricing from historical, current and future perspectives 515

H. K. Gorfine

Post harvest weight loss has iniporlant implications for abalone i.|uotc management 515

H. K. Gorfine and C. D. Dixon

A bclui\ iomal rallicr ihan resourcc-focussed approach may be needed lo ensure suslainabilily of quota managed

abalone fisheries 515

4th International Abalone Symposium. Cape Town. South Africa Abstracts. February 2000 497

H. A. Gorfine, B. L. Taylor and T. I. Walker

Triggers and targets; What are we aiming for with abalone fisheries models? 516

Blair Gray

Variation in mineralogy in the New Zealand blackfoot abalone Halintis iris shell 516

Jose L. Gutierrez Gonzalez, Ana M. Ibarra and Miguel A. del Rio Portilla

Genetic variability of the blue abalone Haliotis fulgens in the west coast of Baja California. Mexico 517

Sergio A. Guzman del Proo, Felipe Salinas. Oleg Zaytsev, Jorge Belmar Perez and Jorge Carrillo Laguna

Dispersion potential of reproductive products and larval stages of abalone {.Haliotis spp.; MoUusca: Gastropoda) in

relation to the hydrodynamics of Bahi'a Tortugas. Mexico 517

Malcolm Haddon

Size-structured models of abalone populations with a cryptic component to the stock 517

A. T. Hancock

Genetic subdivision of the abalone Haliotis roei in south western Australia 518

M. N. Harper, D. W. Keats and R. J. Anderson

The experimental cultivation of the South African kelp Macrocyslis angustifolia 518

J. O. Harris. C. M. Burke, S. J. Edwards and D. R. Johns

Effect of oxygen supersaturation and temperature on juvenile greenlip. Haliotis laevigata, and blacklip, Haliotis

rubra, abalone 518

M. Hauck

An overview of state and non-state responses to abalone poaching in South Africa 518

G. He and K. Mai

Ontogenetic trends of shell biomineraliz.ation in abalone. Haliotis discus hamuli Ino 519

S. Hindrum, C. Burke, S. Edwards and D. Johns

Growth reductions in greenlip (Haliotis laevigata) and blacklip (Haliotis rubra) abalone resulting from chronic

exposure to sublethal combinations of elevated ammonia and low dissolved oxygen levels 519

Z. Hongen

Research into a new technology for artificial abalone breeding 520

T. Horii

Assessment of the effects of fishing intensity on stock levels in the abalone diving fishery 520

D. J. Jackson, K. Williams and B. Degnan

Analysis of the suitability of Australian formulated diets for the aquaculture of the tropical abalone. Haliotis

asiiuiui Lmneus -'^"-'

N. M. J. Kabir and P. V. Mladenov

Spawning induction of Haliotis australis using different chemicals and ganglionic suspensions 521

T. Kawamura. H. Takami and Y. Yamashita

Effects of delayed metamorphosis on survival and growth of newly metamorphosed Haliotis discus haunai 521

R. I. Lewis, E. G. Hall, J. S. Bee and N. A. Sweijd

Non-destructive DNA typing in abalone hatchery management applications 521

A. L. Licona Chavez and M. A. del Rio Portilla

Genetic analysis of a cultured population of the red abalone, Haliotis rufescens. in Mexico 522

M. Litaay and S. S. De Silva

Reproductive performance indices based on physical characteristics of the female blacklip abalone Haliotis rubra L.. . . 522
A. L. Lopata, B. Fenemore and P. C. Potter

IgE and monoclonal antibody binding to abalone and other mollusc allergens 522

A. L. Lopata. T. Luijkx. N. A. Sweijd and P. A. Cook

Immunological detection of various abalone species 523

L. M. Lopez and P. Tyler

Effect of formulated diets, fresh seaweed and temperature on growth rates, gonad development and shell formation of

the European abalone Haliotis tuberculata L 523

A'. C. Loubser and N. Dormehl

The use of ultrasound in the treatment of sabellid infestations in South African abalone 524

498 Ahslmcls. February 2000

4th International Abalone Symposium. Cape Town. South Africa

B. G. Lucas, A. Campbell, B. Clapp and G. S. Jamieson

Growth and ageing of pinto abalone. HalioUs kamtschatkana in Bartcley Sound. British Columbia 524

J. A. Madrones Ladja

The effect of stocking density, temperature and light on the early larval survival of the abalone Haliotis asinhui Linn . 524
K. Mai and G. He

Guaiacol, a powerful modulator of mollusc shell biomineralization 525

S. C. McBride, E. Rotem, D. Ben-Ezra and M. Shpigel

Evaluation of seasonal bioenergetics of Haliotis fuli>ens and Haliotis tuherciilata 525

J. D. Moore, T. T. Robbing and C. S. Friedman

The role of a RickettsiaAike prokaryote in Withering Syndrome in California red abalone. Haliotis rufescens 525

A. Mouton

Health management and disease surveillance in abalone. Haliotis inidae. in South Africa 526

A. Mouton and J. F. Putterill

A comparative scanning electron and light microscopy study of the early life stages of the South African abalone,

Haliotis ntidae 526

D. C. Z. Norman, D. W. Keats and R. J. Anderson

Experimental cultivation of the kelp Ecklonia maxima 526

J. L. O'Loughlin and S. A. Shepherd

Biological reference points for the greenlip abalone {Haliotis laevigata) in different habitats across its

geographic range 527

R. A. Officer

Distance-based abundance estimation for abalone 527

M. Ortiz Quintanilla, G. Lucero M. and J. E. Patron V.

Status of the abalone fishery between 1996 and 1999 on abalone locations of the fishing co-operative production

society "La Purisima", S. C.de R. L., Baja California Sur. Mexico 527

G. Pitcher, J. Franco, K. Whyte and C. Viljoen

Abalone {Haliotis midae) farming and paralytic shellfish poisoning on the coast of South Africa 528

E. E. Plagdnyi and D. S. Buttenvorth

Clues and questions from population models applied to the South African abalone (Haliotis midae) fishery 528

N. L. C. Ragg, H. H. Taylor and J. Behrens

Stress and weight loss associated with handling in the blackfoot abalone. Haliotis iris 528

K. Reddy-Lopata, A. L. Lopata and P. A. Cook

Toxicity and tolerance levels of ammonia in abalone {Haliotis midae) 529

R. Roberts and C. Lapworth

Starvation tolerance of post-larval abalone {Haliotis iris) 529

R. Roberts

A review of larval settlement cues for abalone (Haliotis spp. ) 529

R. Roberts, S. Adams, ./. Smith, A. Pugh, A. Janke, S. Buchanan, P. Hessian and P. Mladenov

Cryopreservation of abalone (Haliotis iris) sperm 530

/,. Rogers-Bennett, P. Haaker and K. Karpov

Selecting and evaluating marine protected areas for abalone in California 530

.S. Sahapong, V. Linthong, S. Apisawetakan, C. Wanichanon, S. Riengrojpitak, V. Viyanant, S. E. Upatham,
N. Kangwanrangsan, T. Pumthong, and P. Sobhon

Morphofunctional slutly ol the hemocytes of Haliotis asiiiiiia: A preliminary report 531

J. Sales, P. J. Britz and I. Shipton

Meal c|ualily characteristics of South African abalone {Ha Hot is midae) 531

R. Sasaki

Post-setlleuicnt observalions o\' \i/o abalone, Haliotis discus liaiiiiai in conjunclion with llora 531

.S'. Sawatpeera, E. Suchart Upatham, M. Kruatrachue, )'. P. Chitramvong, P. Sonchaeng, T. Pumthong and J. Nugranad

Larval development of Haliotis asiiiiiia Linnaeus 532

R. Searcy Bernal, L. A. Velez Espino and C. Anguiano Beltrdn

Effect of biofilm density on grazing rates of Haliotis fiilvciis postlarvae 532

4th International Abalone Symposium. Cape Town. Soiitli Africa Abstracts. February 2000 499

M. J. P. Sehamani. S. M. Degnan. D. Paetkau and B. M. Degnan

Isolation and characterization of microsatellite DNA markers for the tropical abalone, Haliotis asinina 532

E. Serviere Zaragoza, A. Mazariegos Villareal. G. Ponce Diaz and S. Montes Magallon

Growth of juvenile abalone. Haliotis fidgeiis Philippi. fed with different diets 533

S. A. Shepherd and K. R. Rodda

A chronicle of collapse: the dynamics of two overfished greenlip abalone population 533

S. A. Shepherd and K. M. Rodda

Serial decline of the South Australian greenlip and blacklip abalone fishery: time for a requiem mass or a

revival hymn? 533

T. A. Shipton and P. J. Britz

Partial and total substitution of fishmeal with plant protein concentrates in formulated diets for the South African

abalone. Haliotis midae 534

M. Shpigel. I. Lupatsch and A. Neori

Protein content determines the nutritional value of the seaweed Ulva lactuca for the abalone Haliotis tiibercitlata, H.

discus hannai. and H. fulgens 534

J. A. Simental Trinidad, M. P. Sanchez Saavedra and J. G. Correa Reyes

Biochemical composition of benthic marine diatoms using as cultured media a common agricultural fertilizer 534

J. A. Simental Trinidad, M. P. Sanchez Saavedra, J. G. Correa Reyes and N. Flares Acevedo

A novel mass culture system for benthic diatoms 534

N. Sweijd, B. Evans, N. G. Elliott and P. Cook

Molecular tools for compliance enforcement — the identification of southern hemisphere abalone species from

abalone products 555

L. Tai-wu, J. Xiang and R. Liu

Studies on phage control of pustule disease in abalone Haliotis discus hannai 555

H. Takami, T. Kawamura, R. D. Roberts and Y. Yamashita

Morphological changes in the radula of abalone, Haliotis discus hannai and Haliotis iris, in relation to the transitions

in their feeding 555

B. Tan and K. Mai

Availability and dietary requirements for phosphorus in juvenile abalone. Haliotis di.icus hannai Ino 536

K. Mai and B. Tan

Zn and Fe in the forms of methionine chelation or sulphates as sources of dietary minerals for juvenile abalone,

Haliotis discus hannai Ino 536

R. J. Q. Tarr

The South African abalone {Haliotis midae) fishery: a decade of challenges and change 537

R. J. Q. Tarr, P. V. G. Williams, A. J. Mackenzie, E. Plaganyi and C. Moloney

South African Fishery Independent Abalone Surveys 537

N. A. Taylor (nee Dowling), R. McGarvey and S. J. Hall

A parameter estimation model for greenlip abalone (.Haliotis laevigata) population dynamics 537

N. A. Taylor (nee Dowling), S. J. Hall and R. McGar\ey

Simulations or random fishing behaviour as an independent test for active targeting of greenlip abalone {Haliotis

laevigata) aggregations 538

M. J. Tegner, P. L. Haaker, K. L. Riser and L. I. Vilchis

Climate variability, kelps, and the Southern California red abalone fishery 538

M. J. Tegner, K. A. Karpov and P. Kalvass

Abalones and sea urchins: biological and fisheries interactions 539

A. Thongkukiatku, M. Kruatrachue. E. Suchart Upatham, P. Sobhon, C. Wanichanon, Y. Chitramvong and T. Piitnthong

Ultrastructure of neurosecretory cells in the cerebral and pleuro-pedal ganglia of Haliotis asinina Linnaeus 539

M. Tokley

Abalone 539

P. H. Toledo, R. Haroun, H. Fernandez Palacios, M. Izquierdo and J. Peiia

First culture experiences of Haliotis coccinca canariensis in a biofilter system 541

500 Abstracts. February 2000 4th International Abalone Symposium. Cape Town, South Africa

M. E. Vandepeer, P. W. Hone, R. J. van Barneveld and J. N. Havenhand

The digestibility of raw. autoclaved and phytase treated legumes in greenlip abalone, Haliotis laevigata 540

L. A. Velez Espino, R. Searcy Bernal and C. Anguiano Beltrdn

The effect of starvation on grazing rates of Haliotis fulgens postlarvae 540

G. Zhang, Z. Wang, Y. Chang, J. Song, J. Ding, S. Zhao and X. Guo

Tetraploid induction in the Pacific abalone Haliotis discus hannai Ino with 6-DMAP and CB 540

4tli Imeriiational Abalone Symposium. Cape Town. South Africa

Ahslracts. February 2000 501

THE USE OF STIMULANTS AS AN AID TO WEAN FISH-
ERY-CAUGHT BLACKFOOT ABALONE {HALIOTIS IRIS)
TO ARTIFICIAL FOOD. V.J. Allen. L D. Marsden, and
N. L. C. Ragg. Zoology Department, University of Canterbury,
Private Bag 4800. Christchurch. New Zealand.

Abalone use a combination of tactile and chemosensory feed-
ing cues to detect suspended seaweed in their natural environment.
However, in a commercial situation, adult abalone (Haliotis iris)
caught for broodstock or pearling, show reluctance to start feeding
on stationary artificial food and thus mu,st be either induced to feed
or lapse into a starvation phase that may last several weeks. Adult
H. Iris (125 mm) were collected using SCUBA from Banks Pen-
insula and were held at Pendarves Abalone Farm Ltd (South Is-
land, New Zealand). Multiple animals were offered a commercial
pellet diet in a tank containing small quantities (0.03-0.05g dry
weight per litre) of suspended seaweed particles (Gracilaria spp)
acting as a tactile stimulant. Feeding and behavioural responses
were monitored over 4 weeks in triplicate treatment tanks and
compared to control tanks lacking the stimulant. Observations of
behaviour over the first 10 hours showed that abalone held with
and without stimulants spent the majority of their time alert (65.0
± SE 7.6% and 75.0 ± 2.2% of time respectively). However, aba-
lone held with stimulants also displayed typical receptive feeding
posture (shell and foot raised) (21.0 ± 6.8% of time) and feeding
on stimulants (2.1 ± 1.3%) whereas the control animals spent the
remainder of their time either quiescent (15.0 ± 3.8%) or moving
(6.0 ± 2.2%). Abalone fed stimulants in conjunction with artificial
food took approximately 9 days to begin feeding on the artificial
food. The abalone in the control experiment took 15 days to begin
feeding on the artificial food and by day 2 1 their ingestion rate was
only half that of the animals with stimulants (0.064 ± 0.005% and
0.127 + 0.012% biomass ingested per day respectively). The col-
lection and experimental procedures were repeated over four sea-
sons to determine any seasonal effects on behaviour and feeding
patterns. Implications for the maintenance of condition and sur-
vival of adult abalone in aquaculture will be discussed.

CHARACTERIZATION OF TRABECULAR CELLS IN
THE GONADS OF HALIOTIS ASINISA. S. Apisawetakan,'
M. Chanpoo,' C. Wanichanon,' V. Linthong,' M. Kruatra-
chue,- S. E. Upatham,-' T. Pumthong,^ and P. Sobhon,' De

partments of 'Anatomy and "Biology, Faculty of Science, Mahidol
University, Bangkok, Thailand 10400, ^Department of Biology,
Faculty of Science. Burapha University. Chonburi. Thailand.
"'Coastal Aquaculture Development Center. Department of Fish-
ery. Ministry of Agriculture and Cooperatives. Prachuapkhirikhun.
Thailand 77000.

Trabeculae are the connective tissue sheets that extend perpen-
dicularly from capsules of both testis and ovary to make contact at
their innermost ends with the loose connective tissue capsule of

hepatopancreas. Thus they divide the gonads into small compart-
ments, and each trabecula forms the axis for the spermatogenic or
oogenic unit, from which maturing germ cells are generated. When
studied using light and electron microscopes, each trabecula is
shown to be composed of a central capillary, surrounded by a pack
of smooth muscle cells and collagen fibers that are intermingled
with small cells exhibited dense ellipsoid nuclei. Some of these
cells are fibroblasts, while others are follicular or supporting cells
that surround and may play a nurturing role for the developing
germ cells. In addition, there are 3 types of granulated cells ap-
pearing in the trabecula connectives: the first type contains elec-
tron dense rugby-shaped granules with a diameter about 270 x 550
nm; the second type contains electron-dense spherical-shaped
granules with diameters of about 165 nm; and the third type con-
tains electron-lucent spherical-shaped granules with diameters of
about 150 nm. These granulated cells may be the endocrine cells
of the gonads, producing certain gonadotrophic factors yet to be
identified.

THE EFFECT OF IRRADIANCE ON THE SURVIVAL AND
GROWTH OF ABALONE POSTLARVAE HALIOTIS FUL-
GENS FED WITH NAVICULA INCERTA. C. Anguiano Bel-
tran, R. Searcy Bernal, and A. Esparza Hernandez. Instituto de
Investigaciones Oceanologicas, Apartado Postal 453, 22860
Ensenada, Baja California, Mexico.

Survival and growth of Haliotis fulgens postlarvae (lid old)
were evaluated at four different irradiance levels, 6, 24, 47 and 75
microeinsteins-m~"-s~' (p,E). Experimental vessels comprised 11
containers supplied with seawater fiow and aeration. Four repli-
cates per treatment were considered. Postlarvae were fed Navicula
incerta. a benthic diatom. Survival and growth were evaluated.
Shells were counted to detennine mortality. To estimate shell size,
video recorded images were processed by digital analysis. The
highest survival (89.4% after 28d) and growth (37.1|jLmd ') were
found at 6 |jlE. whereas the lowest survival (3.57f) and growth
(21.1|jLm-d'') occurred at 47 |xE. These results suggest that aba-
lone postlarvae have better survival and growth in the low light
intensities under the particular conditions used in the present study.

DIET DEVELOPMENT AND EVALUATION FOR JUVE-
NILE DONKEY'S EAR ABALONE, HALIOTIS ASININA
LINN.: LIPID LEVELS. M.N. Bautista Teruel and O. M.
Millamena. Aquaculture Department, Southeast Asian Fisheries
Development Center, Tigbauan, Iloilo, Philippines 5021.

Juveniles of donkey's ear abalone, Haliotis asinimi w ilh mean
initial weights of 0.71 ± 0.02g and shell lengths of 15.4 ± 0.04 mm
were fed practical diets for 90 days. Diets contained 27% protein

502 Abstracts. February 2000

4th International Abalone Symposium, Cape Town, South Africa

with graded levels of lipid (0.25%, 1.5%, 3%, 4.5%) from a 1:1
ratio of tuna fish oil and soybean oil. Total lipid content of the diets
were 2.2%, 4.69%, 7.7%, 10.7%. The fatty acid profile and proxi-
mate analyses of muscle meat of the test animals and published
nutrient requirements of other Haliotid species were used as a
basis for the practical diets developed. The diets were fed to aba-
lone at 2-5% body weight once daily ( 1600h) for biological evalu-
ation in terms of weight gain (WG). increase in shell length (SL),
specific growth rate (SGR). The main effects of lipid levels on
WG, SL, and SGR were significant at the 0.05 level with 5% total
lipid significantly higher than all other lipid levels tested. The fatty
acid composition of abalone juveniles reflected that of dietary
lipids. Highly unsaturated fatty acids (HUFA) were noted to be
incorporated more into the polar lipid fraction. Diet which con-
tained the least amount of lipid had an increase in 16:0 and 16:1
fatty acids. The ratio of n3/n6 fatty acids was high in abalone fed
diet containing 4.69% total lipid. Diet 2, containing 1.5% lipid
from a 1 : 1 ratio of a tuna fish oil: soybean oil as lipid sources with
a total lipid content of 4.69%, may be used as a basal diet for
abalone juveniles.

as a sole diet. In this study, enrichment of wild U. rigida increased
the algal protein content from 1 1.4 ± 2% to 32.2 ± 1.5%, perhaps
partially explaining the difference between this and other research.
A comparison of survival under salinity stress for abalone from the
different dietary treatments will also be discussed.

COMPARATIVE GROWTH PERFORMANCE OF JUVE-
NILE HALIOTIS ROE! FED ON ENRICHED ULVA RIGIDA
AND VARIOUS ARTIFICIAL DIETS. S.J. Boarder Fre

mantle Maritime Centre, 1 Fleet St.. Fremantle. Western Australia;
and M. Shpigel, Israel Oceanographic and Limnological Research,
National Center for Mariculture, P.O. Box 1212, Eilat, Israel.

The growth rates of juvenile Haliolis rod fed various artificial
diets were compared with growth achieved from the consumption
of inorganically enriched lllvci rigichi. Juvenile abalone (20— tO
mm S.L.) were collected from reef platforms off the Perth metro-
politan area and assigned to one of seven different dietary treat-
ments. All diets were fed <«/ lihitinn (3'/f b.w. day"' ) every second
day and growth rates were quantified over a three month period.
Specific growth rale (SGR). measured on a whole wet weight {/; <
0.01) and shell length (/> < 0.01 ) basis, indicates that abalone fed
enriched Ulva grew at comparable rales to growth achieved from
the best performing artificial dicls. Reduced mortality rates in both
the Ulva treatment and the best artificial diet also indicate a dietary
advantage to general health through consumption of these diets.
These results indicate that Ulva is a suitable feed for H. roei.
providing comparable growth to that achieved from several com-
mercially available diets. In contrast, other researchers have found
Ulva to be a nutritionally poor food source for abalone when fed

THE POTENTIAL RISK OF HARMFUL ALGAE TO ABA-
LONE FARMING ON THE SOUTH COAST OF SOUTH AF-
RICA. L. Botes,' ^ G. C. Pitcher and P. A. Cook," 'Marine
Biology Research Institute, Zoology Department. University of
Cape Town, Rondebosch, 7701, Cape Town. South Africa; •'Ma-
rine and Coastal Management, Private Bag X2, Rogge Bay, 8012,
Cape Town, South Africa.

Toxic algal blooms are common world wide and pose a serious
problem to the aquaculture and fishing industries. Of the di-
noflagellates, species such as Cymnodiniiim breve. Gymnodinium
mikimotoi and Gyrodinium aureolum are recognised tlsh-killers,
implicated in various faunal mortalities. Toxic blooms of G. cf.
mikimotoi were observed on the south coast of South Africa for the
first time in 1988 and have subsequently been responsible for wild
and fanned abalone (Haliotis midae) mortalities. Attempts to iso-
late an culture G. cf. mikimotoi revealed the presence of several
gymnodinioid species on the south coast, namely: G. pyrenoi-
dosiim. G. piilchelliim. G. sangiiiiieiim. Gyrodiniitm cf. cnr.ticiim.
and Lepidodiiiiiim viride. Two other fish-killing species, namely:
Hctcrosiiiimi iikasliiwo and Chatonella sp. have also been isolated,
as well as four more species that are common in this region
namely. Scripsiella trocoidea. Prorocentnim micans. Proroceii-
irum firacile. and Prorocentrum roslvatitm. The species have been
tested for toxicity by means of an ,\rtvmia bioassay (ARTOXKIT).
a routinely-used method in marine and aquatic toxicology. A simi-
lar experimental procedure was used to assess the toxicity of each
culture and its filtrate on both abalone larvae and spat (3 mm
animals). Similar experiments were conducted on Gyrodinium au-
reolum (l.solation site: Norway: Obtained from: Department of
Phycology, University of Copenhagen, Denmark) for comparative
purposes. None of the species tested was toxic to Anemia larvae,
bill G. sanf^uineum was toxic to abalone larvae and spat, G. pul-
chcllum was toxic to abalone larvae, and G. aureolum was toxic to
abalone larvae and spat.

4th Inlemational Abalone Symposium. Cape Town. South Africa

Ahsrmcts. February 2000 503

RIKETTSIALES-LIKE PROKARYOTES IN CULTURED
AND NATURAL POPULATIONS OF THE RED ABALONE
HALIOTIS RUFESCENS, BLUE ABALONE HAUOTIS
FULGENS, AND THE YELLOW ABALONE HALIOTIS
CORRUGATA FROM BAJA CALIFORNIA. MEXICO. J.
Caceres Martinez. C. Alvarez Tinajero, and Y. Guerrero Rent-
eria. Centre de Investigacion Cientifica y de Education Superior
de Ensenada. Laboratorio de Biologia y Patologia de Moluscos.
Apdo. Postal 2732. 2800 Ensenada Baja California; Mexico, and
J. G. Gonzalez Aviles, Sociedad Cooperativa de Produccion
Pesquera. Pescadores Nacionales de Abulon. S. C. de R.L. Av.
Ryerson 117. Ensenada. B.C. Mexico.

Since 1995, Rikettsiales-like prokaryotes (RLP) in the black
abalone. Haliotis carcherodii. have been considered as a presump-
tive causative agent of Withering Syndrome (WS) and they infect
the ephitelial cells of the digestive tract of the host. Posterior field
observations in California. USA. showed that other abalone spe-
cies could present the characteristic symptoms of WS: shrunken
appearance of foot muscle, retracted visceral tissues, and an in-
ability to adhere tightly to the substrate. Recently, highly signifi-
cant correlations between the presence of RLP and WS symptoms
were found in farmed red abalone. In Baja California. Mexico, no
studies on the presence of RLP and WS in cultured or wild abalone
populations have been carried out. To determine whether RLP are
present in cultured and natural abalone populations in Baja Cali-
fornia. Mexico, a survey was carried out in WS symptomatic and
non-symptomatic abalone obtained from commercial catches and
aquaculture facilities in Isla de Cedros, Islas San Benitos and
Bahi'a de Todos Santos B.C. It is important to note that this study
was carried out during the occurrence of the "El Nifio" phenom-
enon, between 1997 and 1998. when temperatures rose 2.5 °C
above the normal range. Results showed the presence of RLP in
cultured red and blue abalone. The prevalence of RLP in cultured
red abalone was around 90%. infecting both WS symptomatic and
non-symptomatic abalone. In cultured WS symptomatic blue aba-
lone. its prevalence was 37.5%. These bacteria were also present in
blue and yellow abalone from wild population, in which a preva-
lence of RLP of around 70% was recorded in both blue and yellow
WS symptomatic and non-symptomatic abalone. However, the
prevalence of RLP in blue abalone was greater (80.7%) than in
yellow abalone (68.2%).

There was an increase in the prevalence of RLP and sympto-
matic WS abalone in samples taken in the middle and end of the
"El Nifio." when compared to the start of the phenomenon. The
increase in RLP prevalence during the middle and end of "El
Nino" when the effect of high temperature was evident (death of
kelp beds, degradation in the sea floor) suggests that these condi-
tions could interact as a synergic factor for the RLP presence and
WS development. This observation supports the hypothesis that
temperature-enhanced RLP infection plays a direct role in the eti-
ology of WS. However, the presence of RLP and its association

with WS symptoms remain confused and suggest that in addition
to the role of temperature, the virulence of RLP could be affected
by different strains or species of these bacteria. It is also probable
that the susceptibility of different abalone species or individuals is
different, or that an unknown condition-pathogen is also involved
in WS development.

SYMBIONTS OF RED ABALONE HALIOTIS RUFESCENS
FROM BAJA CALIFORNIA, MEXICO. J. Caceres Martinez
and G. D. Tlnoco Orta. Centro de Investigacion Cientifica y de
Educacion Superior de Ensenada. Laboratorio de Biologia y Pato-
logia de Moluscos. Apdo. Postal 2732. 2800 Ensenada Baja Cali-
fornia. Mexico.

Although culture of commercial abalone started around 1973 in
Baja California, no studies have been carried out on the symbionts
of this species. In order to determine the symbionts of red abalone,
Haliotis nifescens. a survey of healthy and moribund abalone from
a cultured stock was carried out during "El Nifio", 1997. Healthy
abalone had a greater size (34 mm) than moribund abalone (25
mm). Macroscopical and microscopical analysis of the shell re-
vealed the presence of two polychaetes belonging to the families
Spionidae and Serpulidae. the former having a prevalence of about
10%. and the latter 100%. No serious ill-effects associated with the
relationship between abalone and these worms were however ob-
served. The histopathological evaluation showed the presence of
the renal coccidia Pseudoklossia haliotis. its prevalence was 72%
in moribund and 10% in healthy abalone. Rikettsiales-like
prokaryotes were found in epithelia of the digestive tract of both
groups with prevalence around 90%. Protozoan species were found
in the branchial cavity of healthy abalone (17%) and moribund
abalone (88%). A trend of high symbiotic prevalence and intensity
in moribund rather than healthy abalone was recorded. Differences
were not however statistically significant.

MICROHABITAT, DISTRIBUTION AND ABUNDANCE OF
JUVENILES OF HALIOTIS FULGENS AND H. CORRU-
GATA IN BAHIA TORTUGAS, MEXICO. L. Carreon Palau,
S. A. Guzman del Proo. J. Belmar P.. J. Carrillo L., R. Herrera
F.. and A. Villa B., Laboratorio de Ecologi'a. Departamento de
Zoologia. Escuela Nacional de Ciencias Biologicas, Instituto
Politecnico Nacional. Prol. Carpio y Plan de Ayala s/n, Mexico,
D.F. 11340.

The distribution and abundance of juveniles of green and pink
abalone (Haliotis fulgens and Haliotis corrugata) were studied at
Bahi'a Tortugas. B. C. S. Mexico, during the autumm and spring of

504 Abstracts. February 2000

4th International Abalone Symposium. Cape Town. South Africa

1996-98. Two sites with different depths, relief and wave expo-
sure were surveyed at three different depths between 0.5 and 12 m
deep; nine stations with 30 m" transects were sampled at each
bank.

Four different habitats were recognized in each site: 1 ) .shel-
tered, low bottom relief. 2) semiexposed. moderate bottom relief.
3) exposed, high bottom relief and 4) exposed sites, moderate
bottom relief. Juveniles were not restricted to shallow waters, but
were patchily distributed throughout the bank from 0.5 to 12 m
depth, with higher densities being found in the shallow waters of
the sheltered bank.

Emergent juveniles (3 to 13 mm length shell (l.s.)) were ob-
served on small rocks, pebbles and flat stones not longer than 15
to 20 cm. always cryptic on the crustose coraline algae Lithophyl-
hiiii imitaiis. Juveniles between 15 and 90 mm 1. s. occurred under
flat, rhombic or irregular shaped rocks, found primarily in imbri-
cated arrangements.

Both species showed an aggregated distribution pattern, al-
though this was not true of all environments. Green abalone juve-
niles were the more abundant species on both banks (0.15 ± 0.013
ab m~"); pink abalone juveniles had a lower abundance (0.06 ±
0.01 ab ni~"). Significant seasonal changes in density were ob-
served, primarily in green abalone, which declined in 1997 and
recovered during spring 1998. Juveniles (20-90 mm) and adult
forms share the habitat with briozoan, ascidians, chitons, anellids,
gasteropods and echinoderms, while juveniles less than 13 mm
share their habitat with tiny juveniles of Fissiirclla. chitons, Crepi-
patella, annelida and sea urchins. Size distributions suggest that
both species have two cohorts, one from the summer/fall spawning
season and another from the early spring season. The recruitment
patterns of the species differ, suggesting that H. fidf^ens has an
advantage under adverse climatic conditions such as during the
1997 El Niiio event.

LOCALIZATION OF THE EGG-LAYING HORMONE
(ELH) IN THE GONADS OF A TROPICAL ABALONE,
HALIOriS ASININA LINNAEUS. M. Chanpoo,' S. Apisawe-
takan,' A. Thongkiikiatkul/ C. Wanichanon,' V. Linlhong,'
M. Kruatrachuoe- S. E. Upatham,-' T. Punithong,^ P. J.
Hanna,'^ and P. Sobhon' Dcparlmcnts ol 'Anatomy and 'Biology,
Faculty of Science, Mahidol L'niversity. Rama VI Rd, Bangkok.
Thailand 10400. 'Dcparlnienl of Biology. Faculty ol Science.
Burapha University, Chonburi. Thailand. ^Coastal Aquaculture
Development Center. Department of Fishery, Ministry of Agricul-
ture and Cooperatives, Prachuapkhirikhun, Thailand 77000,
■''School of Biological & Chemical Sciences, Deakin Universit\.
Geelong. VIC 3217. Australia.

The connective tissue scaffold of the gonads of Halioiis iisinina
consists of the outer gonadal wall of fibro-muscular tissue, which
forms a capsule-like sirucliuc. This capsule forms connective tis-

sue trabeculae that partition the gonad into compartments. Each
sheet of trabeculae contains a small capillary in the center, sur-
rounded by a pack of smooth muscle cells and collagen fibers
intermingled with small cells exhibiting dense ellipsoid nuclei.
Some of these cells are fibroblasts, follicular cells and granulated
cells that may synthesize hormones that induce spawning. Local-
ization of the egg-laying hormone (ELH). which can induce the
ovulation of the oocytes, was performed by immunofluorescence,
immunoperoxidase and immunogold with silver enhancement
techniques. Anti-ELH of Haliotis rubra exhibited strong staining
in the trabeculae and the capsules, especially in the granulated cells
within the trabeculae and the innercapsule. and the cytoplasm of
oocytes stages 1 to 3. while the cytoplasm of oocytes stages 4 to
5 were only weakly stained.

THE PALLIAL ORGANS OF HALIOTIS ASININA LIN-
NAEUS. 1758 (GASTROPODA : HALIOTIDAE) IN THAI-
LAND. Y. P. Chitramvong, M. Kruatrachue, E. S. Upatham,
S. Singhakaew, and K. Parkpoomkamol, Department of Biol-
ogy. Faculty of Science. Mahidol University. Bangkok 10400.
Thailand.

The pallial organs are compo.sed of the gills, osphradiuni. hy-
pobranchial gland, kidney, heart and rectum. The bipectinate
paired gills are on the left and right sides of the mantle cavity. The
left gill is smaller than the right one. They are light yellow in color.
The paired osphradia are long, slender and lie anteriorly on each
side of the mantle near the gills. Each has a long ridge with a
central pleated groove. The cells are very tall and closely packed.
The epithelial cells are ciliated cells. The paired hypobranchial
glands arc in the postero-dorsal view of the mantle near the rectum
and tlic heart. They arc usually composed of tall mucous cells and
triangular ciliated cells. The right hypobranchial gland is belter
developed than the left one. The paired kidneys are glandular in
structure. The left kidney is very well developed. Il is bright or-
ange-yellow in color and lies almost along the entire length of the
mantle. Il has a cuboidal epitlieliiun and ihc secretory epithelium
oi the lumen ol the kidney is folded inlo hraiiched luhules. The
heart is at the posterior end o\ the mantle connecting to the gills.
Il is composed of one ventricle and two auricles. The wall of the
auricles contains vcrv delicate lace-like strands of muscle and

4th International Abalone Svniposium. Cape Town. South Africa

Abstracts. Februarv 2000 505

fibrous tissue. The ventricle has thick opaque muscular walls. The
rectum is attached to the left kidney. It is light yellow in color. Its
length is about 1/3 of the length of the kidney and it has a ciliated
columnar epithelium.

ISOLATION AND GROWTH OF SEVEN STRAINS OF
BENTHIC DIATOMS. CULTURED UNDER TWO DIFFER-
ENT LIGHT CONDITIONS. J. G. Correa Reyes. M. del Pilar
Sanchez Saavedra. and N. Flores Acevedo. Aquaculture Depart-
ment: Centre de Investigacion Cientifica y de Educacion Superior
de En.senada (C.I.C.E.S.E). Apartado Postal 2732. Ensenada. Baja
California. Mexico. C.P. 22800.

We have isolated seven strains of benthic diatoms from three
different zones close to abalone seed farms in Baja California.
Mexico. We experimented using 10 replicates of non-axenic batch
cultures of each strain in 250 ml Erlenmeyer flasks with 150 ml of
Guillard & Rhyther's "f medium. The cultured conditions had a
salinity of 34 ± I7cc and temperature of 22 ± 1 °C. Each strain was
subjected to two different conditions of light (blue and white light)
at the same irradiance (150 (jlE m"" s~'). Every second day for ten
days we ultrasonicated the cultures and checked the cell concen-
trations, taking two flasks from each condition and strain (without
replacement). The evaluation of cell concentration was measured
by direct counts with a haemacytometer. Under these experimental
conditions, we observed in all strains that the growth rate on the
second or third day of culture had high values of duplication per
day. We compared the growth rates of these strains with those of
Navicula incerta (a strain used in some abalone farms in Mexico),
and found similar growth rates in some cases. For each strain, we
used an analysis of covariance to compare whether there was any
difference between strains grown under white and blue light and
surprisingly found no significant differences. These results showed
that some strains of benthic diatoms can be cultured under high
light irradiances without any photoinhibition and they may have
high growth rates and high cell concentrations (=5.0 x 10'' ceP
ml"').

Erlenmeyer flasks with 150 ml of Guillard & Rhyther's "f me-
dium. The cultured conditions had salinities of 34 ± l%r and tem-
peratures of 22 ± 1 °C. Each strain was cultured under two dif-
ferent continuous light conditions (blue and white light) at the
same irradiance ( 150 |j.E m"" s"')- Biochemical cell compositions
were determined for triplicate sets for each type of analysis, ex-
perimental condition and samples of each strain, on the sixth and
tenth day of the culture. Biochemical composition (protein, lipids
and carbohydrates) were determined using classic spectrophoto-
metric methods; as expected, there were significant differences in
biochemical composition between the eight diatoms strains. When
the effect of light quality and diatom species was analysed using a
two way ANOVA, there were no significant differences in biomass
production. However, important differences in the biochemical
composition of protein and lipids were found. The highest value of
protein concentration was produced by Nitzcliia sp. and the lowest
value was for Navicula sp.

LOCATION OF EGG-LAYING HORMONE IN REPRO-
DUCTIVE STRUCTURES AND NEURONS OF HALIOTIS
USING ANTIBODIES RAISED AGAINST RECOMBINANT
FUSION PROTEINS. S. Cummins and P. J. Hanna, School of
Biological & Chemical Sciences, Deakin University, Geelong,
VIC 3217. Australia: and A. Thongkukiatkul, Department of Bi-
ology, Burapha University, Bangsaen, Chonburi, Thailand.

Recombinant abalone egg-laying hormone was produced using
a bacterial expression vector. This required TA cloning of a 108 bp
abalone egg-laying hormone (aELH) gene sequence using, PCR of
genomic DNA with primers incorporating restriction enzymes
sites, into a pGEX-2T vector. Following transformation into Es-
cherichia coli, a GST:aELH fusion peptide was produced and
subsequently purified. This was used to immunise mice for pro-
duction of polyclonal antibodies, and monoclonal antibodies,
which were tested for specificity and reactivity using ELISA's,
western blots and FITC assays. These antibodies are now being
used in immunocytochemistry to determine expression of aELH
during reproductive cycles and in which tissues.

CHEMICAL COMPOSITION OF EIGHT STRAINS OF
BENTHIC DIATOMS, CULTURED UNDER TWO DIFFER-
ENT LIGHT CONDITIONS. J. G. Correa Reyes, M. del Pilar
Sanchez Saavedra. J. Arturo Siniental Trinidad, and N. Flores
Acevedo. Aquaculture Department. Centre de Investigacion
CientiTica y de Education. Superior de Ensenada (C.I.C.E.S.E.).
Apartado Postal 2732, Ensenada, Baja California, Mexico. C.P.
22800.

We carried out 10 replicates of non-axenic batch cultures of
eight benthic diatom strains (two Nitzchia species, three Amphora
species, two Navicula species and Navicula incerta) in 250 ml

PRELIMINARY TRIALS WITH "GREEN" ARTIFICIAL
DIETS FOR JUVENILE ABALONE AND SEA URCHINS. C.
Cuthbert and G. Burnell. Aquaculture Development Centre,
Dept. of Zoology and Animal Ecology. UCC, Lee Mailings. Pros-
pects Row, Cork, Ireland: and J. Connolly, Wm. Connolly & Sons
Ltd., Red Mills, Goresbridge, Co. Kilkenny, Ireland.

This study was undertaken to evaluate and compare the effect
of five artificial (formulated) diets (four Irish and one Australian
diet) and a natural diet on the growth rates of juvenile abalone.

506 Abstracts. February 2000

4th International Abalone Symposium, Cape Town. Soutfi Africa

Haliotis discus hannai. and sea urchins, Paracentrotiis lividus.
Juvenile abalone with mean weight 0.21 (±0.07) g and sea urchins
with mean weight of 1.22 (±0.03) g were fed artificial macroalgi-
vore diets over approximately 150 days. The four Irish diets used
contained a vegetable protein, instead of fishmeal protein, because
the philosophy of the company was to develop an environmentally
friendly diet. A seaweed (Laminaria spp) was included as a ref-
erence diet and a starved group served as a control. The diets were
fed in excess every second day and all uneaten feed was removed
prior to the next feed. Animals were maintained in 201 tanks (30-
50 animals per tank) in a recirculation system. Temperature and
salinity levels were monitored daily: mean values of 19 (±0.1 ) °C
and 34.8 (±0.5) mg/1 were recorded over summer months for these
parameters. Aminonia, nitrate and nitrite levels in the systems
were monitored weekly and more detailed chemical analysis was
carried out monthly. No adverse build-up of organic wastes was
noted. Biological evaluation was in terms of absolute weight gain
(WG). increase in shell length (SL) or test diameter (TD) and
specific growth rate (SGR). Physical evaluation was in terms of
shell coloration (abalone only) and shell, test and spine (urchin)
quality. Measurement of the above parameters took place during
the first two weeks of each month, over the course of the study.
Initial results show better growth performance in terms of WG, SL.
TD and SGR for both species fed the five formulated diets, com-
pared with those fed the natural diet. Initial poor shell margin
quality of abalone on all formulated diets decreased over time as
percentage growth between measurements decreased.

GROWTH AND SURVIVAL OF HALIOTIS RUBRA POST-
LARVAE FEEDING ON DIFFERENT ALGAL SPECIES. S.
Daunie and A. Krsinich, Deakin University. School of Ecology
and Environment, PO Box 423 Warrnambool. Victoria 3280 Aus-
tralia; S. Farrell and M. Gervis, Southern Ocean Mariculture,
RMB 2068. Port Fairy. Victoria 3284 Australia.

In previous experiments we showed that larvae of the abalone
Haliotis rubra settle well on the encrusting green alga Ulvelia lens.
However, early growth on this alga is poor and settlement plates
have to be inoculated with appropriate diatom species to comple-
ment feeding.

In this study, a flow-through system with large petri dishes was
used to compare growth and survival of posi-larvae feeding on
different algal species. After setilemeni. small plastic sheets with
Ulvelia lens and recently settled post-larvae were transferred into
replicated dishes and fed with different diatom species. The shell
length of all post-larvae was measured before the trial and every

week up to 2 months after settlement. Growth and survival rates
were calculated to evaluate the success of the different diets.
Faeces samples as well as grazed dishes were examined for broken
diatom valves, to establish whether intracellular nutrients are avail-
able to post-larvae as a food source. The diatom species were
chosen by their ability to attach strongly to the substrate and can
therefore be used on vertical settlement plates in a nursery situa-
tion. Growth-rates were significantly higher on all treatments with
additional diatom feed compared to the unfed control of the alga
Ulvelia lens. The best growth-rate was obtained with Navicula sp.
During the first weeks after settlement, the majority of the diatom
valves were not broken and were still alive in faeces samples of the
post-larvae. After 3 weeks, most of the diatom valves in the
samples were broken and intracellular nutrients became available
to the post-larvae at this stage. This indicates that most of the
nutrition for post-larvae during the first weeks after settlement
must derive from extracellular material of the diatoms, which con-
sists mainly of polysaccharides.

SETTLEMENT AND EARLY GROWTH OF THE ABA-
LONE HALIOTIS RUBRA IN RESPONSE TO DIFFERENT
ALGAL SPECIES. S. Daume and A. Krisinich, Deakin Univer-
sity, School of Ecology and Environment. PO Box 423 Warrnam-
bool. Victoria 3280 Australia. S. Farrell and M. Gervis, Southern
Ocean Mariculture (SOM). RMB 2068. Port Fairy. Victoria 3284
Australia.

Five benthic diatom species were isolated from settlement
plates at SOM. Victoria, Australia and maintained in culture (Nav-
icula sp. (small), Navicula sp. (large), Nitzschia sp., Cocconeis sp..
Amphora sp.). The species were grown on plastic sheets and tested
in settlement experiments with black-lip abalone (Haliotis rubra)
larvae. Settlement was very low and varied between \9r-6Vc.
When given a choice between the natural settlement substratum,
the non-geniculate coralline red algae Sporolithon durum and
single species diatom films, settlement was higher on S. durum
than on any of the diatom films tested. High settlement o'i up to
36'/f was also achieved with germlings of the green encrusting alga
Ulvelia lens.

A flow-through system with large petri dishes was developed to
compare growth and survival of post-larvae feeding on different
algal species. One week after settlement, six post-larvae were
transferred to each of four replicate dishes of each algal species.
Four diatom and two macroalgal species were tested. The shell
length of all post-larvae was measured before being transferred,
and everv week up to I I weeks alter settlement. Post-larvae arow

4th Intemational Abalone Symposium. Cape Town, South Africa

Ahstmcls. February 2000 507

better on diatom films than on sheets with Ulvelki lens or on pieces
of Sporolillum durum. The best growth-rate was obtained with the
large size Navicula sp. (39 ± 4 ijim/d) and the lowest with the
macroalga Ulvella lens (13 ± 3 |j.m/d). Overall the larger size
Navicula sp. produced the largest juveniles (>3 mm shell length at
the end of the 1 1 week trial) with the highest survival rate.

Ulvella lens and Sporolithon durum are both good settlement
inducers but are not sufficient to support the rapid growth of young
Haliotis rubra post-larvae. We suggest that plates with U. lens
could be inoculated with diatom strains such as the large size
Navicula sp. to ensure sufficient food for the growing post-larvae.

one animal may be remarkably consistent over longer periods. We
found tentative direct evidence for this at two sites, Gabo Island
and Port Phillip Bay, Victoria, where abalone were recaptured
more than three years after release. However, the fact that only 5
of over 450 released abalone were recaptured at each of these sites
after 3 years, shows that obtaining hard data for such long term age
validation is limited by tag-and-release recapture.

ARE ABALONE SHELL LAYERS DEPOSITED ANNU-
ALLY? VALIDATION USING MANGANESE VITAL
STAINING. R. W. Day. G. P. Hawkes, and V. Gomelyuck Zo

ology Department. The University of Melbourne, Parkville, 3052,
Australia.

Management models of abalone fisheries would be greatly en-
hanced if we could age abalone with known confidence limits.
Ageing of abalone using shell layers under the spire has received
considerable attention since it was first proposed in Mexican aba-
lone, but there has not been any rigorous validation of the method,
or good estimate of ageing error. The manganese staining method
we have developed here provides the first opportunity to validate
properly the timing and frequency of these layers. Cathodolumi-
nescent marks in recaptured tagged abalone identify subsequent
shell growth over the release period. Validation trials were con-
ducted at 3 Victorian and 3 Tasmanian sites for Haliotis rubra, and
at 2 sites within South Australia for H. laevigata, by releasing
400-600 marked abalone at each site. Low recapture rates from
Tasmanian sites and high mortality of H. laevigata from South
Australia reduced the data available, but recaptures at periods up to
one year showed great variability in the number of layers deposited
subsequent to the mark. These results indicate that growth layers
cannot be used to estimate individual abalone ages to within 1-2
years. However, monthly collections of Haliotis rubra from Port
Philip Bay, Victoria, indicated that the timing of layer deposition
within each year is very variable, and this may explain the varia-
tion seen. The monthly sampling also shows spire layers were most
often deposited in March, after peak summer temperatures and
before winter minima. This appears to contradict theories that rings
are formed during non-growth periods in winter or during spawn-
ing events with increasing water temperature. Various sources of
evidence suggest that the number of layers deposited per year in

THE PARASITE TEREBRASABELLA HETEROUNCINATA
(POLYCHAETA) MANIPULATES SHELL SYNTHESIS IN
HALIOTIS RUFESCENS. R. Day, Zoology Department. Univer-
sity of Melbourne, Parkville, Vic 3052, C. Culver, A. Kuris, A.
Belcher, and D. Morse, Marine Science Institute, University of
California at Santa Barbara, CA 93106, USA.

Terebrasabella heterouncinata. the sabellid polychaete that in-
fests gastropod shells, has been introduced into California by aba-
lone from South Africa. Dense infestations in Haliotis rufescens
result in very distorted shells and reduced growth. The polychaete
broods its young, and the juveniles crawl out of the burrow and
into the space between the abalone mantle and the shell margin,
where they secrete a tube. The effect of infection by juvenile
sabellids on shell synthesis by the host was investigated using a
manganese vital stain that allows identification of calcitic and
aragonitic shell layers. Aragonitic nacre is deposited over the sa-
bellid tubes by the host within 12 hours, about 4 to 8 times faster
than normal aragonite synthesis. Simultaneously, extension of the
shell through deposition of prismatic calcite along the margin,
appears to be almost completely suppressed. The extent of this
suppression may depend on the number of juveniles that have
become established. After 2-3 days, a thick aragonite layer over
the tubes extends to the shell edge. Small cuboid blocks of calcite
are then deposited along the inside of the shell between the tubes,
and the spaces between these blocks are later filled by further
calcite deposition, forming a new broad growing edge oriented
downwards, below the previous margin of the shell. These results
explain the distorted shape and reduced growth of infected aba-
lone. Control of shell synthesis is presumably mediated at least in
part by the tube of the juvenile polychaete. Juveniles isolated in
drops of seawater eventually produced tubes, which we stained
with reagents. They apparently consist of muco-polysaccharides
and proteins with both positive and negative groups on the surface.
This differs markedly from the surface groups on the 'greensheet'
protein synthesized by the abalone as a basement that guides shell
formation.

508 Ahsiracts. February 2000

4th International Abalone Symposium. Cape Town. South Africa

QUANTIFYING THE PHYSICAL AND BIOLOGICAL AT-
TRIBUTES OF SUCCESSFUL OCEAN SEEDING SITES
FOR FARM REARED JUVENILE ABALONE {HALIOTIS
MIDAE). S. De Waal and P. Cook, Department of Zoology,
University of Cape Town. Private Bag Rondebosch, 7701 Cape
Town, South Africa.

In short term experiments carried out in Mac Dougalls Bay, on
the northwest coast of South Africa, survival of seeded juvenile
abalone (Haliatis midae) has been shown to be directly linked to
both the size of the abalone at seeding and to the physical com-
position of the seeding site. The presence of the sea urchin
(Parenchinus angulosus.) has been shown to play an insignificant
role in terms of short term, up to two months, survival of juvenile
H. niidae. Within the context of selecting the right seeding site
attributes, short term survival was shown to be up to a minimum
average of 59% for animals of between 24 and 28 mm, compared
to a minimum average of 24% for animals ranging between 1 2 and
16 mm. The fact that the presence of urchins played no significant
role in the survival of juvenile abalone, ranging in size from 12 to
27 mm, changes the idea of what the characteristics of successful
seeding sites might be. A positive correlation has been established
between habitat consisting of stacked boulders of diameter less
than 50 cm and abalone survival, with a negative correlation ex-
isting between the extent to which the area is exposed, when it
offers no protection to juvenile abalone, and their survival.

yellow abalone in these three zones. Allozyme electrophoresis was
carried out with six samples from two years in the three localities.
The average number of allele per locus was 2.3 with a 67% poly-
morphism. Overall mean unbaised heterozygosity was 0.192
(range 0.151-0.251). which is similar to that of other abalone
species, but a little higher to that of the blue abalone, Haliotis
fiilgens, from the same localities. Only one case out of 34 did not
agree with the Hardy-Weinberg model and there was a tendency
towards heterozygote excess, although this was not significant. A
dendrogram with Nei's genetic distance was constructed using
UPGMA analysis. The number of migrants per generation was less
than unity. Fst statistics showed differentiation between localities.
Therefore, these populations should be considered as independent
populations for fishery management.

POPULATION GENETICS OF THE YELLOW ABALONE,
HALIUTIS CORRUGATA, IN CEDROS AND SAN BENITO
ISLANDS. M. A. del Rio Portllla, Centro de Investigacion
Cicnli'fica y de Educacion Superior de Ensenada Km 107 Carr.
Tijuana-Ensenada, Ensenada, B. C. Mexico A. P. 2732. Ensenada.
Mexico. 22800.

The yellow abalone. Hulinlis cornigala. is the second species
in production in Central Baja California. The Cooperative "Pesca-
dores Nacionales de Abulon" has concessions for abalone exploi-
tation off the Cedros and San Benito Islands. Abalone are niainlv
distributed in three large /ones amuiul ihcse islands: a) ihe norlli
(Punia Norte) and b) south (San Aiigustm) of Cedros Island and c)
around ihe small islands of San Benito. The main goal of the
present work was lo characleri/e gcnclically the populations of

USE OF A SPREADSHEET MODEL TO INVESTIGATE
THE DYNAMICS AND ECONOMICS OF A SEEDED ABA-
LONE POPULATION. S. De Waal and P. Cook, Department of

Zoology, University of Cape Town. Private Bag Rondebosch.
7701 Cape Town, South Africa.

Using a simple spreadsheet model, it is possible to investigate
the dynamics of a discrete abalone population. The two variables
used lo dri\e the population are the percentage survival after the
initial trauma of seeding, termed initial survival, and age differ-
ential survival, termed the survival regime. Of the two, initial
survival is the variable that in practice plays the dominant role in
determining the potential yield of any seeded population. There is
experimental evidence that percentage survival in seeded abalone
populations increases per age class; however, very little data exists
for the species Hiiliotis iiiiihic. Seeding size plays a significant role
in initial survival. The larger the animals, the higher the initial
survival rate. Within the same experimental context, 13-mm ani-
mals showed a minimum siuv ival rate ol 24' i while 26-mm ani-
mals showed a mmimuni rale of 56'/(, a non-linear increase in
polenlial yield. The economic implications of both seeding sce-
narios arc modeled. Hovvever. in order to be able lo address the
issue of potential yield on a commercial scale accurately, differ-
ential mortality amongst seeded populaliiins ol abalone needs to be
researched.

4tli International Abalone Symposium. Cape Town. South Africa

Ahslracly February 2000 509

PREFERRED TEMPERATURE AND CRITICAL THER-
MAL MAXIMA OF RED ABALONE HALIOTIS RUFE-
SCENS. F. Diaz, M. A. del Rio Portilla. M. Aguilar, E. Sierra,
and A. D. Re Araujo, Centre de Investigacion Cienti't'ica y de
Educacion Superior de Ensenada Km 107 Carr. Tijuana-Ensenada.
Ensenada, B.C. Mexico, A.P. 2732. Ensenada, Mexico, 22800.

Cultured red abalone Haliotis rufescens from Isla de Todos
Santos Baja California, Mexico, were used in evaluations of pre-
ferred temperature and critical thermal maxima (CTM). Organism
weights ranged from 12.7 to 26.0 g and from 4.7 to 6.0 cm in
length. The red abalone locates its preferred temperature by the
orthothermokinesis mechanism and the preferred temperature was
18.8 °C higher than optima previously reported for this species,
higher than that of the South African abalones, but similar to the
Australian abalones. The CTM of H. rufescens at 50% was 27.5 °C
which is similar to other abalone species (H. cracherodii. H. mi-
dae. H. rubra, and H. laevigata).

whether the abalone enteric bacterial isolates C4 and U5 influence
the growth rate of the host animal. If abalone enteric bacteria are
to be used as probiotics for farmed abalone, it is important to
ensure that the bacteria colonise and persist in the host animal for
a significant period of time. Thus, colonisation experiments are
being conducted where C4 and U5, tagged with the luciferase
enzyme, are tested for their ability to remain in the digestive tract
of H. inidae. Similarly, it is important to investigate whether the
type of seaweed ingested by the host animal influences the com-
position of the microbiota colonising the gut of H. Midae. Thus,
we have employed ribotyping as a tool for characterising the bac-
terial population colonising the abalone digestive tract. This data
will allow monitoring of population changes in future experiments.

CHARACTERISATION OF THE ENTERIC BACTERIA OF
THE ABALONE HALIOTIS MIDAE. AND THEIR ROLE IN
THE DIGESTION OF INGESTED SEAWEED. K. Doeschate,
B. M. Macey, and V. E. Coyne, Department of Microbiology,
University of Cape Town. Private Bag. Rondebosch. 7701, South
Africa.

One of the challenges in farming abalone is to improve the
growth rate of the animal. In many cases, the presence of bacteria
within the digestive system has led to an improvement in the
growth rate of the host animal. We have identified two bacterial
isolates from the abalone digestive tract that exhibit increased
levels of alginase (strain C4) and agarase activity (strain U5).
Since maricultured abalone in South Africa are fed Ecklonia
maxima and Cracilaria gracilis, which possess cell walls com-
posed primarily of alginate and agar respectively, we are investi-
gating whether these bacteria affect the nutrition of H. midae. The
polysaccharolytic activity of H. midae was compared to that of the
bacterial isolates in order to determine the extent to which each is
able to hydrolyse alginate in E. maxima and agar in C. gracilis.
The possibility that abalone use bacteria as a carbon and/or nitro-
gen source has been investigated by radiolabelling isolate C4 with
[U-''*C] L-amino acids. Radiolabelled bacteria were orally intro-
duced into abalone. Subsequently, various organs were dissected
and '■'C-incorporation determined using a scintillation counter.
Semi-artificial food, based on E. maxima and G. gracilis extracts,
has been devised for use in srowth rate studies in order to ascertain

IDENTIFICATION OF EXPRESSED HSPs IN BLACKLIP
ABALONE (HALIOTIS RUBRA) DURING HEAT AND SA-
LINITY STRESSES. B. Drew. Dean Miller. T. Toop, and P.
Hanna, School of Biological & Chemical Sciences. Deakin Uni-
versity, Geelong, VIC 3217, Australia.

Both prokaryotes and eukaryotes express a set of highly con-
served proteins in response to external and internal stress. The
stressors include tissue trauma, anoxia, heavy metal toxicity, in-
fection, changed salinity, and the most characterised, heat shock.
The result is an expression of stress proteins or heat shock proteins
(HSP's) which lead to protection of protein integrity, and also to
tolerance under continued heat stress conditions. The Australian
blacklip abalone [Haliotis rubra) is found principally in southern
coastal waters and also in estuarine/bay environments. Estuarine/
bay environments have greater fluctuations in environmental con-
ditions, especially those of salinity and water temperature, than are
found along oceanic coasts. Abalone from estuarineAaay and oce-
anic coastal environments were subjected to either increased tem-
peratures (2 °C/d, total of 10 °C) and lower salinity (75% seawa-
ter), in aerated tanks containing seawater and a local food source.
Estuarine/bay abalone were less affected than the oceanic animals
by temperature increase and also demonstrated the ability to regu-
late volumes 3h after the initial salinity shock. SDS-PAGE and
Western blotting techniques, together with dot blots of total pro-
tein, using HSP70 specific antibodies, were used to visualize
HSP70s in the foot muscle of the animals. Analysis of HSP70
mRNA expression, and cDNA library construction, are currently
in progress to study the molecular basis of HSP response in aba-
lone.

510 Abstracts. February 2000

4th International Abalone Symposium. Cape Town. South Africa

ONTOGENETIC CHANGES IN THE ACTIVITY OF MAIN
DIGESTIVE ENZYMES DURING THE LARVAL AND JU-
VENILE STAGES OF ABALONE, HALIOTIS DISCUS
HANNl INO. S. Du and K. Mai, Aquaculture Research Labora-
tory, Ocean University of Qingdao. Qingdao 266003. China.

This study was conducted to determine the ontogenetic changes
in digestive enzyme activities during the larval and juvenile stages
of abalone. Haliotis discus hcmiuil Ino. Activities of protease. 11-
pa,se, amylase, cellulase, laminarinase, carrageenase and alginase
were analyzed in seven stages (before fertilization and 133 days
after hatching). The results showed that activities of protease, li-
pase and caiTageenase increased from fertilization to day 10 after
hatching, then decreased; while activities of amylase, cellulase,
laminarinase and alginase increased substantially and were fully
developed at the 133 day. The changes in all these enzyme activi-
ties coincided with the development of the digestive system and
the changes in natural diets of abalone.

recover from a suppression of oxygen uptake. Benzocaine and KCl
treated animals recovered most rapidly. There was no apparent
recovery from clove oil in the time period studied. Growth trials
showed healthy growth rates for control H. laevigata ( 1 1 6 ± 3 iJim,
78 ± 4 mg per day) and all treatments indicated a suppression of
growth rate as a result of removal from the tanks (48-83 |j.m,
19-70 mg per day).

For H. rubra, control growth rates were much lower (24 ± 1
(i,m/day) and weight gain was erratic (34 ± 10 mg/day). Nonethe-
less, lower growth rates (length 1.4-12.1 n-m/day) were obtained
for all treatments, while all but one treatment also had lower
weight gain than control animals.

Animals subjected to clove oil had the lowest weight gain and
this was the only treatment that resulted in significant mortalities.

RECOVERY AND GROWTH EFFECTS OF ANAES-
THETIC AND MECHANICAL REMOVAL ON GREENLIP
(HALIOTIS LAEVIGATA) AND BLACKLIP (HALIOTIS RU-
BRA) ABALONE. S. Edwards, C. Burke, S. Hindrum, and D.
Johns, School of Applied Science. University of Tasmania. PC
Box 1214, Launceston 7250 Australia.

Haliotis laevif-ata (39.7 ± 0.2 mm, 8.2 ± 0.1 g) and Halitos
rubra (41.9 + 0.1 mm, 1 1.3 ± 0.1 g) were acclimatised to condi-
tions over 3-5 weeks (80 animals per 80 cm diameter fibreglass
tank, flow-through sand-filtered seawater 17 °C, artificial diet ad-
lib). Animals were then removed from the tanks using elhanol
(3%), 2-phenoxyethanol (I mL/L), benzocaine ( 100 ppm), clove oil
(0.5-1.5 mL/L) and mechanical removal (metal spatula), mea-
sured, and returned to clean water for a further six weeks. At
treatment, a sample of the animals was transfened to u multi-
channel flow-through respiromeler for analysis of oxygen uptake,
which lasted at least 3 days. All treatments were duplicated for
both species. One set (control) remained undisturbed from the
beginning of the acclimation period to the end of the trial. Addi-
tional respirometry trials were conducted on the same cohorts for
KCl (10 g/L), Aqui-S (50 ppm) and Tabasco ( 10 ml/L) thai didnl
fit in the growth trial.

Apart from first hour suppression (elhanol) or slimulalion
(clove oil & Aqui-S) of oxygen uptake, most agents showed
shifted normal patterns of oxygen uptake settling to a normal value
(-55 mg O^.kg'' h~') over 3-5 days. Increases in oxygen uptake
were seen in first day averages for clove oil (156%), Aqui-S
(154%) and KCl (127%). Mechanical removal gave first day sup-
pression (50'/f) of oxygen usage, returning lo normal with the
evening activity cycle. Tabasco treated animals look longest lo

APPLICATION OF MOLECULAR GENETICS TO THE
UNDERSTANDING OF ABALONE POPULATION STRUC-
TURE—AUSTRALIAN AND SOUTH AFRICAN CASE
STUDIES. N. G. Elliott,' B. Evans.' - N. Conod,- J. Bartlett,'

R. Officer.' and N. Sweijd.'' 'CRC for Aquaculture. CSIRO Ma-
rine Research, GPO Box 1538, Hobart, Tasmania 7001, Australia;
"School of Zoology, University of Tasmania, Australia; 'Tasma-
nian Aquaculture and Fisheries Institute, Hobart, Australia; ''De-
partment of Zoology, University of Cape Town. South Africa.

The structure of abalone populations is still poorly understood
worldwide, and yet it is a major input to many management issues:
wild fishery management, control of illegal fishing, selection of
aquaculture broodstock and assessment of translocation or en-
hancement programs. Molecular genetic techniques have been ad-
vancing rapidly and there are now a number of techniques suitable
for use in examination of population structure, each with relevant
advantages and disadvantages. We report here on the application
of two different types of molecular markers — mitochondrial
RFLPs and nuclear microsatellites. The discriminatory power of
the mitochondrial genome will be reported from studies conducted
on both Australian {Haliotis rubra) and South African (Haliotis
initlac) abalone species. In addition, preliminary results will be
presented from an on-going industry-funded microsatellite study
on the major Australian hlacklip abalone (H. rubra) population
aroinul the island ol Tasmania. One aim of this two-year study is
to apply a suite oi polymorphic microsatellite loci to samples
collected from 19 selected sites within the fishery, plus several
sites along the southern Australian coastline. In addition, micro-
satellites developed from the blacklip abalone library have been
tested for amplification and variation with other key Australian
and overseas species to provide an initial suite of markers for use
on populations of other species. The sampling plan and early re-
sults will be presented and discussed in context with other studies
and our niiloclionilrlal sliidv.

4th Iiilematioiial Abalone SymposiLiin. Cape Town. South Africa

Ahsimcts. February 2000 511

IN VITRO DIGESTION OF CELLULOSE WITH STOMACH
EXTRACTS FROM ABALONE (HALIOTIS FULGENS). A.
Enn'quez,' A. Shimada,' C. Vasquez,"' and M. T. Viana/ 'Pac-
ullad de Ciencias Marinas. Universidad Autonoma de Baja Cali-
fornia. Ensenada, B. C "Facultad de Estudios Superiores-
Cuautitlan, Universidad Nacional Autonoma de Mexico. Ajuchit-
lan. Qro. 'Direccion Tecnico Administrativa. Unidad de
Zoologicos de la Ciudad de Mexico. Chapultepec, D.F., ""instituto
de Investigaciones Oceanologicas, Universidad Autonoma de Baja
California. Ensenada. B.C. PO Box 450. Ensenada. B.C. 22860.
Mexico.

It had been reported that abalone are capable of digesting cel-
lulose efficiently and that both bacterial and endogenous enzymes
take part in the process. Stomach extracts from adult abalone col-
lected in the vicinity of the "Emancipacion" Fishery Cooperative
were used to assess their capacity to degrade pure cellulose (al-
phacel) in the presence of a phosphate buffer (pH 5.5). The dif-
ference in glucose concentration before and after incubation was
considered as a measure of cellulolytic activity (I Unit mg'' =
relative increment of glucose after 2 h incubation). It appears that
bacteria play an important role, as stomach extracts showed sig-
nificantly decreased cellulolytic activity in the presence of antibi-
otics. A mixture of three antibiotics (chloranifenicol. cephalospo-
rine and ampiciline) appeared to be the most effective growth
inhibitor of stomach bacteria. Throughout a series in time, cellu-
lolytic activity was detected in stomach extracts with antibiotics,
even after 72 h incubation; the possible bacterial origin of the latter
enzymes, is discussed.

Relatively new DNA markers, microsatellites and AFLPs ap-
pear to offer the best potential for parentage analysis due to the
level of variation available. Microsatellites are likely to be more
useful as they are a dominant marker, whereas AFLPs show dom-
inant/recessive inheritance, which would preclude identification of
heterozygotes.

In order to establish a selective breeding program, known pedi-
greed family lines must be established and their progress com-
pared. The infrastructure required to produce and maintain a large
number of lines in isolation is high. The ability to identify the
parents of all progeny from a mixed spawning event would alle-
viate the need for single pair crossing to produce the pedigree
population.

Microsatellite DNA markers have been developed from a par-
tial genomic library of the Australian Blacklip abalone, Haliotis
rubra, and have been used to identify contributing broodstock in a
Tasmanian abalone farm. The utility of the markers is demon-
strated on known family lines and then tested on a batch of spawn-
ing animals. This technology is an essential part of the continued
expansion of abalone culture worldwide, and the transfer of this
technology for use on other species will akso be discussed.

THE USE OF MICROSATELLITE MARKERS FOR PAR-
ENTAGE ANALYSIS IN AUSTRALIAN BLACKLIP AND
HYBRID ABALONE. B. Evans.' " R. W. G. White,' and N. G.
Elliott.' 'CRC for Aquaculture, CSIRO Marine Research, GPO
Box 1538, Hobart. Tasmania 7001, Australia. "School of Zoology,
University of Tasmania. Hobart. Australia.

The understanding of mating processes in natural and cultured
populations of abalone is required for a number of ecological and
aquacultural questions. Mating and reproductive success are af-
fected by behavioral, ecological and genetic aspects, all of which
ultimately determine the transfer of genotypes from generation to
generation. Parentage analysis can be used to either estimate the
likely pair of parents for each progeny or to determine patterns of
inheritance at the population level.

COMPENSATORY GROWTH AFTER INTERMITTENT
FOOD DEPRIVATION AND REFEEDING IN THE DON-
KEYS EAR ABALONE, HALIOTIS ASININA (LINNAEUS
1758). A. C. Fermin and S. Mae Buen, Southeast Asian Fisheries
Development Center Aquaculture Department (SEAFDEC/AQD),
Tigbauan 5021, Iloilo, Philippines.

Compensatory growth was determined after intermittent food
deprivation and refeeding at different times during grow-out cul-
ture of abalone. Haliotis asinina in suspended mesh cages in flow-
through tanks. In experiment 1, abalone were intermittently
starved for 5 or 10 days and refed for the same duration during a
140-day period. Control groups were fed continuously on seaweed
(Gracilariopsis bailinae). Fasted groups showed significantly
lower feeding rates (DFR, 15-16% day"') than the control (22%
day"') resulting in lower growth rates (DOR = 73-82 jjim and
63-70 mg day"'). Control groups had a DOR of 1 15 |jim and 142
mg day"'. When fed continuously over 60-days following the
intermittent fasting and refeeding cycles, the fasted groups showed
increased feeding rates (24-25% day"') resulting in improved
daily growth rates (112-115 p,m and 176-194 mg day"'). The
control group showed similar DFR (26% day"') but had lower

Absrnicts. February 2000

4th International Abalone Symposium. Cape Town. South Africa

DGRs (42.4 p.m and 7 1 .2 mg day" ' ). After 200 days, abalone in all
treatments measured 46-48 mm SL and 25-28 g BW with no
significant differences. The same animals were again subjected to
a 5- or 10-day starvation with longer refeeding periods at 10 and
20 days respectively over 86 days, followed by continuous feeding
for another 83 days. The control group fed continuously on sea-
weed. Results showed that abalone maintained high DGRs (90-93
\xm and 169-183 mg day"') which were comparable with the
control (89 jjim and 183 mg day"'). These findings were confirmed
by another trial showing that abalone deprived of food for 10 or 20
days showed compensatory growth after refeeding for 20 or 40
days, respectively. Harvest size after 372 days ranged between
58-60 mm SL and 49-52 g BW with no significant differences
among treatments. Likewise, percent survival (88 to 90%) was
generally high and was similar for all treatments.

Abalone that were re-fed on artificial diets showed the lowest
growth rates, comparable to the artificial diet-control. However,
percent survival (79%) was significantly higher in the seaweed-
control than in the rest of the treatments (range:33-53%). In Ex-
periment 2. abalone fed artificial diets at 60- and 90-day rearing
periods showed growth depression as indicated by significantly
lower growth rates (24-59 ixm and 14-16 mg day"') than the
control (94 |xm and 118 mg day"'). However, animals exhibited
compensatory growth after resumption of seaweed feeding, and
fed similarly to the control (34 |xm and 126 mg day"'). At the end
of a 150-day culture period, abalone fed artificial diets at shorter
duration (60 days) had significantly higher growth rates (117 |jLm
and 207 mg day"' ) than the control. Percent survival was generally
high (93-97%) with no significant differences among treatments.
Abalone exhibited growth depression when fed artificial diets.
However, animals showed compensatory growth upon resumption
of feeding on fresh seaweeds. Results of both experiments indi-
cated that fresh seaweed using C IxtiUnae proved to be advanta-
geous and more convenient to use than artificial feeds.

EFFECTS OF SEQUENTIAL FEEDING WITH SEAWEED
AND ARTIFICIAL DIETS ON GROWTH DYNAMICS AND
SURVIVAL DURING ON-GROWING OF ABALONE,
HALIOTIS AS/NINA (LINNAEUS 1758). A. C. Fermin, M.
Bautista Teruel, and S. M. Buen. Southeast Asian Fisheries De-
velopment Center Aquaculture Department (SEAFDEC/AQD).
Tigbauan 5021. Iloilo. The Philippines.

The effects of sequential feeding with seaweed and artificial
diet on the growth dynamics and survival rates of Hiilioiis asinina
were determined as part of a feeding management protocol for
on-growing of abalone in tanks. In Experiment 1, abalone juve-
niles (25 mm SL. 3 g BW) were initially fed either on seaweed
(Gracitariopsis hailliuu') or fish meal and soybean meal-based
artificial diet (277r crude protein) during the first 12 weeks, and
then interchanged with opposite feeds for the final 12 weeks. Con-
trol groups were fed cither seaweed alone or artificial feed alone
throughout the duration ol culture. In Experiment 2. juveniles (33
mm SL. 9 g BW) were led artificial feeds (277? crude protein) for
60 or 90 days, after which seaweed was given for the rest of the
l5()-day culture period. Control groups were led seaweed only.

Results of Experiment I showed that groups that fed on sea-
weed during the first 90 days had significantly higher daily growth
rates (179-180 |a.ru ;ukI 84-89 mg day ' I llian Iliose fed with
artificial diets (-6.25-29 |jim and 23-38 mg das ' ). When feeds
were interchanged, animals that were re-fed on seaweed showed
the highest growth rates ( 220 \i.m and 1 75 mg day ' I after 70 days.

EXAMINATION OF THE GEOGRAPHIC DISTRIBUTION
OF A RICKETTSIA-LIKE PROKARYOTE IN RED ABA-
LONE. HALIOTIS RUFESCENS. IN NORTHERN CALI-
FORNIA. C. A. Finley'- and C. S. Friedman,- 'Humboldt State
University, Fisheries Department. Areata. CA 95521-8299. -Cali-
fornia Department of Fish and Game and Bodega Marine Labora-
tory. P.O. Box 247. Bodega Bay. CA 94923 USA.

Withering syndrome (WS) is a chronic wasting disease respon-
sible for mass mortalities in wild populations of black abalone.
Haliiilis craclwriiilii. in southern California. A recently discovered
Rickcllsici-Vike prokaryote (RLP) has been identified as the caus-
ative agent of the disease. The RLP has been positively identified
in both wild populations of black abalone with WS and cultured
red ahalone. //. ii(fi'scciis. which displays signs characteristic ol
WS. Concern has risen that the culture facilities, as well as out-
plantings that took place prior to the identification of the RLP. may
have served as vectors for the disease into northern California red
abalone populations, the only remaining healthy populations of
abalone in the State. In this study. fi\e point source locations and
lour reference locations north ol Carniel (where the RLP was
previously obser\ed) were chosen using a stratified random de-
sign. Strata were defined either by their proximity to an out-plant
location or an abalone facility (both point sources) or as reference

4th Inlernational Ahalone Symposium. Cape Town. South Africa

Abstracts, February 2000 51.^

locations (found between point sources). At each site 60 animals
were randomly collected to achieve a 5% detection level. Animals
were examined for the presence of WS and the RLP. and the shells
were also examined for the presence of an exotic sabellid. Tere-
brasahella heteroimcinala. another aquacultural pest of concern.
Preliminary data show the RLP has spread north to San Francisco
(at both point and reference sites). The RLP has not been observed
in a point source and a reference site examined north of San
Francisco. In addition, no sabellids have been identified in any of
these samples. At this point, the distribution of the RLP does not
appear to have been influenced by either culture facilities or out-
plantings, but several sites have yet to be examined.

LIFE HISTORY OF AN EXOTIC SABELLID POLY-
CHAETE, TEREBRASABELLA HETEROUNCINATA: IN-
FLUENCE OF TEMPERATURE AND FERTILIZATION
STRATEGY. C. A. Finley,' " C. S. Friedman,- and T. J. Mul-
ligan/ 'Humboldt State University. Areata. CA 95521-8299,
"California Department of Fish and Game and Bodega Marine
Laboratory, P.O. Box 247. Bodega Bay, CA 94923 USA,

The California abalone aquaculture industry has been strug-
gling to rid itself of an exotic sabellid, Terehrasahetla heteiounci-
nata, following its accidental introduction from South Africa in the
late 1980s. Intensive culture conditions and periodically elevated
seawater temperatures, associated with El Nifio events, have cre-
ated conditions in which rapid spread and intensive infestations
have occurred in culture facilities. These observations have raised
concerns over the potential threat that the sabellid may pose to
native invertebrate populations throughout California, following
an accidental introduction from an aquaculture facility. Califor-
nia's seawater temperatures range from a low of 8 °C to 2 1 °C and
exceed these averages during EI Nifio years, A need has arisen to
improve understanding of the life history of this sabellid, including
generation time and whether it is capable of self-fertilization. In
the present study, uninfested red abalone, Haliotis rufescens. were
exposed over a 24 hr period to heavily sabellid-infested abalone at
10. 16, and 20 °C. The larvae were subsequently observed as they
developed to specific life stages: initiation of feeding, sexual matu-
ration and the completion of their life history or the production of
a motile, infestive, larva. Approximately 509c of the sabellids ex-
amined had developed the ability to feed by day 6, 5, and 4, and
became sexually mature by day 83, 68, and 49 for 10, 16, and 22
°C, respectively. Preliminary results indicated that 50% of the
sabellids had produced larvae by day 111 at 22 °C. In a separate
study, uninfested abalone were exposed as above, and abalone
with single infestations were held in individual containers at 18 °C
(single host and sabellid per container). This first, parental gen-
eration was held in isolation until individuals self-fertilized to
produce F, larvae. The F, larvae were allowed to infest new aba-
lone (single sabellid per host) and were then isolated as above. We
subsequently observed second-generation, F, larvae. This research
demonstrates that the life history and generation time of T. heter-

ouncinala are highly temperature dependent and that its products
of self-fertilization are fully functional organisms.

"CANDIDATUS XENOHALIOTIS CALIFORNIENSIS," A
NEWLY DESCRIBED BACTERIAL PATHOGEN AND
ETIOLOGICAL AGENT OF WITHERING SYNDROME
FOUND IN ABALONE, HALIOTIS SPP., ALONG THE
WEST COAST OF NORTH AMERICA. C. S. Friedman,' "
K. B. Andree,^ T. T. Robbins,- J. D. Shields,^ J. D. Moore,^ K.
Beauchamp,- and R. P. Hedricli' 'California Department of Fish
& Game, Bodega Marine Laboratory. P.O. Box 247, Bodega Bay,
California 94923, "Department of Medicine & Epidemiology,
School of Veterinary Medicine, University of California, Davis.
California 95616, ""Virginia Institute of Marine Science, Glouces-
ter Point, VA 23062,

Withering syndrome (WS) is a fatal disease affecting wild and
cultured abalone, Haliotis spp., which inhabit the west coast of
North America. A previously undescribed bacterium observed in
abalone with WS has recently been identified as a member of the
family Rickettsiaceae in the order Rickettsiales. Using a combina-
tion of morphological, serological, life history and genomic (16S
rDNA) characterization, we have identified this bacterium as a
new genus and species. Due to the inability to culture the bacte-
rium we propose the provisional status of "Candidatus Xenohali-
otis californiensis." The Gram negative, obligate intracellular,
pleomorphic bacterium is found within membrane-bound vacuoles
in the cytoplasm of abalone gastrointestinal epithelial cells. The
bacterium is not cultivable on synthetic media or in fish cell lines
(e.g., CHSE-214) and may be controlled by tetracyclines (oxytet-
racycline) but not by chloramphenicol, clarithromycin, or sara-
tloxicin. Phylogenetic analysis based on the 16S rDNA of "Can-
didatus Xenoluiliolis californiensis" places it in a subclass of the
class Proteobacteria. The bacterium can be detected in tissue
squashes stained with propidium iodide, microscopic examination
of stained tissue sections, PCR or in situ hybridization. ""Candida-
tus Xenohaliotis californiensis" can be differentiated from other
closely related Proteobacteria by its unique I6S rDNA sequence.
We tested the hypothesis that this bacterium is the etiological agent
of WS in two long term clinical trials using oxytetracycline as the
therapeutant. In two separate trials asymptomatic red and. in a
separate trial, black abalone -I-/-WS administered a series of sham
injections (3% saline as controls) or oxytetracycline injections (21
mg/kg Liquamycin LA-200 as the experimental treatment) over a
9 wk period. Survival and feeding rates were monitored for 6 mo.
Both survival and feeding rates were higher in treated abalone
relative to control animals {p < 0.001, /> < 0.023 for red and black
abalone, respectively). All red abalone and -50% of the black
abalone that received the therapeutant survived, while -40% of the
red and 100% of the black abalone controls died during this time.
These studies indicate that WS is caused by "Candidatus Xeno-
haliotis californiensis" and that losses can be minimized by ad-
ministration of oxytetracycline.

514 Abstracts. February 2000

4th International Abalone Symposium, Cape Town, Soutin Africa

THE CELLULAR IMMUNE RESPONSE OF BLACK ABA-
LONE, HALIOTIS CRACHERODII LEACH. WITH AND
WITHOUT WITHERING SYNDROME. C. S. Friedman.' T.
Robbins,' J. L. Jacobsen,^ and J. D. Shield,"' 'California Depart-
ment of Fish and Game and Department of Medicine and Epide-
miology. Bodega Marine Laboratory. P.O. Box 247. Bodega Bay,
CA 94923, -Bodega Marine Laboratory, P. O. Bo.\ 247, Bodega
Bay, CA 94923, Virginia Institute of Marine Science, Gloucester
Pt., VA 23062.

Withering syndrome (WS) is a chronic disease that has resulted
in dramatic declines in black abalone abundances along the south-
ern and central California coast. A Rkkettsiii-hke procaryote has
recently been identified as the etiological agent. We hypothesized
that the nonspecific immunity function of abalone hemocytes may
be affected by WS and that these changes may serve as early
indicators of disease. We examined the chemotactic, phagocytic
and chemiluminescent abilities of hemocytes from abalone with
and without WS. Although hemocytes from abalone with WS were
more chemotactically active than those from asymptomatic aba-
lone (n = 35, p < 0.01), they were compromised in other key
functions. Hemocytes from diseased abalone were less able to
engulf foreign particles (;? = 59, p < 0.01), engulfed fewer par-
ticles (n = 52, p = 0.00). and produced a reduced respiratory
burst in = 26, /) = 0.00) relative to those from asymptomatic
abalone. The immune capability of the hemocytes correlated with
the degree of WS. Thus, hemocytes from abalone with WS may be
more chemotactically active as a result of degeneration of the
digestive gland and utilization of the foot muscle as an energy
source. However, the capability of these stimulated cells to engulf
and destroy foreign particles appears to be compromised and may
contribute to mortality associated with this disease.

out study to determine the suitability of Roe's abalone for culture.
The study is evaluating three culture systems-Australian raceways,
California round settlement tanks, and sea-based barrels-for grow-
out of juvenile Roe's abalone. These culture systems are being
used at several sites within WA to determine whether there are
differences in growth and survival by location. Greenlip abalone
iH. laevigata) are being reared alongside the Roe's abalone to
provide a direct comparison of growth and survival. Variables
being investigated in replicated land-based tanks include tank de-
sign, location (over the range 29-35 °S), current speed, turbulence
and shading.

SEEDING COMPETENT CULTURED LARVAE OF THE
BLUE ABALONE HALIOTIS FULGENS INTO SOME WILD
STOCKS OFF THE ISLAND OF CEDROS BAJA CALIFOR-
NIA. MEXICO. O. J. G. Gonzalez Aviles. S C P P.. P N A , Av

Ryerson 117. Ensenada, B. C. Mexico.

Due to high poaching pressure on natural abalone populations
and with the additional negative effect of "El Nifio" (1997) aba-
lone stock density has declined in some areas. In our areas and
given our resources, larval .stock enhancement is more viable than
abalone juvenile seeding. Therefore, for several years, our co-
operative has cultured viable larvae for stock enhancement using
wild broodstock collected directly from natural stocks. In this work
we describe the larval releasing process. A semiautomatic di\'ing
"hooka" system is used for seeding. Competent larvae are placed
in siiii from no mixed stocks to avoid genetic exchange. Data from
five years of larval releasing is shown and analysed.

EVALUATION OF ON-SHORE AND SEA-BASED CUL-
TURE SYSTEMS FOR ROE'S ABALONE (HALIOTIS
ROEI) IN WESTERN AUSTRALIA. K.J. Friedman. G.
Maguire. and K. O. Hahn. Fisheries WA. Western Australian
Marine Research Laboratories. PC Box 20, North Beach. Perth.
Western Australia 6020.

Fisheries Western Australia is conducting research to evaluate
the potential of local abalone species for culture. .Although the
state has a healthy wild abalone fishery, there is great interest in
producing abalone through aquaculture techniques. To date, aba-
lone aquaculture in Australia has concentrated on greenlip iHali-
otis kievii>ata). blacklip (Haliotis nibia) and hybrid abalone. Un-
fortunately, these animals are coldwater species and unsuitable lor
the warm water along the western coast of Australia. The Roe's
abalone (Haliotis rod), fished commercially and found in great
abundance, offers an opportunity to culture abalone in the warmer
waters of Western Australia. Fisheries WA is conducting a grow-

WORLD ABALONE SUPPLY. MARKETS & PRICING. H.
Roy and Gordon N. Qing, China and Taiwan, N. Ujki, Japan, R.
Fields, USA, R. Flores, Mexico, A. Ziomi, South America. M.
Tokley. Australi;i/Tasmania. R. Roberts. New Zealand. P. Cook
and A. du Plessis. South Africa, G. Burnell. Ireland and Europe.
This session will comprise a country by country overview of
both wild fisheries and cultured abalone. Each speaker has gath-
ered important tonnage and pricing data along with details of spe-
cies caught or grown, cultivation methods, feed information and
disease implications, and introductions which ma\ include past
and present trends as well as locations of particular interest in each
country. Species discussion may include locations and in the case
of cultured abalone, hybrids. Types of cultivation may include a
discussion of various systems including both land and ocean cul-
li\alion. Feeding discussions will include manufactured food as
well as available or cultured algae. Pathology comments will in-
clude an overview of past and current problems and may include
order of magnitude impact of each disease.

4th International Abalone Symposium. Cape Town. South Africa

Ahslnicts. February 2000 515

WORLD ABALONE SUPPLY. MARKETS AND PRICING
FROM HISTORICAL, CURRENT AND FUTURE PRO-
SPECTIVES. H. R. Gordon, Fishtech Inc. Box 6886 San Rafael.
California 94903.

The world supply of wild fisheries catch and cultured abalone
production are considered with implications of the past, present
and future. Much of the data available in recent years from various
go\ernnient sources and even FAQ have often been inadvertently
misleading, as quantities reporting is developed using differing and
sometimes conflicting base lines. Catch and production data in
some countries have been either nonexistent or with substantial
gaps in reporting. Reporting has often combined a number of
dissimilar abalone products (fresh in shell, frozen meat, canned,
dried etc) with misleading results. As a further complication, ex-
port numbers in some cases inadvertently include illegal catch
abalone. Reporting from the cultured or farmed abalone "world""
has been limited and much of the available information has been
reported using differing standards. Surprising numbers result from
this effort to standardize the production and export information for
both the commercial catch and cultured product. Comparative
charts and order of magnitudes have been developed. The decline
in total world supply from the 1980"s is given an order of magni-
tude in relation to poaching, pollution, pathology, predators and
climate. Discussion will include factors affecting abalone prices
worldwide, a review of market form (live, fresh, frozen, canned,
dried) and how pricing is affected by processing and packaging as
well as economic conditions, followed by a review of suggestions
for value added abalone product. The implications of Asian tradi-
tions are also described and explained. Ethnic preparation and
cooking methods are summarized along with their impact on world
market prices.

POST HARVEST WEIGHT LOSS HAS IMPORTANT IM-
PLICATIONS FOR ABALONE QUOTA MANAGEMENT.
H. K. Gorfine. Marine & Freshwater Resources Institute. PO Box
1 14. Queenscliff, VIC, Australia.

Regulations forming part of the abalone quota management
system in Victoria, Australia, require that abalone processors no-
tify fisheries authorities of quantities of abalone consigned from
commercial divers within 25 hours of landing. The regulations also
require that the abalone are to be landed whole in the shell, and
transported and stored in sealed bins until one half hour after
official notification. Whilst the bins cannot be opened prior to
notification, the 25 hour limit for notification provides a window
of opportunity to make potential gains through weight loss in those
abalone destined for canning. This arises because notified weights,
rather than weights estimated upon landing, are used to decrement
the quota allocations of individual divers.

1 investigated the potential for post-harvest weight loss by sub-
jecting abalone to three experimental treatments selected to simu-
late a range of possible transport and storage regimes. My results
demonstrated that substantial weight loss can occur in whole aba-
lone during both ambient and refrigerated storage. Loss of weight
in abalone during storage results from the release of water and
body fluid associated with physiological responses to hypoxic
stress caused by exposure to air. In Victoria, abut 60% of the
landed catch is used to produce canned product. At present, weight
losses during storage are not accounted for in setting the Total
Allowable Catch and divers supplying abalone for canning have to
harvest more abalone to achieve their quotas than those supplying
abalone for live export. Losses in weight of 10-20% observed
during this study equate to 350,000-700,000 more abalone har-
vested than if beach weights were deducted from quotas. These
additional quantities of abalone harvested may exceed desired fish-
ing mortalities for long-term population sustainability.

A BEHAVIOURAL RATHER THAN RESOURCE-
FOCUSED APPROACH MAY BE NEEDED TO ENSURE
SUSTAINABILITY OF QUOTA MANAGED ABALONE
FISHERIES. H. K. Gorfine and C. D. Dixon, Marine & Fresh-
water Resources Institute, PO Box 1 14, Queenscliff, VIC, Austra-
lia.

The Victorian blacklip abalone fishery is Australia"s second
largest fishery and accounts for about one eighth of reported global
abalone catch. Most indicators of stock status for this fishery are
favorable, with relatively high daily catch expectations (about 500
kg) among divers. The fishery is subdivided into three manage-
ment zones, each spanning several hundreds of kilometers of coast,
and within each zone divers are restricted to harvesting an equal
share of the total allowable catch. During 1998 we initiated an
on-board observer program to gain a better understanding of spa-
tial and temporal patterns in catch and effort. Although average
CPUE has been increasing, there has also been significant spatial
contraction of the fishing grounds away from reefs of low produc-
tivity, consistent with the effects of quota introduction during
1988/89 and with subsequent serial depletion. It is this shift away
from reefs of low productivity rather than an increase in abalone
abundance that is responsible for the trend in CPUE. From our
on-board observations, divers do not operate in an area if they
believe that they will not meet their daily catch expectations; they
have a relatively high catch rate threshold for deciding when to
shift to another reef Catch rates per bag of abalone are several
times higher than the daily reported CPUE rates, but vary substan-
tially. We conclude that under quota management, spatial re-

516 Abstracts. February 2000

4th International Abalone Symposium. Cape Town. South Africa

allocation of effort and incentives to maintain high catch rate
thresholds for cessation of fishing at the reef scale in an environ-
ment where competition for prime fishing grounds is reduced
through restricted entry have been the keys to sustainability of the
Victorian fishery during the past 35 years. However, contemporary
changes in the fishery, such as reductions in the number of divers
who own access entitlements, may lead to unfavourable fishing
behaviour patterns among divers. Managers need to be aware that
regulations affect diver behaviour and that, despite increased in-
terest in resource manipulation through restoration and sea ranch-
ing, it is the diver and not the resource that is managed. Our studies
shows that it is important to focus on identifying and promoting
behaviour among divers that is desirable in terms of sustainable
production within the context of contemporary management strat-
egies.

TRIGGERS AND TARGETS: WHAT ARE WE AIMING
FOR WITH ABALONE FISHERIES MODELS? H. K. Gor-
fine, B. L. Taylor, and T. I. Walker, Marine & Freshwater Re-
sources Institute, P.O. Box 1 14, Queenscliff. VIC, Australia.

A variety of quantitative measures have been applied as refer-
ence points in the management of Australian abalone fisheries. In
New South Wales, changes in legal-sized and mature biomass will
trigger management responses; in South Australia, catch rates, size
composition and abundance indices provide target reference points
and in Tasmania, catch rales are used to provide triggers for man-
agement decisions. However, Victoria and Western Australia have
yet to determine their reference points for abalone stock assess-
ment. Victoria has been developing length-based fisheries models
similar to those applied in NSW, and is now confronted with the
necessity of converting model outputs into decision-making crite-
ria. A Victorian fishery management plan is also under develop-
nienl in which reference points will be specified within a risk-
based matrix of catch control rules for TAC adjustment. Recent
biodiversity conservation legislation, compelling fisheries man-
agement agencies in Australia to demonstrate that export fisheries
managed under their jurisdictions are ecologically sustainable, has
increased the urgency to establish these reference points. The ap-
plication of this legislation draws upon the "Principles and Criteria
for Sustainable Fishing' of the Marine Stewardship Council in
London. We considered a range of alternative measures for refer-
ence points that may be useful as triggers and targets applied in a
stochastic tramework for management decisions. Although not a
modeling output, one of the more consistent signals of localised
depletion in the Victorian fishery relates to spatial allocation of
effort at the scale of reef complexes. Reductions in annual effort
applied to a particular reef system invariably precede significant
decreases in abundance indices with typically large coefficients of

variation and catch rates characterised by hyperstability. Victorian
abalone divers have high daily catch expectations and allocate
their effort accordingly. Empirical reference points such as effort
allocations provide utility for fishery management, and can be
readily assimilated and adopted by industry. Consequently, we
conclude that maintenance of reef-scale effort allocation and daily
catch expectations should form part of a suite of fishery pert'or-
mance indicators and target criteria related to modelling outputs
for the Victorian blacklip abalone fishery.

VARIATION IN MINERALOGY IN THE NEW ZEALAND
BLACKFOOT ABALONE HALIOTIS IRIS SHELL. Blair
Gray, Department of Marine Science, University of Otago, P.O.
Box 56, Dunedin, New Zealand.

The blackfoot paua (abalone) Haliotis iris is the most common
of the three species of abalone occurring in New Zealand, and has
been commercially fished for its meat and shell for many decades.
In more recent times, there has been increasing interest in pearl
production using the paua shell.

The shells of the majority of gastropod species consist of ara-
gonite, and only a few species belonging to 13 families, utilise
calcite as part or the whole of the shell. Haliotis iris has the ability
to control the growth of two crystal polymorphs of calcium car-
bonate (CaCO,). These biologically formed polymorphs, calcite
and aragonite, have the same chemistry but vary in their crystal-
lographic arrangement, giving them different physical and chemi-
cal properties. The location and thickness of these mineral layers
was examined in Haliotis iris, using both Feigl's Solution and
Scanning Electron Microscopy (SEM), These techniques con-
firmed the presence of an outer calcitic layer and inner aragonitic
layers. These are separated by both calcified and non-calcified
organic layers running longitudinally through the shell.

Many of the classical studies on the mineralogy of Molluscs
have only used a small sample size to assess the mineralogy of a
species. Even fewer studies have examined the variation within the
shells of individuals. This study examined the varying airounls of
calcite and aragonite within individual shells and within popula-
tions using X-ray diffraction (XRD). It was found that there is a
significant difference both within individual shells (/> < 0.01 1 and
individuals within a population (/) < 0.01 ). Within a population, the
variation in mineralogy may be as high as 40-93% aragonite. This
finding may have important implications for the classification of
mineralogy for not only Haliotids. but Molluscs in general.

This paper will also make an attempt to correlate the variation
in mineralogy between populatioiis and cinironinental parameters
such as wave exposure, seawater teinperatine. and salinity, as well
as to estimate the effects of erosion and biotic interactions e.g.,
epiphyte growth and shell boring.

4th International Abalone Symposium. Cape Town. South Africa

Abstracts. February 2000 5 1 7

GENETIC VARIABILITY OF THE BLUE ABALONE HALI-
OTIS FULGENS IN THE WEST COAST OF BAJA CALI-
FORNIA. MEXICO. Jose L. Gutierrez Gonzalez.' Ana M.
Ibarra.^ and Miguel A. del Rio Portilla,' 'Universidad Au-
tonoma de Baja CaUfornia Sur/Centro Regional de Investigaciones
Pesqueras La Paz, "Centre de Investigacion Biologicas del No-
roeste. A.P. 128. La Paz B.C.S. 23000 Mexico. 'Centro de Inves-
tigacion Cientifica y de Educacion Superior de Ensenada Km 107
Carr. Tijuana-Ensenada, Ensenada. B.C. Mexico A.P. 2732.
Ensenada, Mexico 22800.

Even though on the west coast of Baja California Peninsula
there are five commercially exploited species of abalone, of which
Haliotis fiilgeiis (blue abalone) produces more than 50% of the
catch, little research has been done on population genetics. Thus,
the main goal of the present work was to characterize genetically
the blue abalone populations in Baja California. The Peninsula is
divided into four abalone exploitation zones. Therefore, an area
with high abalone density from each zone was sampled, as follows:
a) Isla de Cedros, zone I; b) Bahia Tortugas, zone II: c) Bahia
Asuncion, zone III and d) Isla Magdalena, zone IV. Allozyme
electrophoresis was carried out for a total of 377 organisms at 16
loci of which 1 1 were polymorphic. The proportions of polymor-
phic loci were 37.5% in Bahia Asuncion and 43.7% for the other
three populations. Twenty-five cases did not adjust significantly to
the Hardy-Weinberg (H-W) model, and all of them had heterozy-
gote deficiencies. It was considered that the four populations as a
whole were not in H-W. having high heterozygote deficiency.
Mean unbiased heterozygosity ranged from 0.173 tp 0.197, where
Bahia Tortugas had the smallest value. Cluster analysis first joined
Bahia Asuncion and Isla Magdalena as one group, then linked
them with Isla de Cedros and finally with Bahia Tortugas. Most of
the F values were positive and the mean Fst was 0.022 showing a
low genetic diversity.

DISPERSION POTENTIAL OF REPRODUCTIVE PROD-
UCTS AND LARVAL STAGES OF ABALONE {HALIOTIS
SPP.; MOLLUSCA:GASTROPODA) IN RELATION TO
THE HYDRODYNAMICS OF BAHIA TORTUGAS,
MEXICO. Sergio A. Guzman del Proo. Felipe Salinas. Oleg
Zaytsev. Jorge Belmar Perez, and Jorge Carrillo Laguna.
Laboratorio de Ecologi'a, Departmento de Zoologi'a, Escuela Na-
cional de Ciencias Biologicas. Institute Politecnico Nacional. Prol.
Carpio y Plan de Ayala s/n, Mexico, D. F. 1 1340.

Field observation of currents and water mixing were made in
autumn 1996. at four coastal sites close to Bahi'a Tortugas, on the
central part of the Baja California Pacific coast, to evaluate the

influence of hydrodynamics on the transport of abalone larvae
(Haliotis spp.). Cun'ent measurements and full-scale Lagragean
experiments on surface-water transport were carried out during the
main spawning season of abalone in the area. Wind and wave-
driven currents appear to be the most important factors affecting
larval transport in this coastal area, and tidal currents do not appear
to play a dominant role. Additional echo sounding and aerial sur-
veys confirmed that the reef topography and kelp beds attenuate
current velocity. The hypothesis of larval dispersion is that during
a typical 3 to 5 day pelagic period, larval and postlarval stages
might be retained primarily in areas close to parental reefs. Flush-
ing time in Bahi'a Tortugas was evaluated as 5 semi-diurnal tidal
periods. Sufficiently intensive currents at the mouth of the bay (up
to 25 cm s"') may complicate larval interchange between the
northern and southern vicinities of the bay.

SIZE-STRUCTURED MODELS OF ABALONE POPULA-
TIONS WITH A CRYPTIC COMPONENT TO THE
STOCK. Malcolm Haddon. University of Tasmania, TAFI/ Ma-
rine Research Laboratories, Nubeena Crescent, Taroona, TAS
7053, Australia.

Size-structured models are often used when attempting to
model the population dynamics of commercial stocks of abalone.
These models are based around applying a projection matrix (P) to
a vector of the population size distribution for each time period of
growth (N,^.| = PN,). The projection matrix is generated by mul-
tiplying a growth transition matrix (G) by a survivorship matrix (S)
and adding a recruitment matrix (R) thus: N,^, = (OS + R)N, or
N,^., = (GS)N, + R, depending on the timing of processes in the
model. The survivorship matrix is a combination of natural mor-
tality and fishing mortality as applied through the filter of a se-
lectivity curve. The repeated application of such a projection ma-
trix and recruitment enables the expected size distribution of the
modelled stock to be followed through time. Such simplistic mod-
els attempt to model the total population but cannot accommodate
the often significant proportion of the abalone population that is
cryptic (non-emergent, perhaps under rubble or boulder fields).
Assessing only the emergent population would tend to underesti-
mate both survivorship and recruitment. Alternative models, which
include this non-emergent sector of the population, are described,
with their respective strengths and weaknesses. Unfortunately, the
field observations needed to fit these alternative models suggest
that successful modelling of both the cryptic and emergent com-
ponents of each population will require more than size information
alone.

518 Abstmcts. February 2000

4th International Abalone Symposium. Cape Town, South Africa

GENETIC SUBDIVISION OF THE ABALONE HALIOTIS
ROEI IN SOUTH WESTERN AUSTRALIA. A. T. Hancock.

WA Marine Research Laboratories. PC Box 20. North Beach.
Western Australia.

Population structure of the abalone Haliotis roei in .south west-
em Australia was investigated genetically using starch-gel electro-
phoresis. Eight polymorphic enzymes were examined in 624 ani-
mals from 10 populations. Samples covered 3000 km of coastline,
from Shark Bay in Western Australia to West Island in South
Australia. Replicate samples were collected from 2 sites at 12
month intervals. The average Fst was 0.009 with 5 of the 8 loci
showing significant variation in allelic frequencies. Sites showed
no striking geographic trends in allelic frequencies or apparent
clustering of sites using multidimensional scaling of Gst as a mea-
sure of genetic dissimilarity. A population structure of isolation-
by-distance was evident when pairwise measure of Gst between
populations were plotted against geographic distance. This rela-
tionship was evident beneath relatively high levels of variability in
some pairwise comparisons of Gst for sites separated by small
distances. The area of complete genetic mixing, or neighbourhood
size, was estimated from pairwise Gst calculated for replicate
samples at the same site. This technique estimates the size of the
genetic neighbourhood to be less than the distance between the two
nearest sites, or 13 km. The apparent contradiction between rela-
tively high levels of gene flow across the species distribution, as
indicated by a low average Fst. and substantial heterogeneity be-
tween sites separated by lO's of kilometers, is discussed in the
context of the species biology. Conclusions highlight the common
conceptual difficulty presented by relatively high levels of gene
flow maintained despite the apparent isolation of local populations,
which are primarily dependent on local recruitment for their con-
tinuation.

THE EXPERIMENTAL CULTIVATION OF THE SOUTH
AFRICAN KELP MACKOCYSTIS ANGVSriFOUA. M. N.
Harper, D. W. Keats, and R. J. Anderson, Botany department.
University Western Cape. P.O. Box XI 7. 7.'i3.'i. South Africa.

Kelps are the basis of a number of commercial products, in-
cluding alginate, plant growth substances, beauty products, and
food additives. More recently, in South Africa, kelps have become
highly sought after as feed for the abalone maricullurc industry.
The potential of low volume, high value products such as medical
alginate, as well as the available local market lor abalone feed
suggests that the commercial cultivation of kelps may be economi-
cally viable in some areas. This study examines the experimenlal
cultivation of the kelp Macrocyslis aiiyKslifoliu. The kelp is cul-
tivated using various techniques such as tank. rati, mist, spray, and
laboratory cuUivalion. Al present. oiil\ laboratory cultivation ol

gametophyte stage to sporophyte stage has been completed, using
nutrient enriched seawater as a growth medium. Growth was con-
sistent and favourable. Kelp will be cultivated on rafts in St Helena
Bay and harvested kelp will be tested for alginate quality following
purification using (a) viscometry, (b) mannuronic acid:guluronic
acid ratios and (c) protein determination. In addition, properties of
kelp important in terms of food quality for abalone will be inves-
tigated. These experiments will be used to determine which culti-
vation method would offer the highest quality of kelp for either
alginate production or abalone feed.

EFFECT OF OXYGEN SUPERSATURATION AND TEM-
PERATURE ON JUVENILE GREENLIP. HALIOTIS LAE-
VIGATA, AND BLACKLIP, HALIOTIS RUBRA, ABALONE.
J. O. Harris, C. M. Burke, S. J. Edwards, and D. R. Johns.

School of Aquaculture. University of Tasmania. P.O. Box 1214.
Launceston. Tasmania. Australia.

The growth and survival of greenlip and blacklip abalone were
investigated in terms of their responses to high levels of dissolved
oxygen (98-123% saturation). Blacklip abalone. Haliotis rubra,
held at 17 °C and 99% oxygen saturation, grew significantly faster
than all other treatments of blacklip abalone held at 19 "C, and
significantly faster than blacklip abalone maintained at 107% oxy-
gen saturation and 17 °C. Both temperature and oxygen saturation
significantly affected the survival for this species. Blacklip abalone
held at 19 °C had significant mortalities for both 98% oxygen
saturation and 123% oxygen saluration when compared with mor-
tality at 106% oxygen saturation. Oxygen consumption rates were
depressed in supersaturated conditions for both species. No sig-
nificant differences were noted for greenlip abalone. Haliotis lae-
vigata, within the range tested in terms of growth rates, food
consumption rates or survival, indicating more tolerance for these
conditions than blacklip abalone.

AN OVERVIEW OF STATE AND NON-STATE RE-
SPONSES TO ABALONE POACHING IN SOUTH AFRICA.
M. Hauck, Institute of Criminology, University of Cape Town,
South Africa.

Abalone poaching has escalated in South Africa over the last
ten years, contributing to controversial political debates and
heightening concern over the future of the abalone fishery. With
ongoing conflict between the informal fishers, the commercial
sector and the government, the last fi\e years has spurred a
suite of diserse responses. Slale-inlervention has focused on

4tli International Abalone Symposium, Cape Town. South Africa

Ahstracls. February 2000 519

methods of policing, ranging from reactive to proactive strategies.
These have included the implementation of training in the Western
Cape province as a means of building capacity and increasing the
effectiveness of law enforcement partners. In addition, non-state
initiatives have been developing to mobilise communities to take
action and to investigate other alternatives for fisheries compli-
ance. The organisation of Seawatch has developed with one com-
munity as a iTieans of addressing poaching activity at a local level.
Residents have decided to take responsibility for building relation-
ships with the authorities, compiling information and exploring
local mechanisms for managing marine resources in its area. In-
dependent research has also investigated the socio-political cir-
cumstances involved in poaching activity and has explored alter-
native possibilities for resource management. These strategies,
which focus on both deterrence and voluntary compliance, seem to
encompass important dynamics highlighted by fisheries compli-
ance theory. However, the effectiveness of these initiatives has not
yet been suitably evaluated. The appropriate balance between this
'carrot and stick' approach is still being explored in South Africa
and important loopholes have been identified in each. One ques-
tions whether the political climate is conducive, and the economic
resources available, to find this balance before the imminent col-
lapse of the fishery.

sainple. 23.3 kDa and 46.4 kDa bands were detected in addition to
bands detected in the 25-mm sample. The expression amount of
28.5 kDa protein was found to increase dramatically with devel-
opment. These results demonstrate a hierarchical change in shell
biomineralization in abalone development.

ONTOGENETIC TRENDS OF SHELL BIOMINERALIZA-
TION IN ABALONE, HALIOTIS DISCUS HANNAI INO. G.
He and K. Mai, Aquaculture research laboratory. Ocean Univer-
sity of Qingdao 266003. China.

In the present study, the abalone Haliotis discus hannai, with
different shell lengths (8. 14, 25, and 55 mm) was used for min-
eralogy and minor element composition study by X-ray diffraction
and ICP. Besides calcite and aragonite, dolomite (CaMg(C03)2)
was detected for the first time in abalone shells. No calcite was
detected in the 8-mm shell. From 14 to 55 mm shells, the ratio of
calcite increased steadily from 1.6% to 13.6%. Correspondingly,
the ratio of aragonite decreased from 95.3% to 83.9%. Generally,
from 8-mm to 55-mm shell length, the concentration level of Mg,
Mn, and Fe increased, while Zn and Cu dropped. Al and Na
increased slightly with shell growth. The possible reasons for these
variations are discussed in this paper. A characteristic comparison
of shell matrix proteins among the different sizes of abalone was
also conducted to reveal the changes in shell biomineralization
with abalone growth. The apparent molecular weights of matrix
proteins increased with shell growth when determined by gel-
filtration. When separated by SDS-PAGE. four bands were de-
tected in the 14-mm sample, i.e. 7.5 kDa, 18.3 kDa, 28.5 kDa, and
30.9 kDa. In the 25-mm sample, a 34.9 kDa band was detected in
addition to bands detected in the 14-mm sample. In the 55-mm

GROWTH REDUCTIONS IN GREENLIP {HALIOTIS LAE-
VIGATA) AND BLACKLIP {HALIOTIS RUBRA) ABALONE
RESULTING FROM CHRONIC EXPOSURE TO SUBLE-
THAL COMBINATIONS OF ELEVATED AMMONIA AND
LOW DISSOLVED OXYGEN LEVELS. S. Hindrum, C.
Burke, S. Edwards, and D. Johns, School of Aquaculture, Tas-
manian Aquaculture Fisheries Institute, University of Tasmania,
PC Box 1214, Launceston, 7250, Tasmania, Australia.

Six groups of cultured abalone juveniles ( 1 control = Treat-
ment I, 7.61 ppm dissolved oxygen (DO), 5.62 ppb free ammonia
nitrogen (FAN)) of two species were held in a flow-through bio-
assay system for 6-8 weeks and exposed to various combinations
of dissolved oxygen and FAN in a factorial design as follows:
Treatments 2, 3, 4 = 6.0 ppm DO and 32. 50 and 196 ppb FAN
respectively. Treatments 5, 6 = 4.3 ppm DO and 32 and 50 ppb
FAN respectively (actual measured values). Sand-filtered oceanic
water was used to supply all treatments, ammonia being added to
Treatments 2-6 as ammonium chloride, mixed into individual res-
ervoirs for each treatment. Each reservoir supplied three replicate
tanks through a 1.8 m constant head column in which DO was
adjusted using a mixture of oxygen and nitrogen. Each tank held
1 5-20 abalone of each species in two separate cages, which were
suspended in the water column.

For both species, at both oxygen levels, growth in terms of
either specific growth rate for length (SGR-L) or specific growth
rate for weight (SGR-W) tended to decline as FAN increased, with
a significant interaction between DO and FAN for both species (P
< 0.005). For greenlip abalone. SGR-L was significantly higher for
Treatment I than for Treatments 2-6 (0.12% d'' vs. 0.106, 0.058,
0.043, 0.065, and 0.081% d"' respectively). For SGR-W. Treat-
ments 5 and 6 were significantly higher than Treatments 1^. and
Treatments I and 2 were significantly higher than Treatments 3
and 4 (0.44 and 0.43% d~', 0.32 and 0.28% d"', 0.09 and 0.16%
d"' respectively). For blacklips. Treatments 1 and 2 grew signifi-
cantly faster in terms of SGR-L than Treatments 3-6 (0.034 and
0.036% d~' vs. 0.024, 0.013, 0.022, 0.014% d"' respectively), with
Treatment 4 significantly lower than Treatments 3, 5 and 6. In
terms of SGR-W, Treatments 3 and 4 were significantly lower than
Treatments 1, 2, 5 and 6 (-0.025 and -0.051% d"' vs. 0.16, 0.074,
0.19 and 0.1 1% d"' respectively).

These results confirm the sensitivity of these species to chronic
exposure of even modest reductions in water quality.

520 Ahslmcts. February 2000

4th International Abalone Symposium, Cape Town, South Africa

RESEARCH INTO A NEW TECHNOLOGY FOR ARTIFI-
CIAL ABALONE BREEDING. Z. Hongen, Director of Dalian
Fisheries Institute. Fujiazhuang 267, Xigang District, Dalian,
China.

A hybridization technique is used to increase resistance against
disease in cultured abalone, by cross-breeding of Haliotis discus
Reeve broodstock from Japan with Haliotis discus Lannai from
Dalian, China. Half of the total breeding area is applied to collect
seeding. The other half of the area is used to culture benthic
diatoms so that juvenile abalone transferred to the plates will have
sufficient diatoms to feed.

The use of the hybridisation technique extended the time during
which abalone feed on diatoms, and improved the growth of ju-
veniles. Survival rates increased form 20% to 80%. and production
of abalone from 1500 m"" to 5000 m"", accompanied by the best
growth rates. Average shell length also increased, from 1.5 cm to
2.0 cm. The growth rates increased by 33%.

ing year. It is therefore necessary to adjust fishing intensity, de-
pending on the stock level within the fishing period, to avoid
over-exploitation in the subsequent year of limited abalone re-
sources.

ASSESSMENT OF THE EFFECTS OF FISHING INTEN-
SITY ON STOCK LEVELS IN THE ABALONE DIVING
FISHERY. T. Horii, National Research Institute of Fisheries Sci-
ence. 6-31-1 Nagai, Yokosuka. Kanagawa, 238-0316, Japan.

Based on catch-effort data on the abalone diving fishery over a
20-year period (1978-1998) in Ojika Island off Nagasaki, the ef-
fects of fishing intensity in a particular year on the stock level in
the subsequent year were examined. Owing to the extensive range
of the annual total effort of 2156-3798 worker-days, it was prac-
tically dilTicult and unreliable to assess the stock level with mean
annual values of catch per unit effort (CPUE). As such, a cumu-
lative catch when the cumulative eflbrt reached 2000 worker-days
(CooDo,,) was defined as an alternative index of stock abundance in
t year. In addition, the ratios of C2()(i(i, to mean values of C,|||„|,_|,
C2(K)o,,_2 and C,|„,|,,_, (last three years) were calculated as a rela-
tive index of stock abundance in i year {Nil). Furthermore, the
ratios of lolal effort in / year to mean values of total elfcirl in the
last three years were calculated as a relative index of fishing in-
tensity in t year (/;'//). Here, At is denoted NIt/Eli: the lower the At
value, the higher the tendency of over-exploitation. Relationships
between At-I and Nit showed a highly positive correlation (less
than 1% level of significance); the slock level on any particular
year was markedly affected by the fishing intensity of the precetl-

ANALYSIS OF THE SUITABILITY OF AUSTRALIAN
FORMULATED DIETS FOR THE AQUACULTURE OF
THE TROPICAL ABALONE, HALIOTIS ASININA LIN-
NEUS. D. J. Jackson, K. Williams, and B. Degnan, Department
of Zoology and Entomology, University of Queensland, Australia
4072.

The tropical abalone, Haliotis usinina Linnaeus, has recently
been recognised for its potential as a new aquaculture species
within Australia. However it is not known whether any of the
commercially available diets formulated for temperate species and
temperate conditions are suitable for this species. A growth assay
testing four commercial Australian diets and a seaweed, Cracilaria
edulis. was conducted to investigate the suitability of temperate
formulated diets for H. asiiiina. The trial was run for 6 months at
28 °C following a nutritional acclimation period of 41 days. Aba-
lone with a starting shell length of 18.3-2.76 mm and weight of
1 .32-0.577 g were used. A formulated diet that performed well
under the experimental conditions was identified by measuring
growth (shell length and weight) and survival. The presence or
absence of gonad tissue was monitored from the third month of the
trial onwards, and the gonad index (GI) was measured externally
at the end of the trial. Animals maintained on the four formulated
diets matured precociously while only one female maintained on
the G. edulis diet developed gonad tissue by the end of the trial.
There were no significant differences in GI between formulated
diets. However, histological examination of female gonads re-
vealed significant structural differences that reflected diet quality
(as measured by growth and survival). Animals maintained on
poorer quality diets showed evidence of degenerated gonads with
large populations of macrophages suggesting resorption of gonadal
tissue. Animals maintained on higher quality diets had normal
oocytes and gonad structure, and did not displav this macrophage
activity. The efficiency of dry matter food conversion by abalone
fed each of the diets was measured during a two week period of the
growth assay, revealing no significani differences. During this pe-
riod, the nightly weight of food ingested per experimental unit was
measured. A gradual increase in the weight of food ingested (ex-
pressed as a percentage of wet body weight) over the nights lead-
ing up to a full moon (coinciding with spawning patterns observed
in adults) and a subsequent decrease was observed for animals fed
the highest qualilN artificial diet.

4tli Intcmatioiial Abalone Symposium. Cape Town. South Africa

Ahslnwts. February 2000 521

SPAWNING INDUCTION OF HALIOTIS AUSTRALIS US-
ING DIFFERENT CHEMICALS AND GANGLIONIC SUS-
PENSIONS. N. M. J. Kabir and P. V. Mladenov, Department of
Marine Science. University of Otago, P.O. Box 56. Dunedin. New
Zealand.

Injections of serotonin (5-hydroxytryptamine. 10^'M). dopa-
mine (3-hydroxytyramine lO'-^M). prostaglandin E, (lO^'^M), de-
ionised fresh water, filtered seawater. cerebral (CG) and pleural-
pedal (PPG) ganglionic suspensions ( 10 ganglia per mL of water)
were assayed as inducers of spawning in the yellowfoot abalone.
Haliotis australis. Injections were made three times, once a day.
into the haemocoel near the cerebral ganglia of males and females
at a dose of 0. 1 mL per day. A batch of 98 animals with fully ripe
gonads (n = 6-10 per trial) was selected. Changes in the body
weight and release of gametes were monitored regularly for each
individual for four days. 100% of the females and 67% of the
males injected with filtered seawater spawned. 50% of the females
and 25% of males injected with serotonin spawned. Prostaglandin
E, did not induce the release of gametes. 50% of the females
treated with dopamine spawned a small number of eggs while the
males did not respond. Males did not respond to injection of sus-
pensions from any kind of ganglia and 20% of the females
spawned a few eggs in response to CG from females and PPG from
females. 40% of females spawned in response to PPG from males.
Injection of de-ionised fresh water caused no weight changes
whereas filtered seawater caused a reduction in weight. Prosta-
glandin treated animals gained weight on the 2nd day but lost
weight over subsequent days. Only the females gained weight in
the dopamine treated group, and both males and females gained
weight in the serotonin treated group. In the case of ganglionic
injection, males treated with male CG or PPG gained more weight
than their female counterparts and the females treated with female
CG or PPG gained more weight than males. The increase in mean
body weight of animals was followed by a swelling and softening
of the ovaries, possibly due to an increased water content in the
ovaries. It seems likely that uptake of water in the ovary is a
physiological precursor to spawning.

EFFECTS OF DELAYED METAMORPHOSIS ON SUR-
VIVAL AND GROWTH OF NEWLY METAMORPHOSED
HALIOTIS DISCUS HANNAI. T. Kawamura, H. Takami. and
Y. Yamashita. Tohoku National Fisheries Research Institute,
3-27-5 Shinhama. Shiogama. Miyagi 985-0001. Japan.

Swimming larvae of Haliotis species delay metamorphosis if
they fail to contact an appropriate environmental stimulus. The
effects of delayed metamorphosis on survival and growth of post-
larval Haliotis discus hainiai were examined. Competent larvae
were induced to metamorphose at 5, 10. 15, and 19 days after

fertilization by the addition of 1 p,M 7-aminobutyric acid (GABA).
Larvae in another group were maintained until individuals meta-
morphosed spontaneously. Metamorphosed individuals (post-
larvae) were reared in the laboratory and fed on a benthic diatom
Cyiindrotheca closterium. Starved post-larvae were reared in
equivalent conditions, but without any food. All experiments were
conducted at 20 °C. Metamorphosis, survival, and growth rates
(determined from shell length) were measured.

The percentage of metamorphosed individuals 2 days after the
addition of GABA increased with the length of larval swimming
period; larvae that were 5 and 19 days old when induced to meta-
morphose showed 19 (±5.6; SE) and 96 (±3.6)% metamorphosis
rates, respectively. The percentage of post-larvae that metamor-
phosed spontaneously increased after 17 days from fertilization
and reached 96 (±3.0)% at 24 days post-fertilization.

Survival rates of fed post-larvae depended on the larval swim-
ming period. For larvae that swam for <15 days, more than 80%
of post-larvae survived the 20 day experimental period. In contrast,
survival was only 57 (±2.9)% for 19 day old larvae. Post larval
growth rates did not differ significantly for larval swimming pe-
riods <I5 days. However, the growth rate of post-larvae from 19
day old larvae was significantly lower than that of 5 and 10 day old
larvae. In the starved treatments, survival rate was lower, and the
final shell lengths of the dead animals were less, as larval period
became longer.

These results indicate that post-larval H. discus Iniiuuii are able
to survive and grow normally within a larval period of 15 days (at
20 °C) if given adequate food. Nineteen days of larval swimming
reduced post-larval survivorship and growth rate, suggesting the
diminishment of larval yolk as an initial energy source for the
metamorphosed post-larvae.

NON-DESTRUCTIVE DNA TYPING IN ABALONE
HATCHERY MANAGEMENT APPLICATIONS. R. I. Lewis.
E. G. Hall, and J. S. Bee, Division of Aquaculture, Dept. of Ge-
netics, University of Stellenbosch, Private Bag XI. Stellenbosch
7602, South Africa, N. A. Sweijd, Dept. of Zoology, University of
Cape Town, South Africa.

Genetic management of hatchery stocks is often given a low
priority, since initially the most dramatic production gains can be
achieved through improved husbandry practices. In the long term
however, genetic management becomes highly desirable, not only
to evaluate and exploit the productivity increases available from
selection programmes, but to avoid productivity losses from inad-
vertent inbreeding and loss of diversity (and hence reduction in
improvement potential) through genetic drift. Genetic management
is considerably enhanced by the use of molecular tools that can
estimate such characteristics as genetic variance, relatedness
among individuals and populations, and levels of inbreeding. We

522 Abstracts. February- 2000

4th International Abalone Symposium. Cape Town. South Africa

used PCR amplification of mtDNA to estimate levels of genetic
diversity within and between two broodstock collections to evalu-
ate their potential for short term heterotic exploitation. DNA was
extracted from small tissue samples taken from mantle tentacles of
sexually mature animals, which were later spawned — i.e. sampling
was non-destructive facilitating future broodstock management
based on known genetic background. Eggs, sperm, and subse-
quently larvae from pair matings were collected to investigate
DNA extraction procedures, and to confirm the mode of mitochon-
drial DNA inheritance. Fl hatchery stocks were sampled to evalu-
ate the genetic effects of current broodstock management practices.

GENETIC ANALYSIS OF A CULTURED POPULATION
OF THE RED ABALONE, HALIOTIS RUFESCENS, IN
MEXICO. A. L. Licona Chavez and M. A. del Rio Portilla,

Centro de Investigacion Cienti'fica y de Educacion Superior de
Ensenada. Km 107 Carr. Tijuana-Ensenada. Ensenada. B.C.
Mexico. A. P. 2732. Ensenada, Mexico. 22800.

The red abalone. Haliotis rufescens. is one of the species that
has been cultivated and successfully commercialized in the USA
and Mexico. However, there are not many known genetic studies
on the cultured populations. Allozyme electrophoresis was used to
characterize genetically the population of red abalone cultured by
the "Abulones Cultivados. S. A." company. Five batches (range
1.6-9.5 cm) were sampled, with a total of 298 abalones. from the
grow-out facilities in the Isia de Todos Santos, B. C. and another
two batches were from the inland facilities in Ejido Erendira, B. C.
(range 0.49-1.42 cm) were also sampled, with a total of 298 aba-
lones. As a result of farm procedures, these two batches (J and K)
were divided into three (6. 8. and 10 mm) and two (6 and 8 mm)
groups, and so were genetically analysed separately.

Thirteen allozyme loci were scored for all organisms: three
were monomorphic and the others were polymorphic, although in
some batches few loci were monomorphic. Only three loci did no!
adjust significantly to the Hardy-Weinberg mode, of which two
showed hetero/ygote excess and one showed heterozygote defi-
ciency. From a total of 74 cases, 25 showed heterozygote defi-
ciencies, while 43 had heterozygote excess. Thus, in general, there
was not heterozygote deficiency. Unbiased heterozygosity (//) was
evaluated for each subpopulalion and. on average, H was slightly
larger than that of the red abalone cultured in California. H showed
a tendency to increase with size. J6 heterozygosity was smaller
than J8 and the latter was smaller than .110. and, also, K6 heterozy-
gosity was smaller than KIO. However, there was no significant
positive correlation between individual heterozygosity and size in
any case. In general. "Abulones Cultivados" population was con-
sidered to be in equilibrium and with high genetic variability.

REPRODUCTIVE PERFORMANCE INDICES BASED ON
PHYSICAL CHARACTERISTICS OF THE FEMALE
BLACKLIP ABALONE HALIOTIS RUBRA L. M. Litaay and
S. S. De Silva, School of Ecology & Environment, Deakin Uni-
versity PC Box 423, Warmambool. Victoria, Australia 3280.

Selection of abalone broodstock from the wild is often based on
external appearances. The criteria used are size, colour and shape
of the gonad. However, perforinances such as egg fertilisability,
hatching rates and larval survival of selected broodstock vary
widely. The present study, instigated to develop easily useable
indices based on physical characteristics for assessing the potential
reproductive performance of female abalone was conducted on
wild-caught, artificially propagated blacklip abalone, obtained
from coastal waters (142°15'E, 38°2rS), Australia. Shell charac-
teristics, length (SL), width (SW), height (SH). and total weight
(W) were determined. Feinales were spawned using a combination
of desiccation and UV irradiated methods. The fertilised eggs from
each spawning were incubated, hatched, and larvae reared sepa-
rately in a flow-through system. The criteria used for assessing
reproductive performance were: fecundity, fertilisability. hatch-
ability, and pre-settlement survival. The results showed that shell
characteristics can be used as a predictor of the reproductive per-
formance. In addition, a combination of physical characters, such
as SL and SW appeared to be good indicators of reproductive
performance predictability. Accordingly, a number of highly non-
linear regressions incorporating egg characteristics to reproductive
performance were developed. A ratio between some of the shell
characteristics can also be used as indices for broodstock assess-
ment. We also found that fertilisability was positively correlated
with hatchability and larval survival (P < 0.01 ).

IgE AND MONOCLONAL ANTIBODY BINDING TO ABA-
LONE AND OTHER MOLLUSC ALLERGENS. A. L. Lo-

pata, B. Feneniore, and P. C. Potter, Allergology Unit. Groote
Schuur Hospital, Observatory 7925, South Africa.

World-wide, seafood represents one of the most important
groups of allergens in the induction of food allergy. With the
increased consumption o( seafood, the rate of adverse reactions is
believed to be rising. In recent years, patients with adverse reac-
tions to abalone ( 'perlemoen'. Hiiliotis inidiie) have been reported
to the .Allergology Unit with increasing frequency. Several immu-
noglobulin E (IgE) binding sites have been identified in extracts of
abalone using SDS-gel electrophoresis and Western blotting. Two
proteins w ith molecular weights of 38 kDa and 45 kDa were found
to he the major allergens and appeared to very heat-stable. The
later unique seafood allergen has been named Hal m 1 (according
to the WHO/lnlernational Union of Immunological Societies). Hal
m I -like bands were also detected in other abalone and mollusc

4th International Abalone Symposium. Cape Town. South Afiica

Abstracts. February 2000 523

species. In addition, allergens with different molecular weights
that varied between the analysed subjects were demonstrated.

To be able to detect the same or similar abalone allergens
specifically in other mollusc species, we generated monoclonal
antibodies (MoAbs). using hybridoma technology. For this pur-
pose, several abalone proteins were extracted after SDS-gel elec-
trophoresis and used for the immunisation of mice. Monoclonal
antibodies have the advantage that they are homogenous in immu-
noglobulin subclass specificity and bind to the same epitope.

Two cross-reacting proteins, with 42 kDa and 45 kDa. were
recognised by MoAb 1. 10 in most of the analysed mollusc species
such as snail, limpet, and various mussel species. In contrast.
MoAb 1.4 demonstrated binding only to the 42 kDa protein in
abalone but not in other molluscs. These results demonstrated
clearly that the two MoAbs, developed against the same purified
protein from abalone. recognised different epitopes on the same
protein. Furthermore, these MoAbs differentiate between closely
related species, such as between the two South African abalone H.
midae and H. spadicea and even between subspecies such as H.
discus hannai and H. discus discus. This immunological technique
can therefore be used for the differentiation of closely related
mollusc species.

Further sequence analysis and epitope mapping of the aller-
gens/antigens in abalone and other molluscs will advance our un-
derstanding of the molecular basis and pathogenesis of mollusc
allergy.

thermore, species-specific binding patterns distinguished between
very closely related subspecies, such as between H. discus discus
and H. discus hannai as well as between H. diversicolor diversi-
color and H. diversicolor supertexta. These immunological tech-
niques present many advantages, among them their relative sim-
plicity and accuracy in identifying different abalone species.

IMMUNOLOGICAL DETECTION OF VARIOUS ABA-
LONE SPECIES. A. L. Lopata and T. Luijkx. Allergology Unit.
Groote Schuur Hospital. Observatory 7925, South Africa, N. A.
Sweijd and P. A. Cook, University of Cape Town, Department of
Zoology, Rondebosch 7701.

Proteins of any given organism are an expression of its genetic
composition. Therefore, many of the methods employed in differ-
entiating various animal species rely on different techniques of
protein analysis. Electrophoretic separation of proteins according
to their molecular weight was conducted using SDS-polyacryl-
amide gels. Species-specific banding patterns were observed.
However, the differentiation of very closely related species did not
always give satisfactory results. Novel immunological techniques
were applied to allow clear identification between subspecies. Fur-
thermore, the development of a field-based identification kit was
anticipated.

Monoclonal antibodies were generated in mice against species-
specific proteins of Haliotis midae. Over ten commercially used
abalone species from South Africa. Australia and Japan were
analysed by Western blotting. Specific antibody binding identified
mainly proteins with about 38 kilodalton (kDaj and 45 kDa. Fur-

EFFECT OF FORMULATED DIETS, FRESH SEAWEED
AND TEMPERATURE ON GROWTH RATES, GONAD DE-
VELOPMENT AND SHELL FORMATION OF THE EURO-
PEAN ABALONE HALIOTIS TUBERCULATA L. L. M. Lo-
pez and P. Tyler, School of Ocean and Earth Science. University
of Southampton. Southampton Oceanography Centre. European
Way, Southampton. SOU 3ZH, United Kingdom.

Formulated diets have been shown to improve the growth rates
of juveniles and young adults of species of Haliotis. When juvenile
Haliotis tuberculata were fed on formulated diets, and cultured at
18° and 22 °C, our research showed early development of the
gonad and deformation of the shell. Three different diets were
evaluated and the growth rates (shell length and body weight) of
the Juveniles were obtained for each. The first diet was formulated
with fishmeal (FM) as the main protein source, the second was an
abalone commercial (CO) diet containing casein as the main pro-
tein source and the third was a mix of fresh seaweed (Palmaria
palmata and Ulva lactuca. SW) used as a control. Seven-month-
old juveniles (4.16-5.97 mm 14.33-30.12 mg) reared in our labo-
ratory, were fed over a period of seven months and cultured at 15°,
18''. and 22 °C temperature.

During the first three months, the daily growth rates (shell
length and body weight) were similar between formulated diets
(FM and CO). A maximum growth rate of 136 |j.m day"' and
3,091 |jig day"' was attained when cultured at 22 °C. From the
fourth month, the growth rates decreased to 41 (jim day"' and 229
[j-g day"'. At the same time the gonad began to develop in all
animals (FM and CO) cultured at 18° and 22 °C. Whitest growth
rates decreased, the gonad development, was notably more rapid,
showing that a great part of the metabolised energy was diverted to
reproduction. In our study, gonad development started at 1 .09 cm
shell length, and at eleven months old. The first juveniles spawned
at the end of the first year. On the other hand, abalone fed on all
three diets and cultured at 15 °C plus those on the SW diet cultured
at 18° and 22 °C did not show gonad development. Their growth
rates were low and constant throughout the experimental period.

87% of the population presented shell deformation after four
months feeding on CO diet. In this context, a specific nutrient
deficiency may occur when there is an imbalance in the proportion
of that nutrient in an otherwise adequate diet.

524 Abstracts, February 2000

4th International Abalone Symposium. Cape Town. South Africa

THE USE OF ULTRASOUND IN THE TREATMENT OF
SABELLID INFESTATIONS IN SOUTH AFRICAN ABA-
LONE. N. C. Loubser and N. Dormehl, I&J Abalone Culture
Division, PO Box 522, Gansbaai, South Africa.

Gill tissue of Haliotis inidae and different life stages of the
sabellid worm were exposed to ultrasound to investigate any de-
structive effects of micro-cavitation. Live abalone infected with
the sabellid worm were also exposed to varying periods of ultra-
sound treatment to investigate the long-term effect of ultrasound
cavitation on the reproductive rate of the sabellid worm.

Ultrasound treatment for one minute was enough to destroy
isolated sabellid adults in seawater media in a test tube. The feed-
ing crown of the sabellid adult was destroyed after a thirty second
treatment. Ultrasound treatment for one minute was not adequate
to destroy isiolated sabellid larvae, sabellid eggs or the abalone gill
tissue.

Individually marked abalone. 60 in total, were treated either
completely submerged during the ultrasound treatment or partially
submerged with only the shell in the water column during the
treatment. Replicates were exposed for time intervals varying from
1 minute to 10 minutes in one minute increments. The abalone
showed severe stress behavior during all treatments, with two of
the shells coming off during the shell-only treatment. One rnonth
after the treatment, no sabellid eggs or larvae were present in the
abalone shells. There were still sabellid adults present, with the
greatest proportion of the adults having their feeding crowns de-
stroyed. A very small proportion of the adults near the shell edge
retained their normal size and colour and still had active feeding
crowns. After three months, the treated abalone had an average
growth rate of 3.16 nim/mth. whereas the untreated control still
had an average growth rate of 1.32 mm/mth which was similar to
the historical growth rate of the batch, viz. 1 .26 mm/mth.

We speculate that the ultrasound micro-cavitation destroys the
sensitive feeding crown of the worm, either completely or par-
tially, and is therefore interfering with the worm's ability to feed
properly and reproduce. A second treatment may be necessary to
destroy the newly recruited adults which matured from the larvae
and eggs. This research is now being continued on a much larger
scale.

GROWTH AND AGEING OF PINTO ABALONE, HALI-
OTIS KAMTSCHATKANA IN BARKLEY SOUND, BRITISH
COLUMBIA. B. G. Lucas. A. Campbell, B. Clapp, and C;. S.
.laniieson, I-ishcrics and Oceans Canada, Pacific Biological Sta-
tion, Nanaimo, BC. Canada V9K 5K6.

A long-term lag recapture study of Haliotis l<amtschatkana was
conducted at three islands in Barkley Sound on the West Coast of
Vancouver Island. British Cohnnbia between l^'^l and 1998. A

total of 5 627 abalone were tagged, and 742 were recaptured. Time
at liberty ranged from one day to 5 years, with the percent of total
recaptures being 12% for less than one year, 72% for one year,
14% for two years and 2% for more than 2 years. Preliminary
analyses indicated some differences in growth rates between sites
and for different one-year periods, but not between males and
females.

Instantaneous natural mortality rates for pinto abalone were
0. 1 29 at Hankin Island, 0. 1 57 at Turret Island and 0. 1 39 at Demp-
ster Island. The spire ring technique was used to age abalone shells
from Dempster Island and provided an average growth rate of 6.25
mm per year, which was similar to rates determined from tag
recapture growth increments. Von Bertalanffy growth parameters
derived from tag recapture growth increments were most similar to
those derived from age data when each prismatic ring was counted
as one year of growth per ring (simple brown rings were not
counted).

Damage from boring parasites and erosion had destroyed an
average of 60% of the shell spires, limiting the region of the
hori/<intal section of the spire where rings could be counted. Al-
though H. kamtscliatkana appeared to deposit one prismatic ring
per year in British Columbia, further research is needed to deter-
mine the significance of fine rings, brown rings, boring parasites
and erosion on the number of apparent rings.

THE EFFECT OF STOCKING DENSITY. TEMPERATURE
AND LIGHT ON THE EARLY LARVAL SURVIVAL OF
THE ABALONE HALIOTIS ASININA LINN. J. A. Madrones
Ladja, Aquaculture Department. Southeast Asian Fisheries Devel-
opment Center, Tigbauan 5021 lloilo, Philippines.

Newly hatched early veliger larvae of the abalone Haliotis
asinina were stocked at densities of 1000, 3000 and 5000 larvae/I
at low (20-25 C) and high (ambient, 28-30 °C) water temperature
levels, in transparent (light) and black cloth-co\ered (dark) glass
containers. Larvae were reared in UV light-inadiated seawater
until pre-settlement stage. Aeration was not provided during the
20-h incubation period. A 3 x 2 x 2 factorial design with three
replicates per treatment was followed.

The three-way ANOVA showed a significant interaction
among the factors tested. Analysis showed that at a stocking den-
sity of 1000 larvae/I. there were no significant differences between
temperatures, and between light or dark conditions. However, at
densities of 3000 or 5000, significantl)' high survival rales were
obtained at lower temperatures [F < 0,001 ), but no difference was
detected hetucen the light and dark conditions. At high tempera-
tures, better survival rates iP < 0.05) were obtained at a slocking
density of 1000 than at higher densities, and at light than at dark
coiulition. .Xt low temperatures, no significant difference between

4lli Imernatiiinal Ahalonc Syniposiuiii. Cape Town. South Africa

Ahslracrs. February 2000 525

densities or between light and dark conditions was detected.
Analysis of data from the light or dark condition showed thai at
any of these conditions, larval survival was always higher at a
stocking density of 1000 than at other densities. The stocking
densities of 3000 and 5000 larvae/1 were not significantly different
from each other.

Therefore, during incubation of hatch-out larvae of H. asinina
to pre-settlement stage, the optimum stocking density at high tem-
peratures is 1000/1, in a light-penetrable rearing container. But
when reared at higher stocking densities of 3000 or 5000, higher
survival rates can be obtained at temperatures of 20-25 °C, in
either rearing conditions tested.

rate of 2-mg d"' in all size classes while H. tubemilata lost weight
but showed a small amount of shell growth in the early summer.
Respiration was greater during summer for both species and all
size classes. Assimilation efficiency was highest for small and
medium sized abalone of both species, compared to large abalone
(CO. 75% vs 60%) but did not differ significantly (P > 0.05) be-
tween seasons. On an energy basis, respiration, ammonia excre-
tion, and mucus production accounted for 30% ± 2; 37% ± 1 ; and
57% ± 1 of the total energy (joules d~') absorbed by small, me-
dium and large abalone, respectively.

These results suggest that the prevailing conditions in the Red
Sea are more suitable for the culture and growth of H. fiilgens and
H. tiiberciilata.

GUAIACOL, A POWERFUL MODULATOR OF MOLLUSC
SHELL BIOMINERALIZATION. K. Mai and G. He, Aqua
culture research laboratory. Ocean University of Qingdao Qingdao
266003, China.

As a modulator of eicosanoids metabolism and an inhibitor of
calcium flux, guaiacol was examined for its influences on shell
biomineralization in abalone, Haliotis discus hannai. Juvenile aba-
lone (14 mm in shell length) were fed on artificial diet with guai-
acol at 10 mg/kg diet for 100 days. The concentration of calcium
in shells decreased from 38.7% to 32,1%-a level similar to that in
adult shells (55 mm in shell length). The concentration of zinc
decreased from 53.4% to 39.3%: but other elements showed no
significant difference. Compared to the control, the fraction of
calcite increased dramatically from 1.5% to 1 1.5% similar to that
in adult shells (13.2%). Similarly, the fraction of aragonite de-
creased from 93.2% to 85.1%. Compared to the control, the acidic
amino acid content in shell soluble matrix proteins decreased.

EVALUATION OF SEASONAL BIOENERGETICS OF
HALIOTIS FULGENS AND HALIOTIS TUBERCULATA.
S. C. McBride, University of California Sea Grant Extension Pro-
gram. 2 Commercial St. Suite 4 Eureka, California 95501, E.
Rotem, D. Ben-Ezra, and M. Shipgel, Israel Oceanographic and
Limnological Research, National Center for Mariculture P.O. Box
1212. Eilat 88112, Israel.

Bioenergetics (food ingestion and absorption, respiration, am-
monia excretion, mucus production and growth) of the temperate
water abalone species. Haliotis tuberculata. and warm water spe-
cies Haliotis fulgens. were studied under summer and winter con-
ditions in the Gulf of Eilat. Three sizes (mean ± S.D) of H. fulgens
and H. tuberculata (0.2 -I- 0.03 g; 0.5 ± 0.02 g; 1.9 ± 0.1 g) were
cultured for one year (July 1995 to March 1996) where they were
fed ad libitum with the seaweeds Ulva lactuca and Gracilaria
conferta. Growth was highest for both H. fulgens and H. tubercu-
lata during winter (3 to 7 mg DW d"' and 3 to 6 mg DW d~',
respectively). In summer, H. fulgens increased in dry weight at a

THE ROLE OF A RICKETTSIALIKE PROKARYOTE IN
WITHERING SYNDROME IN CALIFORNIA RED ABA-
LONE, HALIOTIS RUFESCENS. J. D. Moore, T. T. Robbins,
and C. S. Friedman, Bodega Marine Laboratory, 2099 Westside
Road, Bodega Bay CA.

Withering syndrome (WS) is a chronic, progressive disease
responsible for mass mortalities in wild populations of black aba-
lone Haliotis cracherodii in southern California since the 1980s.
Although the red abalone, Haliotis rufescens. appears more refrac-
tory to WS, farmers nevertheless suffered severe losses of red
abalone with WS clinical signs during the 1997-1998 El Nino
event. Our studies investigated the roles of a gastrointestinal Rick-
ettsia-Xikt prokaryote (RLP) and elevated temperature in WS. In
one experiinent, 60 red abalone were obtained from a culture fa-
cility with cool (14 °C) ambient water that had not experienced
WS. although animals were known to harbor low-level RLP in-
fections. One half of these animals were maintained at 14.7 °C
(control) and the other half were brought to 18.5 °C (elevated
temperature) to attempt induction of WS. After 220 days, those
held at the elevated temperature showed higher mortality, more
severe signs of WS and more severe RLP infections than those
held in cool water. Signs of WS were strongly correlated with RLP
infection intensity among the elevated temperature animals. To
investigate this relationship under typical farm conditions, 70 red
abalone were sampled from five farms before and during the
1997-1998 El Nifio, and severity of WS clinical signs and asso-
ciated histopathology were strongly associated with RLP infection
intensity. In a separate study, the lack of requirement for physical
contact between abalone for RLP transmission was shown by suc-
cessful transmission to RLP-free red abalone held in separate tanks
downstream from infected abalone. One year after initiation of a
second, ongoing transmission study conducted at 18 °C, RLP-free
red abalone that were injected with RLP-infected gastrointestinal
tissue experienced 80% mortality with nearly all showing signs of
WS and severe RLP infections, while those injected with a 0. 1 (j.ni
filtrate of infected tissue as well as saline-injected and uninjected

526 Ahstracts. February 2000

4th International Abalone Symposium. Cape Town. South Africa

control animals remained healthy. Collectively, these .studies sup-
port the hypothesis that the RLP is the etiologic agent of WS. with
temperature influencing expression of the disease. Cool water may
provide a refuge from the pathogenic effects of the RLP although
it is also possible that .stressors additional to elevated temperature
may influence the occurrence of WS.

HEALTH MANAGEMENT AND DISEASE SURVEIL-
LANCE IN ABALONE, HAUOTIS MIDAE, IN SOUTH AF-
RICA. A. Mouton, Onderstepoort Veterinary Institute. Private
Bag X05, Onderstepoort, 0110, South Africa.

Abalone production units in South Africa have participated in
a health management programme since March 1999. The aim of
the programme is to identify potential threats to the health of the
animals and to determine suitable ways of managing such threats
so as to minimise the impact on production. This aim is achieved
by a comprehensive system of sampling for each production unit.
Samples typically include animals from the weaning and grow-out
sections, as well as eggs, newly hatched larvae and post settlement
larvae. Animals which are seen to be suffering from disease are
also submitted for examination. Methods of examination include
histopathology, bacterial culture and scanning and transmission
electron microscopy. Regular consultation with production man-
agers is an essential component of the programme. Disease sur-
veillance for the entire cultured abalone population is facilitated by
the large number of animals examined together with the wide
distribution of sample sites. By August 1999, approximately 4.^0
weaner and adult animals were being examined each month. This
figure is likely to increase to over 800 towards the end of 1999.
Interesting findings from the programme include the occurrence of
renal coccidia in Haliotis inidac. the presence of an unknown
rickettsia-like organism in the digestive gland, and protozoan para-
sites affecting various sections of the gut. Problems and challenges
experienced by the health management programme include lack of
information on abalone diseases in general and H. inidac in par-
ticular, reliable sample transport over long distances, and stan-
dardisation of processing techniques for eggs, newly hatched lar-
vae and post settlement larvae.

A COMPARATIVE SCANNING ELECTRON AND LIGHT
MICROSCOPY STUDY OK THE EARLY LIFE STAGES OF
THE SOUTH AFRICAN ABALONE, HAUOTIS MIDAE. A.

Mouton and J. F. Puttcrill, Onderstepoort Veterinary Institute,
Private Bag X05, Onderstepoort. 01 10, South Africa.

The early life stages of abalone are often neglected when it
comes to diagnosis of disease. Although mortalities in the hatchery
and nursery account for90';f' or more of the total mortalities diiririL:

the production cycle, the reasons for these losses are not com-
pletely understood. A comparative study of scanning electron and
light microscopy of the early life stages of the South African
abalone, Haliotis midae, was undertaken to determine normal
characteristics for the species. Animals processed for scanning
electron microscopy (SEM) were fixed in 4% gluteraldehyde in 0.2
M sodium cacodylate buffer (pH 7.2 to 7.4). Samples were rinsed
twice in sodium cacodylate buffer, after which they were routinely
dehydrated through an ascending series of ethanols (50. 70. 90, 96,
100, 100, and 100%, 30 minutes per step). The samples were then
critical point dried from 100% ethanol through liquid carbon di-
oxide in a Polaron Critical Point Drier (Watford. England). Dried
samples were mounted onto SEM viewing stubs and sputter coated
with gold. The samples were viewed at 3 to 8 kV acceleration
voltage in a Hitachi S-2500 Scanning Electron Microscope. Ani-
mals processed for light microscopy were fixed in either 4% glu-
teraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2 to 7.4) or
Davidson's fixative. Samples were rinsed twice in distilled water
to remove adhering detritus. A modified double embedding tech-
nique (Feist & Bucke 1983 as described in Austin 1989, Methods
for the Microbiological Examination of Fish and Shellfish ) was
used prior to processing routinely for light microscopy. Sections
were cut at 6 |jim and stained with haematoxylin and eosin. Due to
inaccessibility of SEM. light microscopy is commonly used. Ad-
vantages and disadvantages of both methods are discussed.

EXPERIMENTAL CULTIVATION OF THE KELP ECKLO-
NIA MAXIMA. D. C. Z. Norman. D. W. Keats, and R. J.
Anderson, Botany Department. University of the Western Cape,
P.O. Box XI 7. Bellville. 7535.

Kelps form the basis of a number of commercial products,
including alginate, plant growth substances, beauty products, and
food additives. L'nprocessed kelps are also used as feed in the
abalone maricullure industry. .Alginate has potential for the de\'el-
opment oi biomedical implants, but the product must be of a very
high purity and reproducible quality. Therefore, it may be desir-
able to grow kelp under mariculture conditions to improve alginate
quality and reproducibility of medical implants. Laboratory, lank,
spray and raft cultivation techniques were investigated. The labo-
ratoi'y cultivation of gamelophytes and sporophytes was success-
ful, using enriched seawater medium, and the young sporophytes
have been grown in spray and mist cultivation prior to out-planting
on a raft in the sea. Characteristics of the mist and spray system
thai affect growth are currently being investigated. The growth of
sporophytes on rafts in St. Helena Bay is reported here.

4th International Abalone Symposium. Cape Town. South Africa

Abstracts. February 2000 527

BIOLOGICAL REFERENCE POINTS FOR THE GREEN-
LIP ABALONE (HALIOTIS LAEVIGATA) IN DIFFERENT
HABITATS ACROSS ITS GEOGRAPHIC RANGE. J. L.
O'Loughlin'"" and S. A. Shepherd, 'South Austrahan Research
and Development Institute. GPO Box 120. Henley Beach. Austra-
lia, 5022, ^Australian Maritime College. PC Box 2 1 . Beaconsfield.
Australia. 7270.

The greenlip abalone {Haliolis laevigata) is an exploited sed-
entary benthic gastropod endemic to southern Australia, with a
geographic range from eastern Bass Strait to Cape Naturaliste in
Western Australia. A sub.stantial number of greenlip abalone popu-
lations have been declining over the last 25 years despite manage-
ment techniques such as minimum legal sizes and total allowable
catches which control fishing mortality.

Using aging techniques to estimate growth and mortality, egg
production levels in metapopulations of Haliotis laevigata were
estimated from western, central and eastern populations in the
species' range. Comparisons were made between egg production
in stable and declining populations, with a view to estimating
threshold levels of egg production necessary for sustainable ex-
ploitation. Results confirm previous suggestions that small
metapopulations are more vulnerable than large ones. Threshold
egg production levels of small metapopulations appear to be
around levels of 60% whereas thresholds for larger metapopula-
tions are about 45%. Populations occuning in bays or around
islands that constitute larval traps are more resilient to fishing than
those on low relief, rocky bottoms, distant form shore. Our results
suggest that up to 207f higher levels of egg production need to be
conserved in the latter kind of habitat.

DISTANCE-BASED ABUNDANCE ESTIMATION FOR
ABALONE. R. A. Officer, Tasmanian Aquaculture and Fisheries
Institute. University of Tasmania. Nubeena Crescent, Taroona.
Tasmania 7053, Australia.

Indices of abundance are an important component of stock
assessment models. Unfortunately, conventional attempts to esti-
mate the abundance of abalone are hampered by the patchy spatial
distribution characteristic of abalone. Fine scale mapping of aba-
lone positions was used to evaluate alternative distance-based
abundance estimators for abalone that better accommodate the
aggregated distribution of abalone. Simulated quadrat sampling of
a real abalone population was used to examine the accuracy, bias
and sensitivity of the distance-based approach. Within each quad-
rat, the distances from a randomly selected point and abalone to the
nearest abalone were used as the basis for an overall estimate of
abundance. An iterative randomisation procedure was used to ob-
tain confidence limits about abundance estimates. This ability to
re-sample the observed population reduces the need for the exten-

sive field sampling normally required to narrow the confidence
limits of abundance estimates. The distance-based method was
found to under-estimate consistently the true abundance of the
population and therefore may not be useful as an indicator of
absolute abundance. However, this bias does not diminish the po-
tential of the method as a relative abundance indicator because the
method is sensitive to changes in abundance. Sensitivity to
changes in abundance was examined by simulating the effects of
fishing at varying rates of exploitation above specified size limits.
Simulated reductions at moderate levels of exploitation (10-20%
overall reduction) showed that the method was able to detect
changes in abundance. The ability of the distance-based method to
detect fishing-induced changes in abundance that could occur in
reality is discussed.

STATUS OF THE ABALONE FISHERY BETWEEN 1996
AND 1999 ON ABALONE LOCATIONS OF THE FISHING
CO-OPERATIVE PRODUCTION SOCIETY "LA
PURISIMA", S. C. DE R. L., BAJA CALIFORNIA SUR,
MEXICO. M. Ortiz Quintanilla, G. Lucero M., and J. E. Patron
v., S. C. P. p. "La Purisima". S. C. de R. L. Calle Segunda y Soto No.
2048, Col. Obrera. CP.22830. Ensenada, Baja California. Mexico.

This work describes the status of the abalone fishery (Haliotis
spp) during the commercial seasons between 1996 and 1999 on
abalone diving areas held by the "La Purisima", S. C. de R. L.,
fishing co-operative production society. From the season of 1996,
total quotas officially suggested by National Fishing Institute
(I.N.P-CRIP), and based on the results of an annual survey, were
subject to additional modifications imposed internally by the co-
operative itself as a measure towards improving wild abalone
stocks, through a more rigorous resource management process.

These measures included the aquaculture production and re-
lease of competent green abalone H. fulgens veliger larvae into the
wild, as well as leaving a portion of the recommended commercial
stock as part of the fishery reserve, and the designation of pro-
tected areas. The 1996 abalone catch, with a quota of 3 861 kg
(shelled abalone weight) was 814 kg; as a measure of effort, this
catch derived from 74 fishing trips, with catch per unit effort
(CPUE) data, measured as kg/diving team/day. of 1 1 kg per 12
abalone divers per day.

Up until 1996, two abalone species were included in the fish-
ery, namely H. cormgata Wood and H. fiilgens Philippi. Between
1997 and 1999, catches of H. fulgens were small. However, be-
tween 1997 and 1999. catches gradually increased from 2 029 kg
in 1997 to 3 097 kg in 1999. and with changes in fishing efforts
from 74 to 102 trips in 1999, and the 1997 average of 26.7 kg/
diving team/day increasing to 33.22 kg/diving team/day in 1999.
based on four abalone fishing units. CPUE based on the average
mass of abalone captures per minute was also compared.

528 Abstracts, February 2000

4th International Abulone Symposium. Cape Town. South Africa

ABALONE iHALIOTIS MIDAE) FARMING AND PARA-
LYTIC SHELLFISH POISONING ON THE COAST OF
SOUTH AFRICA. G. Pitcher.' J. Franco.' K. Whyte,' and C.
Viljoen,'* 'Marine and Coastal Management, Private Bag X2,
Rogge Bay. 8012. Cape Town. South Africa. "Instituto de Inves-
tigaciones Marinas. Eduardo Cabello. 6. 36208 Vigo. Spain. 'West
Coast Abalone, P.O. Box 185, Stompneusbaai. 7382. South Africa.
^Jacobsbaai Sea Products. P.O. Box 837. Vredenberg. 7380. South
Africa.

The abalone Haliotis inidae forms one of the oldest fisheries on
the South African coast, with present-day operations including
recreational, subsistence and commercial activities. During the
1990s, land-based farming of this species also developed and has
recently attained coinmercial scale production. In April 1999. rou-
tine monitoring provided evidence of the presence of PSP toxins in
cultured abalone. Subsequent analysis of wild abalone collected
from the West Coast also revealed the unexpected accumulation of
PSP toxins in these non-filter feeding shellfish. Toxicity, as mea-
sured by the mouse bioassay, showed considerable variation be-
tween individual animals, with inaximum values exceeding 1000
(xg STXeq 100 g"'. The observation of PSP toxins in abalone
coincided spatially and temporally with blooms of the dinotlagel-
\iAe Alexandriuin calenella. Toxicity as measured by High Perfor-
mance Liquid Chromatography was notably higher than that mea-
sured by the mouse bioassay. The toxin composition of the abalone
was dominated by saxitoxin and therefore differed significantly
from the toxin profile of ,4. catenelta, indicating either a high
capacity for transformation of PSP toxins by abalone or that A.
catenelta was not the source of the toxin. Investigation of the
anatomical distribution of toxins revealed that they were not
evenly distributed throughout the abalone tissues. The muscular
foot, which contributes substantially to the total weight of the soft
tissues and is the organ marketed for human consumption, makes
a disproportionately low contribution to the total toxin content of
the mollusc. To date, the inability of abalone to detoxify accumu-
lated PSP toxins below the regulator) level threatens the future of
the established abalone fishery and the nev\ly developed aquacul-
ture operations on the West Coast.

CLUES AND QUESTIONS FROM POPULATION MODELS
APPLIED TO THE SOUTH AFRICAN ABALONE {HALI-
OTIS MIDAE) FISHERY. E. E. Plaganyi and D. S. Butter-
worth, Marine Resource Assessment and Management Centre.
Department of Maths & .Applied Maths. University of Cape Town.
Private Bag 7701. Rondebosch. South Africa.

South Africa's commercially important abalone fishery de-
pends on a single species Haliotis midae and is managed by allo-
cating total allowable catches (TACs). with a minimum si/e limit,
in each of seven specific fishing zones (zones A-Gl. Uncertainty

and concern has been expressed regarding the status of the re-
source because several of the zones are subject to particularly high
levels of illegal fishing. The magnitude of the catch taken by the
illegal sector is difficult to esfimate because of the evasive behav-
iour of the illegal fishing community. A population modelling
approach has been used to estimate the magnitude of the illegal
catch as well as to assess the status and productivity characteristics
of the resource. The model applied is a deterministic age-
structured production model. By projecting abundance trends un-
der alternative future catch levels, the model is currently used as a
basis for developing management advice in four (zones A-Dl of
the seven fishery zones. Attention has focused on zone C in par-
ticular and model results suggest that the annual poaching catch
has exceeded the commercial catch in recent years. Moreover,
results suggest that continued depletion of the resource in this and
other zones is likely unless takes by all sectors of the fishery are
reduced in future. This paper summarises current progress in the
application of an age-structured production model basis for man-
aging the abalone fishery, discusses problems associated with the
use of both CPUE and fishery independent indices in the model-
ling process, and offers suggestions for simulating a postulated
recruitment decrease in zones C and D.

STRESS AND WEIGHT LOSS ASSOCIATED WITH HAN-
DLING IN THE BLACKFOOT ABALONE, HALIOTIS IRIS.
N. L. C. Ragg. H. H. Taylor, and J. Behrens. Department of
Zoology. Unixersity of Canterbury. Private Bag 4800.
Christchurch; New Zealand.

During the course of physiological in\ estigations carried out at
the University of Canterbury, it became apparent tliat the simple
act of handling an abalone markedl\ affected the animal's physi-
ological state. Thus we routinely include handling as a control
treatment in experiments; the responses of these control animals
form the basis of this presentation. Adult Haliotis iris, subjected to
standardised handling involving gentle detachment from the tank
bolloni followed by 2 minutes inversion on blotting paper to drain
branchial water before weighing, consistently lost wet weight. The
weight of animals handled repeatedly in this way. at intervals of
0.5 hours or less, continued to fall over 3 hours to a plateau. 7-17'^
below their undisturbed weight. The rate of weight loss varied
directly with handling frequency. When left undisturbed, initial
weight was recovered over several days. Thus, the operational
body weight and volume of an abalone is a function of the fre-
quency of weighing and of the intensity of the associated distur-
bance. Volume reduction of a number of fiuid pools could poten-
tially contribute to the weight loss. e.g. extra-corporeal water be-
tween the mantle and shell, fiuid within the lumen of the kidneys,
crop and other regions of the gut. nuicus release, cellular dehy-

4tli Imernalional Abalone Symposium. Cape Town. South Africa

Ahstrocts. February 2000 529

dration. or extracellular fluid (blood) loss. More than half of the
volume of an undisturbed H. iris is blood (inulin space = 52.2 ±
S.E 3.0 ml/ 100 g wet tissue, or about 31*^ of total weight, includ-
ing shell). Surprisingly, repeated handling caused a 27.5 ± 5.79!^
increase in the concentration of the abalone" s oxygen carrying
pigment, haemocyanin. A tiny fraction of this increase (about 039c
elevation of total haemocyanin) could be attributed to release of
concentrated haemocyanin stored in the vasculature of the left
kidney. The major increase in haemocyanin concentration was
quantitatively consistent with a decrease in blood volume equiva-
lent to the weight lost. We conclude that the principal effect is a
handling-induced ultrafiltration of the blood, perhaps via the kid-
neys. Attention is therefore drawn to the fact that, in addition to the
stress associated with air exposure (desiccation, hypoxia, thermal
shock) and manipulation (possible injury), handled abalone also
endure a substantial alteration in blood composition and volume,
which must profoundly perturb circulatory function, oxygen up-
take and transport to the tissues. The commercial implications of
reduced meat weight and compromised growth and survival will
be discussed, as well as techniques to avoid direct handling of
abalone.

TOXICITY AND TOLERANCE LEVELS OF AMMONIA IN
ABALONE {HALIOTIS MIDAE). K. Reddy-Lopata,' A. L.
Lopata. and P. A. Cook,' 'University of Cape Town. Department
of Zoology. Rodenbosch 7701. South Africa. "Allergology Unit.
Groote Schuur Hospital, Observatory 7925.

Ammonia is the major end-product of protein metabolism.
Therefore, it is important to determine the accurate levels of tox-
icity and tolerance of ammonia in abalone (Halioris midae) to help
improve the water quality in South African abalone farms. Spe-
cifically, this study looks at ammonia tolerance and toxicity levels
of abalone in relation to body size. pH and temperature. Growth
experiments show that high concentrations of ammonia retard
growth.

Toxicity tests revealed that when abalone are exposed to sub-
lethal levels of ammonia, the LC50 value (ammonia concentration
that kills 507r of experimental animals) increases. A possible
mechanism for this increased tolerance of ammonia involves the
so-called stress or heat shock proteins (hsp's). Hsp's are activated
not only by heat but by other physiological stress (such as the
toxicant ammonium chloride used in these experiments). Hsp's are
designated according to their molecular weights. The 70 kilodalton
hsp has been reported in a number of invertebrates and a few
molluscs (limpets and mussels) but not in abalone.

The presence of hsp's were determined using biochemical and
immunological techniques such as gel electrophoresis and immuno
blotting. In addition, species-specitlc low molecular weight pro-
teins in abalone were also investigated. Hsp's are of importance as
they may be indicators of chemical stress and could be utilised to
control water quality in abalone farms.

STARVATION TOLERANCE OF POST-LARVAL ABA-
LONE {HALIOTIS IRIS). R. Roberts and C. Lapworth, Caw-
thron Institute, Private Bag 2, Nelson, New Zealand.

Competent larvae ( 16 days old) were induced to metamorphose
with 2 |jlM GABA. Post-larvae were either fed diatoms (Nitzscliia
longissima) or starved. In Experiment 1, post-larvae were starved
immediately after settlement, for periods of 1, 2, 4, 8, 15, 20, 25,
and 30 days. Starved post-larvae grew relatively well for several
days after metamorphosis (averages of 10.4 and 17.8 p,m shell
length per day after 8 days, for two batches). Subsequent growth
was minimal, averaging 1.7-0.7 txm/day over 6-7 days. There was
no clear relationship between period of starvation and growth rate.
Mean daily growth rate over 20-25 days after return to food
ranged from -15-22 |xm/day.

The duration of starvation had a significant effect on survival.
Survival of post-larvae fed after just 1-2 days of starvation was
90-100'7f after 3 weeks of feeding. Longer starvation periods gave
progressively lower survival rates. Survival probabilities were
standardised to a 22 day period, and the relationship between
survival (in our experimental conditions) and starvation period was
estimated by linear regression (r-square = 0.861. P < 0.001):
Probability of survival to 22 days post settlement = 0.943 -
0.029x. where x is the period of starvation in days.

In Experiment 2. post larvae were fed for 3 weeks after settle-
ment, then starved for 0, 3, 7, 14, or 21 days. Growth rates of
starved post-larvae dropped dramatically, averaging only 5-6 \x.ml
day in the first week (versus 30 (xm/day in controls), and later
declining to zero. Growth resumed within a week following return
to food, but the 14- and 21 -day starvation treatments took two
weeks to reach growth rates comparable to controls. Survival rates
after return to food were strongly influenced by the period of
starvation. The no-starvation controls and the 3- and 7-day star-
vation treatments all had > 70% survival over 5 weeks after return
to food. Survival in the 14- and 21-day starvation treatments was
15-207f. with almost all mortalities occurring in the first week
after return to food.

A REVIEW OF LARVAL SETTLEMENT CUES FOR ABA-
LONE (HALIOTIS SPP.). R. Roberts, Cawthom Institute. Pri-
vate Bag 2, Nelson, New Zealand.

Settlement of abalone larvae involves larval attachment (a re-
versible behaviour) followed by metamorphosis {which involves
irreversible physical changes). Coralline algae induce settlement in

530 Abstracts. February 2000

4th International Abalone Symposium. Cape Town. South Africa

all abalone species tested. The speed/strength of the settlement
response differs among abalone species, and some abalone species
prefer certain coralline species. The settlement-inducing chemicals
from corallines have not been identified. In one case, a GABA-
mimetic peptide is implicated, while in another, halomethanes are
thought to be critical. Corallines are generally regarded as unsuit-
able for use in hatcheries, but their potential use has not been fully
evaluated.

Many abalone hatcheries rely on biofilms to induce larval
settlement. The activity of biofilms appears to increase with their
age. Ungrazed films are generally dominated by fast-growing
benthic diatoms, and settlement on these films is variable and often
low. Few diatom strains are consistently good for settlement, and
strains that are excessively mobile, or form 3-dimensional colo-
nies, can prevent successful settlement. The chemistry of settle-
ment induction by biofilms. and the role played by bacteria, are
poorly understood. Bacteria grown in pure culture have limited
settlement-inducing activity.

Pregrazing by juvenile conspecifics improves the settlement-
inducing activity of a biofilm. The mucus trails from the foot of
grazers may contain chemicals (not identified) that trigger settle-
ment. Alternatively, the mucus or the grazing activity may enhance
the biofilm in ways that favour settlement.

Various pure chemicals induce attachment and/or metamorpho-
sis of abalone larvae. They may bind to larval receptors (e.g..
GABA) or act "downstream" of the receptors (e.g.. compounds
that depolarise membranes or alter levels of cyclic AMP or cal-
cium). None of these chemicals is considered to be a natural settle-
ment cue, and only GABA is used in abalone hatcheries.

The timing and end point of the abalone settlement response
varies in cues, and among abalone species. Cues can combine
synergistically to enhance settlement (e.g.. GABA + diatoms, di-
bromomethane -i- mucus). Cues for attachment are more common
than cues for metamorphosis.

sperm cryopreservation. We induced ripe abalone to spawn, using
the hydrogen peroxide methods. Spawning males were removed
from water to "dry spawn", yielding undiluted sperm (1-1.7 x
10'"/ml). Small-scale (3 to 10 ml) fertilisation assays were used to
cope with the large number of samples required. These small as-
says can produce lower fertilisation rates than large-scale fertili-
sations.

There are two published protocols for the cryopreservation of
abalone sperm. Neither produced satisfactory results with Haliotis
iris sperm, so systematic investigation of freezing methods was
initiated. Various cryoprotectants. equilibration periods, diluents,
cooling rates and sperm densities were tested. In the most success-
ful treatments, sperm were viable and highly motile after cryo-
preservation. but their fertility was reduced. The highest fertilisa-
tion rates obtained with cryopreserved sperm were 20— t0'7r. These
percentages were only achieved at very high sperm concentrations
(10^/ml). Corresponding fresh sperm controls gave lOVe fertilisa-
tion at 10'' sperm per ml. The most successful treatments were
those with high sperm densities. DMSO as cryoprotectant. and
relatively slow cooling.

Flow cytometric analysis of sperm stained with SYBR-14 and
propidium iodide was used to examine sperm membrane integrity.
Many sperm were membrane-intact after exposure to cryopro-
tectants. and after some cryopreservation treatments. However.
"Hobson Sperm Tracker" analysis showed that many cryopre-
served sperm were immolile, and that the motile sperm rapidly lost
their motility after dilution. The swimming \elocity and linearity
were lower for cryopreserved sperm than for fresh sperm. We are
investigating possible causes of low fertility of cryopreserved
sperm, including damage to the sperm's mitochondria, fiagellum
or acrosome.

CRYOPRESERVATION OF ABALONE {HALIOTIS IRIS)
SPERM. R. Roberts,' S. Adams,- ,|. Smith,' A. Pugh,' A.
Janke,' S. Buchanan,' P. Hessian,'* and P. Miadenov," 'Caw-
thorn Institute. Private Bag 2. Nelson. New Zealand, "Dcpt. Of
Marine Science. University of Otago. P.O. Box 5(>. Duncdin. New
Zealand. 'AgResearch. Private Bag 3123. Hamilton, New Zealand.
■"Dept. of Physiology, University of Otago.

We are attempting to develop commercially applicable proto-
cols for cyropreserving gametes and embryos of abalone. mussels
and oysters. This paper reports prcliminars findings from abalone

SELECTING AND EVALUATING MARINE PROTECTED
AREAS FOR ABALONE IN CALIFORNIA. L. Rogers-
Bennett,' P. Haaker,- and K. Karpov,' California Department of
Fish and Game, 'Bodega Marine Laboratory, PO Box 247. Bodega
Bay. CA 94923. "330 Golden Shore. Long Beach. CA 90802.
'19160 S Harbor Dr.. Fort Bragg. CA 9.5437.

Abalone populations have declined dramatically in California,
resulting in the closure of the commercial and recreational fisher-
ies south of San Francisco. Marine Protected Areas (MPAs) have
been proposed as a tool to help restore declining abalone popula-
tions. Halidlis spp. but more information is needed to locate MPAs
and to determine the efficacy of such areas. One simple yet prac-
tical plan for siting MP.As is to analyze historical cumulati\e catch

4th International Abalone Symposium. Cape Town, Soutii Africa

Abstracts. February 2000 531

data to identify areas which once supported large populations of
target species. To do this, we examined spatially explicit catch data
from the commercial fishery (1950-1996) to direct the selection of
MPAs for abalone in California. San Clemente Island was the area
of peak abundance of the now endangered white abalone. Haliotis
sorenseni and the soon to be listed black. Haliotis cracherodi,
pink. Haliotis coniigata. and green. Haliotis fulgens abalone.
making this island uniquely suitable as an abalone restoration
MPA. We also examined fishery independent data which included
abundances and size frequency distributions of abalone inside and
outside MPAs to examine the efficacy of existing MPAs. We
found that the Anacapa Island MPA in the Channel Islands, where
abalone fishing is excluded, supports higher populations of aba-
lone than tlshed sites. Furthermore, remote parts of MPA that are
not under the observation of the reserve manager, failed to protect
pink abalone stocks which declined to zero as did neighboring
fished sites. Therefore, we caution that while abalone abundances
may be higher inside MPAs. effective enforcement of these areas
is critical to their success.

MORPHOFUNCTIONAL STUDY OF THE HEMOCYTES
OF HALIOTIS ASININA. A PRELIMINARY REPORT. S.
Sahaphong,' V. Linthong," S. Apisawetakan," C. Wanicha-
non,- S. Riengrojpitak,' V. Viyanant,' S. E. Upatham,'-'' N.
Kangwanrangsan,' T. Pumthong/ and P. Sobhon," Depart-
ments of 'Pathobiology. "Anatomy, and ''Biology. Faculty of Sci-
ence. Mahidol University. Bangkok. Thailand 10400. ''Department
of Biology, Faculty of Science. Burapha University. Chonburi.
Thailand. 'Coastal Aquaculture Development Center. Department
of Fishery. Ministry of Agriculture and Cooperatives, Prachua-
pkhirikhun, Thailand 77000.

The hemocytes of the abalone Haliotis asinina were studied
using light and electron microscopy in order to describe their main
morphological features and to relate these to their roles in immune
defense. The cells comprise two differentiated types: agranulocyte
or hyalinocyte and granulocyte. The hyalinocyte shows the pres-
ence of several filopodia. a large nucleus with dense chromatin, a
moderate amount of cytoplasm, microfilaments, oval and round
shaped mitochondria with a rather dense matrix, a considerable
amount of rough endoplasmic reticulum, a few cytoplasmic gran-
ules, coated pits and vesicles, phagocytic vacuoles and numerous
large and small vacuoles. Like the hyalinocyte. the granulocyte
processes similar cytoplasmic organelles but in fewer numbers.
and has a peripheral organelle-free zone containing numerous
dense granules of various types. The shape of the granules vary
from round, oval to elongated forms. Several dense granules ex-
hibit crystalloid substructures that show a close relationship to the
plasma membrane.

MEAT QUALITY CHARACTERISTICS OF SOUTH AFRI-
CAN ABALONE (HALIOTIS MIDAE). J. Sales, P. J. Britz,
and T. Shipton, Department of Ichthyology and Fisheries Science.
Rhodes University. P O Box 94. Grahamstown. 6140. South Af-
rica.

Some meat quality characteristics related to post-mortem gly-
colysis, drip loss, cooking loss and objective tenderness were
evaluated in adult wild abalone {Haliotis midae) from the Eastern
Cape coast of South Africa. The onset of pH decline was found to
be 1 7 h after shucking when abalone were kept at 7 °C. while the
corresponding figure was 13 h at 16 °C. From the time of onset of
pH decline till 63 h post-mortem pH decline could be modelled by
the fomiula pH = Bg - 6,(1 - exp(B,t). where B„ is the estimated
pH at time (t) = 0. Bo - B, is the asymptotic minimum pH. and
B, is a measure of the rate of pH decline. Asymptotic minimum pH
were 5.73 ± 0.056 (n = 6) at 7 °C and 5.54 ± 0.105 (n = 6) at 16
°C. while the rates of pH decline were -0.149 ± 0.121 and -0.090
± 0.037 respectively. Drip loss (DL) over time could be described
by the exponential model DL = a -i- b(l - exp(-ct)). where a is the
intercept at time (t) = 0 (set to 0 in the present study), a -I- b is the
asymptotic maximum drip loss and c is a measurement of the rate
of drip loss. No differences (P > 0.05) could be found in either b
(7.1 1 ± 3.001 vs 6.52 ± 1.278) or c (0.031 ± 0.031 vs 0.088 ±
0.075) between storage at 7 or 16 °C respectively. Abalone frozen
at -20 °C immediately after shucking had a higher pH^j^^., (P <
0.05), but a lower Instron value (more tender) than those kept at 7
and 16 °C respectively {P < 0.05), indicating that toughness asso-
ciated with rigor in red meat is absent in abalone meat. However,
this has to be investigated regarding crumbling in fast frozen aba-
lone meat. The present study presented the basic post-mortem pH
decline and drip loss incline of South African abalone (H. midae)
meat that could be used as a baseline for successive studies on
manipulation of these parameters.

POST-SETTLEMENT OBSERVATIONS OF EZO ABA-
LONE, HALIOTIS DISCUS HANNAI IN CONJUNCTION
WITH FLORA. R. Sasaki, Miyagi-Prefectural Sea-Farming Cen-
ter. Maeda. Yagawa-hama. Oshika-chou Miyagi-ken. 986-2402.
Japan.

Natural rates of spat settlement of Japanese abalone {Haliotis
discus hannai) on crustose coralline boulders were measured at
various sites in the bay after larval monitoring. Averaged density
was recorded as 220 inds m~", with 510 + 20 |jim shell-length
along the sea-bottom from 150 m (depth 5 m) to 200 m (depth 7
m) distance from the shore. Daily mortality and growth rates were
respectively calculated as 13% and 38 |jim within a month after
settlement. In some cases, dead shells of ca.500 (xm length were
found in the samples from crustose coralline boulders. These were

532 Abstracts. February 2000

4th International Abalone Symposium, Cape Town, South Africa

regarded as the tlrst critical size caused by a starvation. Small H.
discus hannai juveniles were primarily distributed around the
depth layer of 5-6 m at the outer site and 1-2 m at the inner site
of the bay. which coincides with the lowest distribution of Eisenia.
The appearance of 1 year abalone was in the following propor-
tions: 6% in Eisenia algal forest, 49% in the boundary zone and
25% in the crustose coralline algal area. In conjunction with the
flora, the boundary zone between the Eisenia algal forest and the
crustose coralline algal area is considered to be a substantial site
for larval settlement and spat growing.

inoculated with different densities of the diatom {ca. 100-4 000
cells/mm"). Postlarvae (3-7 per dish) were allowed to graze for
2-3 hours and video recordings were taken to estimate postlarval
size and grazing rates by digital image analysis. Seawater was
changed every second day and postlarvae were measured again
after 6-8 days to estimate growth. Grazing and growth rates of
postlarvae older than 15d increased linearly with biofilm density.
The highest grazing rates for 7 and 60 day-old abalones were 79
and 10 999 cells/postlarva/hour, respectively. The most important
increase in grazing activity occurred between ages 45d and 60d,
when postlarvae reached 1 .5-2.0 mm and started the formation of
the first respiratory pore. Implications for the management of pro-
duction systems are discussed.

LARVAL DEVELOPMENT OF HALIOTIS ASININA LIN-
NAEUS. S. Sawatpeera," E. Suchart Upatham,^ M. Kruatra-
chue,' Y. P. Chitramvong,' P. Sonchaeng,' T. Punithong.' and
J. Nugranad,' 'Department of Biology, Faculty of Science, Ma-
hidol University, Bangkok 10400, "Faculty of Science, Burapha
University, Chonburi 20131, Thailand.

The larval development of Haliotis asinina was ob.served from
fertilization to the formation of the fourth tubules of cephalic ten-
tacle under water temperatures of 25, 28, 3 1 , and 34 °C. The larvae
had 42 stages of development. The time period for larval devel-
opment depended on the water temperature, lasting 65. 49, 41, and
41 hours at water temperatures of 25, 28, 31 and 34 °C, respec-
tively. After settlement, the development of postlarvae through to
the formation of the first respiratory pore was observed under
room temperature (28-35 °C). The velum was shed and the mantle
began to secrete a new shell. Mouth, radula and digestive organs
were developed on the third day after settlement. The heart was
seen on the fourth day. A prominent structure apparent on the roof
of the mantle cavity showed the formation of the ctenidium. The
second pair of epipodium tentacles began to form and the eye
stalks were completely developed by the eighth day after settle-
ment. Between days 9 and 24. the postlarvae increased in shell size
and number of epipodia and tubules on the cephalic tentacles. The
ctenidium was more developed. The first respiratory pore began to
form between days 24 and day 30. depending on the temperature,
diatom type and larval density in the settlement tank.

EFFECT OF BIOFILM DENSITY ON GRAZING RATES
OF HAUOTIS FVLGENS POSTLARVAE. R. Searcy Bcrnal.
L. A. Veicz EspiiK). and C. Anguiaiio Bt'ltnin. Inslituto de lii-
vestigacioncs Oceanologicas, Apartado I'ostal 453, Ensenada
22860. Baja California. Mexico. (rsearcy@faro.ens.uabc.mx)

Grazing rates of Hciliiitis ftili;ciis postlarvae of different ages (7.
15, 30, 45, and 60 days), feeding on ihc cultured diatom Navictila
iiiccrta. were estimated m 10 ml sicnlc plastic ilishcs, previously

ISOLATION AND CHARACTERIZATION OF MICRO-
SATELLITE DNA MARKERS FOR THE TROPICAL ABA-
LONE. HAUOTIS ASININA. M. J. P. Selvamani, S. M. Deg-
nan. D. Paetkau, and B. M. Degnan, Department of Zoology and
Entomology, University of Queensland, Brisbane, Australia.

In abalone aquaculture, marker assisted selection for growth
would enhance industry development. Highly variable microsatel-
lite DNA has been identified as a useful marker in assessing the
level of genetic variation in a population. The high abundance and
ubiquitous distribution of microsatellite loci in the genome make it
also an appropriate marker for identifying quantitative trait loci
and parentage and pedigrees. Microsatellites allow the parents of
superior progeny to be identified in mixed family rearing environ-
ments, as is often the case in abalone. thus enabling .selective
breeding in commercial aquaculture farms. This study describes
the isolation and characterisation of a number of highly polymor-
phic microsatellite loci in the tropical abalone. Haliotis asiniiui and
their utility in identifying parents of individual juveniles. A partial
genomic library of H. asinina. was screened for dinucleotide mi-
crosatellite DNA using a biotinylated (AC),^ primer, cloned and
sequenced. Out of 29 sequences containing microsatellites. 1 2 con-
tained microsatellite motifs and priming sequences for detailed
studies on the natural and cultured population. Using tluorescently
labelled primers. PCR analysis of 30 individuals from Heron Is-
land population demonstrated that 10 of the 12 loci are highly
polymorphic with the number of alleles ranging from 7 to 15. The
polymorphic loci were used to test the parentage of juveniles from
a brood stock of three females and four males. These loci were al.so
used to test their ability to amplify microsatellite loci in other
species of abalone.

4th International Ahalone Symposium. Cape Town. South Africa

Abstractx. February 2000 533

GROWTH OF JUVENILE ABALONE. HALIOTIS FUL-
GENS PHILIPPI, FED WITH DIFFERENT DIETS. E.
Serviere Zaragoza.' A. Mazariegos Villareal,' G. Ponce
Diaz,'"^ and S. Monies Magallon/ 'Centre de Investigaciones
Biologicas del Noroeste (CIBNOR). P.O. Box 128. La Paz. Baja
California Sur. 23000. Mexico, "CICIMAR. IPN. La Paz. B.C.S,
""SEMARNAP. Delegacion en Baja California Sur.

Growth rates of juvenile Halioris fiilgen.s (green abalone).
17.33 ± 2.13 mm shell length and 0.44 ±0.16 body weight, were
evaluated with five different diets over a period of 106 days. Three
diets were based on algae, palm kelp Eisenia arborea. giant kelp
Macrocystis pyrifera. and Gelidiwn robustum: one on seagrass
Phyllospadix toneyi. and one was an artificial diet. Shell length
and body weight growth rates varied between 0.0191 mm day"'
and 1 .5 mg day"' for E. arborea and between 0.046 mm day ' and
5.5 mg day"' for M. pyrifera. Higher specific growth rates (SGR)
in length and weight were determined for M. pyrifera: 0.23 and
0.71% day"' and for the artificial diet: .22 and 0.67% day"'. Sig-
nificant differences between these percentages and the rest of the
diets were found. The highest mortality (1 1%) was in juvenile fed
with the red alga G. robustum. Factors affecting abalone growth
are discussed with special reference to protein percentage of the
diets.

concomitantly with the catch, indicating that divers respond to
declining abundance by reducing effort. However, this behaviour
is not sufficient per se to allow population recovery, which may be
achievable only by extraordinary management measures such as
closure.

A CHRONICLE OF COLLAPSE: THE DYNAMICS OF
TWO OVERFISHED GREENLIP ABALONE POPULA-
TIONS. S. A. Shepherd and K. R. Rodda, South Australian Re-
search and Development Institute, PO Box 120 Henley Beach,
South Australia.

Two populations of greenlip abalone [Haliotis laevigata) in
Backstairs Passage and Avoid Bay respectively collapsed over two
decades of fishing. Annual surveys of the populations over >10
years during the period of collapse showed that in open habitats,
recruitment failed when adult densities fell below about 0.2 m"",
whereas at sites in bays or behind headlands recruitment failed
more slowly. The differential failure of recruitment at both sites
led to strong spatial contraction of the two metapopulations to
remnant sites of higher larval retention. Stock-recruitment curves
for the populations were mainly of the Beverton-Holt form and
showed weak density-dependence. As population densities de-
clined, recruitment variability increased, making the populations
even more vulnerable to overfishing. Estimates of Z, the total
fishing mortality rate, derived by ageing catch samples and doing
a catch-curve analysis, showed little change during the decline and
collapse of the populations. Furthermore, fishing effort declined

SERIAL DECLINE OF THE SOUTH AUSTRALIAN
GREENLIP AND BLACKLIP ABALONE FISHERY: TIME
FOR A REQUIEM MASS OR A REVIVAL HYMN? S. A.

Shepherd and K. M. Rodda, South Australian Research and De-
velopment Institute. PO Box 120. Henley Beach 5022, South Aus-
tralia.

Historical catch information over two decades at the scale of
the metapopulation show that 19 out of 45 metapopulations of the
greenlip abalone (Haliotis laevigata) and 9 out of 45 putative
metapopulations of the blacklip abalone (Haliotis rubra) have col-
lapsed or seriously declined in the Western Zone of the fishery. In
the case of greenlip abalone, populations of low initial productivity
tended to decline at a faster rate than ones with high productivity,
in accordance with the model of Shepherd and Baker ( 1998), and
those on open rocky bottoms of low relief faster than those around
islands or in bays. In the case of blacklip abalone, no pattern of
decline was evident, except that sites of blacklip decline were also
those where greenlip abalone declined. Fishing intensity was
higher on populations close to home ports than on more distant
populations. Fishing effort declined as the populations declined —
not enough to allow the populations to recover, rather, of a suffi-
cient intensity to ensure that they would not recover. As catches of
inshore greenlip populations declined, fishing effort increased on
more distant populations and has already caused the decline in
density of adults to levels that presage recruitment decline, unless
prompt action by management is taken to reduce fishing effort.

Management of multiple populations of two abalone species
subject to different vulnerabilities and different intensities of fish-
ing requires efficient detection of, and rapid response to, overfish-
ing at the metapopulation scale. To facilitate focused research and
timely management responses, we propose five fishery indicators
as warning lights to be applied to every metapopulation; each
indicator triggers an appropriate and unequivocal response of in-
creasing management attention and/or severity. The response to all
five triggered warning lights is to close the population in question
to fishing and establish a recovery plan. For effective management,
industry and management would need to agree in advance on the
indicators and the designated responses. The appropriate indicators
are certain to be species-specific and imply a good understanding
of the species" population biology.

534 Abstracts. February 2000

4th International Abalone Symposium. Cape Town, South Africa

PARTIAL AND TOTAL SUBSTITUTION OF FISHMEAL
WITH PLANT PROTEIN CONCENTRATES IN FORMU-
LATED DIETS FOR THE SOUTH AFRICAN ABALONE,
HALIOTIS MIDAE. T. A. Shipton and P. J. Britz, Department
of Ichthyology and Fisheries Science. Rhodes University. PC Box
94. Grahamstown 6140. South Africa.

As proteins are the most expensive constituents in abalone
feeds, it is necessary to evaluate (hem to produce least cost diets.
Sixteen diets were formulated to contain 347f protein and 6% lipid
and fed to juvenile abalone (initial shell length: 10.6 ± 0.1 mm).
Dietary fishmeal was substituted at 30. 50. 75. or 100% with plant
protein concentrates, and the growth and nutritional parameters
recorded over a 180 day growth period. No significant differences
were found in the growth rates between the control diet (lOO*^
fishmeal) and diets in which 30% of the fishmeal component had
been replaced by either soya or sunflower meals, or torula yeast IP
> 0.05). 50% fishmeal substitutions with either soya meal or spir-
ulina did not effect growth rates (P > 0.05). Replacement of either
75 or 100% of the fishmeal with plant protein sources had a sig-
nificant affect on growth {P < 0.05). Pearson product moment
correlations between dietary lysine levels and either growth rates
or protein efficiency ratios revealed positive correlations (r =
0.77. P = 0.0005: r = 0.52. P = 0.04 respectively), suggesting
that lysine may have been the first limiting amino acid in these
diets. Carcass analysis revealed that dietary protein source had no
significant effect on body composition {P > 0.05).

PROTEIN CONTENT DETERMINES THE NUTRITIONAL
VALUE OF THE SEAWEED ULVA LACTUCA FOR THE
ABALONE HALIOTIS TUBERCULATA. H. DISCUS HAN-
NAI, AND H. FULGENS. M. Shpigel, I. Lupatsch, and A.
Neori, Israel Oceanographic and Limnological Research, National
Center for Mariculture. P.O. Box 1212. Eilal 88112. Israel, and
N. L. C. Ragg, Department of Zoology, University of Canterbury,
Private Bag 4800. Christchurch. New Zealand.

The nutritional value to abalone of Ulvu hictiica L. with dif-
ferent tissue nitrogen levels was studied. The seaweed was cul-
tured at two levels of ammonia-N enrichment. Cultures receiving
0.5 g ammonia-N m^'d"' C'Low-N") yielded 164 g nr'd"' of
fresh Ihalli containing 12% crude protein in dry matter and 12 kJ
g~' energy; cultures receiving 10 g ammonia-N m "d ' ("High-
N") produced 105 g of fresh Ihalli nr"d"' containing 44% protein
and 16 kJ g ' energy. High-N and Low-N algae, and a "standard"
mixed diet of 75% U. lactuca and 25% Gmcilaria cdiiferta (w/w)
containing 33% protein and 15 kJ g"' energy, were fed to juvenile
(0.7-2.1 g) and adull (6.9-19.6 g) Holiolis luhvniilala. H. discus
luinnai. and H. fi<li;ciis in a 16 week feeding trial. Voluntary feed
intakes of the High-N and standard diets were significantly lower
than the Low-N diet in all the cases. Clear differences in perfor-
mance between ircalrnents were founil in the ju\cnile and adull

abalone of both species. Juveniles fed High-N and standard diets
grew significantly faster (specific growth rate of H. tuherculata
was 1.03% day"' on High-N algae compared to 0.72% on Low-N
algae; H. discus liannai grew 0.63% and 0.3% day"' on High and
Low-N algae, respectively) and showed much better food conver-
sion ratios. The nutritional value of Uha lactuca to abalone is
greatly improved by a high protein content, attainable by culturing
the seaweed with high supply rates of ammonia.

BIOCHEMICAL COMPOSITION OF BENTHIC MARINE
DIATOMS USING AS CULTURED MEDIA A COMMON
AGRICULTURAL FERTILIZER. J. A. Simental Trinidad,
M. P. Sanchez Saavedra, and J. G. Correa Reyes, Aquaculture
Department. Centro de Investigacion Cienti'fica y de Educacion
Superior de Ensenada (C.I.C.E.S.E.). Apartado Postal 2732.
Ensenada. Baja California. Mexico. C.P 22800.

Three strains of benthic marine diatoms {Navicula incerta.
Navicula sp, and Amplioni sp.) were grown individually in batch
systems with 101 of nonconventional culture medium formulated
with three common agricultural fertilizers. The quantity and qual-
ity of the biomass produced with the nonconventional culture me-
dia were compared to those obtained with the traditional culture
media "f/2" (Guillard and Rhyther 1962). The aim of the present
work was to obtain a low price and alternative culture medium for
benthic diatoms commonly used for commercial abalone culture.
The quantity of biomass produced did not differ as a result of
medium for each diatom culture. The general trends in biochemical
composition evaluated as protein, carbohydrates and lipids content
of each diatom culture showed, as expected, significant differences
through time ( 10 days). The biochemical composition of Amphora
sp. and N. incerta did not differ as result of the alternative medium,
but the culture of Navicula sp. had significantly higher values for
protein and carbohydrate concentrations, in comparison with the
traditional culture medium. We consider that the chemical com-
position of the non-conventional medium does not limit biomass
production, however, depending on the culture age. the quality of
the biomass used as food for abalone postlarxae can change. The
savincs. in terms of cost of chemicals, ramie between 80 and 90%.

A NOVEL MASS CULTURE SYSTEM FOR BENTHIC DIA-
TOMS. J. .\. Simental Trinidad, M. P. Sanchez Saavedra, J. G.
Correa Reyes, and N. Flores Acevedo, Aquaculture Department.
Centro de Investigacion Cientifica y de Educacion Superior de
Ensenada (C.I.C.E.S.E.). Apartado Postal 2732, Ensenada, Baja
California. Mexico. C.P. 22800.

The production of benthic diatoms as food for abalone postlar-
vac is an important consideration in commercial abalone farms.
Common problems in this field include the economic cost of bio-

4th International Abalone Symposium, Cape Town, South Africa

Abstracts. February 2000 S."^?

mass production, biomass quantity and quality. World wide, several
culture techniques are used for the production of benthic diatoms,
each of which has different culture requirements. The aim of the
present work was to offer a new system for the production of
benthic diatoms. This innovative system was designed using a 201
circular white plastic bucket, covered with transparent polycarbon-
ate plastic lid "Lexan", with two orifices through which aeration
was supplied to allow air circulation and to avoid water conden-
sation and temperature increases. The biomass of benthic diatoms
produced with this novel system was compared with that from two
other classic systems. We obtained a higher biomass production
and a reduction in the culture time. Other important considerations
are the lower requirements of culture volume, culture area and
cost. This system was experimented with to produce Nariciila
incerta and Amphora sp. which are commonly used as food for
abalone postlar\'ae.

MOLECULAR TOOLS FOR COMPLIANCE ENFORCE-
MENT—THE IDENTIFICATION OF SOUTHERN HEMI-
SPHERE ABALONE SPECIES FROM ABALONE PROD-
UCTS. N. Sweijd,' B. Evans,-' N. G. Elliott.' and P. Cook,'

'Department of Zoology, University of Cape Town, South Africa,
"School of Zoology, University of Tasmania. Australia, 'CRC for
Aquaculture, CSIRO Marine Laboratories, Hobart, Australia.

Abalone poaching remains a serious threat to the sustainable
utilisation of abalone stocks and presents a diftlcult problem to
resource managers and compliance officials alike. In South Africa,
Australia, New Zealand, Mexico, the USA and Canada, abalone
poaching continues to varying degrees with reported cases involv-
ing significant quantities of abalone in terms of biomass and value.
Due to over-exploitation, many abalone stocks are in decline. In
the USA, one species is commercially extinct and others are threat-
ened, resulting in the closure of the commercial abalone fishery.
Several major abalone producing countries have commercial and/
or recreational fisheries for more than one abalone species. In
many cases, the species distributions traverse regional and even
international boundaries. Thus, different authorities have jurisdic-
tion over a species" range, leading to different regulations (e.g.,
season and size) applying within that range. To complicate these
issues, once the animals have been processed, morphological iden-
tification of the species might be impossible, thereby creating an
opportunity for alleged poachers not only to dispute jurisdiction.
but also species identity.

Of the approximately 25 exploited and potentially exploitable
species globally. 1 1 occur within the waters of South Africa. New
Zealand and Australia. The need for a non-morphological method
to distinguish between these species has led to the development of
a suite of molecular markers to footprint these species genetically.
PCR-RFLP tests, the target between 1 25 and 300 bp fragments of
the lysin gene and the mitochondrial cytochrome oxidase I. cyto-

chrome oxidase 2. and 16s RNA have been developed. In combi-
nation, these markers can consistently distinguish between Haliotis
iniJae. H. spadicea (South Africa). H. rubra. H. conicopora. H. roei,
H. laeviagata. H. scalaris. H. assinbia (Australia). H. iris. H. au.stra-
lis. and H. Virginia (New Zealand). The development of these markers
will contribute to more effective compliance enforcement. The
conserved nature of the genes selected allows for further expan-
sions of this approach to include all exploited abalone species in
order to aid compliance in an increasingly global abalone market.

STUDIES ON PHAGE CONTROL OF PUSTULE DISEASE
IN ABALONE HALIOTIS DISCUS HANNAI. L. Tai-wu, Ma-
rine Biotechnology Key Laboratory. Ningbo University, Ningbo,
31521 1. China. J. Xiang and R. Liu, Institute of Oceanology. The
Chinese Academy of Sciences. Qingdao. 266071. China.

The isolation, purification and propagation of the phage of
Vibrio fluvialis-U were studied. The phage was isolated from 12
different water samples, using the normal isolation procedure. The
Vibrio fliivialis-U can grow well on STA (seawater. tryptone and
agar) medium, which was used as a host and test culture. The
plaques were obtained by the agar bilayer method. Concentrated
phage suspensions were obtained from plates by washing them
with 2% NaCI solution (e.g.. add 4ml 2% NaCI solution into each
plate); they were then put into a refrigerator at 4 °C overnight. The
phage suspensions from the plate were added into a 5ml tube. The
ho.st bacteria were removed by centrifugation at 8000r/min. filtered
and then purified and propagated by picking the single plaque
repeatedly. The bacteria Vibrio fluvialis-ll can be split using the
phage at a high concentration. The effect of phage controlled pus-
tule disease of abalone by muscular injection and infection of the
wound in the abalone foot can raise abalone survival rate by up to
50%. Electron microscopic examination of the material taken from
the plaques of the phage show that they contained simultaneously
two forms of phage particle, with large and small heads, while
their tails were morphologically identical. Numerous successive
passages of the material taken from a single plaque did not allow
the separation of small and large phage particles.

MORPHOLOGICAL CHANGES IN THE RADULA OF
ABALONE, HALIOTIS DISCUS HANNAI AND HALIOTIS
IRIS, IN RELATION TO THE TRANSITIONS IN THEIR
FEEDING. H. Takami,' T. Kawamura,' R. D. Roberts," and Y.
Yamashita,' 'Tohoku National Fisheries Research Institute.
3-27-5 Shinhama. Shiogama. Miyagi 985-0001. Japan. "Cawthron
Institute. Private Bag 2, Nelson. New Zealand.

The radula morphology of Haliotis discus hannai and H. iris
were examined by SEM from larval to adult stages. The overall
length of the radula increased lineariy with shell length (SL) in

536 Abstracts. February 2000

4th International Abalone Symposium. Cape Town, South Africa

both species. The radula of competent H. iris larvae ( 160 days old)
contained -10 transverse rows of teeth. The number of rows in-
creased rapidly to 26-28 by 10 days post-settlement (533 ± 1 1 p-m
SL; mean ± SE). Six days post-settlement (458 ± 10 |xm SL; mean
± SE). H. discus hamuli had 20-26 rows. The number of rows
remained at 25-30 throughout the remainder of the post-larval
period (over -500 p,m SL). and started increasing again at -4 mm
SL. Radula width also increased linearly with shell length, due to
an increase in the number of teeth per row and in the width of
individual teeth. Marginal teeth were added steadily from just one
pair per row in competent larvae to -60-80 pairs per row in the
adult. For both species, post-larvae <-l mm SL contained only the
two pairs of lateral teeth (LI, L2) present in the larval radula. An
additional 3 pairs of lateral teeth (L3-L5) were added progres-
sively between 0.9 and 1 .9 mm SL in H. discus haiiiiai. and be-
tween 1.0 and 1.7 mm in H. iris. The L3-L5 teeth became longer
than the central teeth (R, LI-L2) as abalone grew above 1.5 mm
SL, and the space between adjacent rows of teeth increased. Post-
larvae <1 mm SL had highly curved rachidian and lateral teeth
with clearance angles around or below zero, whereas larger post-
larvae had positive clearance angles. These developinents suggest
that the teeth of post-larvae <1 mm SL function as "scoops" that
slide across the surface, collecting small diatoms and fine, loose
particles. The radulae of post-larvae <1 mm SL become more
suitable for collecting larger particles and gouging feeding sub-
strata. This pattern is consistent with the transitions in their feeding
from microbial to macroalgal diets, and the improved ability of
larger post-larvae to ingest large diatom cells.

AVAILABILITY AND DIETARY REQUIREMENTS FOR
PHOSPHORUS IN JUVENILE ABALONE, HAUOTIS DIS-
CUS HANNAI INO. B. Tan and K. Mai. Aquaculture Research
Laboratory, College of Fisheries, Ocean University of Qingdao,
Qingdao 266003. P. R. China.

Two experiments were performed to determine the availability
and dietary ret|uircmcnls for phosphorus in juvenile abalone. Hali-
olis discus liiininii. Abalone juveniles ol sintilar size were distrib-
uted in a single-pass, How-through system using a completely
randomized design. Abalone were hand-fed once daily at 17:00,
with the appropriate diets in excess. The feeding trials were run for
120 d. In Hxp. I, the availability of phosphorus tojuvenile abalone.
Haliolis discus luimuii from primary, secondary and tertiary cal-
cium phosphate, primary sodium or potassium phosphate sepa-
rately or in combination was determined in a 1 2()-day leeding trial.
The availability of dietary phosphorus was evaluated based on the
growth rates of abalone, chemical analysis of the shells and soft
bodies, and apparent absorption of dietary phosphorus from the
digestive tract. The results indicated that among these inorganic

compounds, primary calcium, potassium and sodium phosphates
separately or in combination could be utilized effectively by Ju-
venile abalone as dietary phosphorus sources (availability ranging
from 87-97%). However, secondary and tertiary calcium phos-
phates were found to be low in availability, the values being 45%
and 77%, respectively.

In Exp. 11, five semi-purified diets were formulated to provide
a series of graded levels of total dietary phosphorus (0.23-1.98%)
from monobasic potassiuin phosphate (KH^POj). The survival,
soft-body to shell ratio (SB/S ratio), and calcium levels in whole-
body (WB). soft-body (SB) and shell (S) were remained constant,
regardless of dietary phosphorus level. However, the weight gain
rate (WGR), daily increment in shell length (DISL), muscle RNA
to DNA ratio (RNA/DNA ratio), carcass levels of lipid and pro-
tein, soft-body alkaline phosphatase (SBAKP), and phosphorus
concentrations of WB, SB and S were significantly (ANOVA, P <
0.05) affected by the dietary phosphorus level. The dietary phos-
phorus requirement of the abalone was evaluated based on WGR,
DISL. and RNA/DNA ratio respectively, by using the second-
order polynomial regression analysis. Based on these criteria,
about 1.0-1.2% total dietary phosphorus, i.e. 0.9-1,1% dietary
available phosphorus, is recommended for the maxiniuni growth of
the abalone.

Zn AND Fe IN THE FORMS OF METHIONINE CHELA-
TION OR SULPHATES AS SOURCES OF DIETARY MIN-
ERALS FOR JUVENILE ABALONE, HAUOTIS DISCUS
HANNAI INO. K. Mai and B. Tan. Aquaculture Research Labo-
ratory. College of Fisheries. Ocean University of Qingdao,
Qingdao 266003. P. R. China.

Two feeding experiments were conducted to determine the di-
etary mineral (zinc and iron, respectively) requirements of juvenile
abalone. Haliolis discus lumnai. with amino chelated forms (zinc
methionine and iron methionine, respectively) and inorganic forms
(zinc sulfate and iron sulfate, respecti\ely) as the mineral sources
and to compare the bioavailability of the two forms oi mineral,
using a premium quality diet based on casein-gelatin as the protein
sources. In Exp. 1, 13 semipurified experimental diets containing
graded levels of dietary zinc (5.6-84.6 mg zinc/kg) provided as
either ZnMet or ZnSOj were fed to juvenile abalone in triplicate
for 16 weeks. The results showed that the growth rate of the
abalone. soft-botl> alkaline phosphatase activity and soft-body
zinc concentrations were significantly (ANOVA. P < 0.01) af-
fected by dietary treatment and responded in broken-line models to
increases in dietary zinc levels from the luo zinc sources. The
requirements of dietary zinc as determined h\ broken-line regres-
sion analysis were: 16-18 mg/kg with ZnMet as the zinc source,
and 35 mg/kg for ZnSOj. This experiment also showed thai the

4th Internationa] Abalone Symposium. Cape Town. South Africa

Ahslracls. February 2000 .537

bioavailability of dietary zinc with ZnMet as the zinc source is
approximately 3 times as high as that with ZnSOj as the zinc
source to juvenile abalone. Haliinis discus Ininiuii Ino.

In Exp. II. experimental diets containing graded levels of di-
etary iron (24.9-212.7 mg iron/kg) provided as either FeMet or
FeSOj were fed to juvenile abalone in triplicate for 16 weeks.
Abalone fed the basal diet without iron supplementation exhibited
significantly (ANOVA. P < 0.0? I lower survival rates and carcass
protein {9c) than did the other groups. The growth rate and soft-
body iron concentration of the abalone were significantly
(ANOVA. P < 0.01 ) affected by dietary treatment and responded
in broken-line models to increases in dietary iron levels from the
two iron sources. The requirements for dietary iron determined by
broken-line regression analysis, was recommended to be 65-70
mg/kg with either FeMet or FeSOj as the iron source. This ex-
periment also showed that the bioavailability of dietary iron with
FeSOj as the iron source is as high as that with FeMet as the iron
source for juvenile abalone. Haliotis discus hannai Ino.

SOUTH AFRICAN FISHERY INDEPENDENT ABALONE
SURVEYS. R. J. Q. Tarr, P. V. G. Williams, A. J. Mackenzie,
E. Plaganyi, and C. Moloney, Marine and Coastal Management,
Private Bag X2. Rogge Bay. Cape Town. South Africa.

New fishery independent abalone surveys (FIAS) were initiated
in 1995 to provide an unbiased index of relative abundance of
abalone (Haliofis iiiidae) for each fishery management zone. Den-
sity data from previous surveys were used to calculate the effect of
varying transect length and transect numbers on the coefficient of
variation (CV). Given constraints over available diving time, a
survey design was chosen that would yield an expected coefficient
of variation of 23 to 26'7f. This required twenty evenly spaced
GPS-located diving stations to be surveyed annually per major
fishing zone. Additional transects were swum in smaller fishing
zones and marine reserves. The coastline of each fishing zone
ranged from 15 km to 30 km in length. The transects are stratified
in a 4m to 6m depth range, and each station comprises a 30m by
2m transect swum perpendicular to the coastline, by two divers. In
addition, four "deep" stations (6 m water depth), with 50 m
transects, are swum per zone. A total of 110 shallow and 14 deep
stations are therefore swum each year.

Results have shown declining trends in abalone in all the major
fishing zones since 1996. Variance has. in general, been within the
predicted ranges. These data are being incorporated into age-
structured models on which resource inanagement decisions are
based.

THE SOUTH AFRICAN ABALONE (HALIOTIS MIDAE)
FISHERY: A DECADE OF CHALLENGES AND CHANGE.
R. J. Q. Tarr, Marine and Coastal Management. Private Bag X2.
Rogge Bay. Cape Town. South Africa.

The South African abalone Hcdiotis midae fishery is now 50
years old. The commercial sector has recently been consolidated
into one category, rights holders, who may both dive and market
their catch. Previously divers and processors were separate enti-
ties. A new overall Total Allowable Catch (TAC) has been initi-
ated, which is 693t (whole mass in shell) for the 1999/2000 season.
This includes a commercial TAC of 500t. a new "subsistence"
TAC of 45t, and a recreational TAC of I48t. Poaching is a major
problem, and large volumes are being taken, of which a high
proportion are sublegal size abalone. This has resulted in serious
reductions in TAC in one zone from 150t to I5t. In addition,
movement of rock lobster y((.v;(.v lakmdii into two of the four major
TAC zones has interrupted the normal recruitment cycle of H.
midae due to lobster predation on sea urchins Parechinus angulo-
sus. on which juvenile abalone depend for protection.

Intensive fishery-independent diving surveys are underway.
Co-management and ranching pilot projects have been initiated.
Modelling of the resource dynamics per zone is being carried out
using an age structured production model. Due to the combined
effect of poaching and ecological changes, the prognosis for the
resource is now poor, with future reductions in TAC likely.

A PARAMETER ESTIMATION MODEL FOR GREENLIP
ABALONE {HALIOTIS LAEVIGATA) POPULATION DY-
NAMICS. N. A. Taylor (nee Dowling), R. McGarvey, and S. J.

Hall, School of Biological Sciences, The Flinders University of
South Australia. G.P.O. Box 2100. Adelaide 5001. South Austra-
lia, Australia.

The degree of abalone aggregation has been found to be a key
factor affecting greenlip (Haliotis laevigata) population sustain-
ability. Larger aggregations contribute most to fertilisation suc-
cess, yet are also most vulnerable to fishing. We have developed a
deterministic model of the fishable population, structured with
respect to aggregation size and incorporating a revised catch equa-
tion and matrix of re-aggregation probabilities.

The modified catch equation, which describes catch as a func-
tion of effort and catchability as a function of aggregation size, was
derived from 1998 and 1999 field data. Catchability parameters
were allowed to vary freely in the model and were fitted using
historical catch by-numbers. The revised catch equation was in-
corporated into a cohort equation for abundance with an annual
time step.

538 Abstracts. February 2000

4th International Abalone Symposium. Cape Town, South Africa

Recruitment (in terms of growth to the legal minimum length)
was allocated equally among all cluster sizes, in accordance with
results from field data, and was allowed to vary freely in the
model. Fitted recruitment values suggested a high degree of den-
sity dependence among the fishable biomass, such that recruitment
to the fishable biomass was suppressed when existing numbers
were high.

Fitting the model to aggregation distributions from four years
showed that recruitment alone did not adequately explain the ob-
served shift into larger aggregation sizes in the absence of fishing.
Re-aggregation was therefore incorporated in the model using a
matrix of probabilities for individuals to move into any cluster size
given their initial cluster size. The matrix was applied to the cohort
equation output. Probabilities were assumed to follow a left-
truncated generalised Poisson distribution.

This is the first model developed for abalone that is structured
in terms of aggregation size. Moreover, the model formulation
incorporates novel but realistic biological features that yield a
better fit to empirical data. As such, this model provides a basis for
assessing alternative management strategies in terms of their effect
on a factor that has been shown to be critical for population sus-
tainability.

SIMULATIONS OF RANDOM FISHING BEHAVIOUR AS
AN INDEPENDENT TEST FOR ACTIVE TARGETING OF
GREENUP ABALONE (HALIOTIS LAEVIGATA) AGGRE-
GATIONS. N. A. Taylor (nee Dowling), S. J. Hall, and R. Mc-

Garvey. School of Biological Sciences, The Flinders University of
South Australia, G.P.O. Box 2100, Adelaide 3001, South Austra-
lia, Australia.

Aggregation patterns, analysed using multi-dimensional scal-
ing analysis (MDS), and diver feedback, have indirectly suggested
that divers actively target large (cluster size >20) aggregations of
greenlip abalone (Haliotis laevigata). However, no independent
analyses have been conducted to test the hypothesis that observed
aggregation distributions may occur purely as a result of random
fishing. We present the results of three Monte Carlo simulations of
diver behaviour, where each simulation tested a different form ot
random fishing.

In the least random strategy, each individual abalone had an
equal probability of capture, but once an individual was selected,
the fishable proportion of the aggregation (cluster) in which it was
found was also removed. Thus, larger aggregations had a higher
probability of capture. The second, intermediate strategy assigned
an equal probability of capture to the fishable proportion of each
aggregation. The third, most random strategy ignored aggregation
structure and randomly selected and removed individuals.

Aggregation data from I'ield surveys conducted immediately

prior to commercial fish-down experiments were used as input to
the simulations. The output was compared, using Chi-Squared
analyses, to the aggregation distributions from surveys undertaken
immediately following the fish-downs. The results showed that the
simulated aggregation distributions were significantly different to
field survey distributions for all three strategies. We conclude that
observed patterns in aggregation structure did not result from a
random fishing strategy, and that divers do indeed target larger
aggregations.

CLIMATE VARIABILITY, KELPS, AND THE SOUTHERN
CALIFORNIA RED ABALONE FISHERY. M.J. Tegner,
P. L. Haaker, K. L. Riser, and L. I. Vilchis, Scripps Institution of
Oceanography, University of California, San Diego, La Jolla, Cali-
fornia 92093-0201, USA.

Declines in landings in Southern California abalone fisheries
and the eventual collapse of many stocks over the last two decades
coincided with a period of greatly increased environmental vari-
ability. This included massive storms, an increase in the frequency
of warm-water El Nifio events after 1977, and an interdecadal-
scale increase in sea surface temperatures. Kelp populations may
be decimated by .severe storms or warm water. Because of the
strong inverse relationship between nitrate availability and water
temperature, temperature is a good indicator of nitrate availability
or stress; kelp growth ceases in warm nutrient-depleted water,
tissue decays, and standing stocks may be greatly reduced. Aba-
lones are directly affected by the availability of the drift kelp on
which they feed, anomalously-warm temperatures may affect re-
production, and altered current patterns may affect larval dispersal.
Because water temperature varies with location in Southern Cali-
fornia and each of the five exploited species has its own thermal
preferences, we chose to evaluate the role of environmental vari-
ability on red (Haliotis rufescens) abalone populations off three
northern Channel Islands spanning a temperature gradient. We
evaluate evidence for poor abalone growth and reproduction dur-
ing El Nino events, water temperature anomalies, and monthly
aerial survey data of giant kelp (Macrocystis pyrifera) canopies.
The severity of El Nino disturbances and long-term changes in
kelp standing stocks both correlated with the temperature gradient.
Despite major long-term changes in kelp populations on the warm-
est island, the time scale of the decline in abalone landings pre-
dates the decline in kelps. The subsequent collapse of many popu-
lations, however, and especially the recovery of these depleted
populations, may be directly related to kelp declines. Southern
California abalones evolved in this disturbance regime, but the
combination of extended periods of increased environmental vari-
ability with intense fishing pressure may have led to the loss of
local populations, especially in warmer areas.

4th International Abalone Symposium. Cape Town. South Africa

Abslracts. February 2000 539

ABALONES AND SEA URCHINS: BIOLOGICAL AND
FISHERIES INTERACTIONS. M. J. Tegner, Scripps Institu
tion of Oceanography. University of CaUfornia. San Diego, La
Jolla. California 92093-0201. K. A. Karpov and P. Kalvass, Cali-
fornia Department of Fish and Game. 19160 S Harbor Dr.. Fort
Bragg. California 95437.

Abalones and sea urchins share similar food and habitat pref-
erences, and both are subject to fisheries. Here we contrast com-
munity changes under different fishing regimes in Southern and
Northern California to consider the ecological interactions of red
abalone (Haliotis rufescens). and red (Strongylocentrofus fran-
ciscanus) and purple (S. Purpuratus) sea urchins and their roles in
kelp forest community structure. In Northern California, a well-
managed recreational abalone fishery allows continued high abun-
dances of red abalone. In contrast, a short-lived red sea urchin
fishery dramatically reduced stocks of that species. In Southern
California, abalone fishing was poorly regulated and many popu-
lations have collapsed, but higher rates of red urchin recruitment
have sustained red urchin stocks. Purple sea urchin fishing is mini-
mal in both areas, and these urchins may have been released from
competition with red urchins and abalones. Here we compare
changes in abundance and size-frequency distribution of these spe-
cies in areas of each region open and closed to fishing, to evaluate
the evidence for competition among the three grazers. Aerial pho-
tos of kelp canopies in Northern California during the period of
intense red sea urchin removal strongly suggest increases in avail-
able food, as well as space, as adult abalones and purple sea
urchins increased in abundance. We consider alternative explana-
tions for these data, and discuss an ecosystem approach to man-
agement of these valuable resources.

ULTRASTRUCTURE OF NEUROSECRETORY CELLS IN
THE CEREBRAL AND PLEUROPEDAL GANGLIA OF
HALIOTIS ASININA LINNAEUS. A. Thongkukiatku, M.
Kruatrachue, E. Suchart Upatham, P. Sobhon, C. Wanicha-
non, Y. Chitramvong, and T. Pumthong, Department of Biology.
Faculty of Science. Mahidol University. Rama VI Road. Bangkok
10400. Thailand.

The ultrastructures of neurosecretory cells (NS,. NS,, and NS,)
in the cerebral and pleuro-pedal ganglia contained a euchromatic
nucleus with a distinct nucleolus. The cytoplasm contained the
usual organelles: RER, Golgi bodies, mitochondria and polyribo-
somes. There were two types of neurosecretory granules in the NS,
of cerebral ganglia: type 1 were large osmiophilic membrane-
bound granules and type 2 were small, electron-dense spherical
granules. The NS, of the pleuro-pedal ganglion contained only one
type of small electron-dense spherical granules. The NS, of the

cerebral and pleuro-pedal ganglia had a nucleus that contained
blocks of heterochromatin resembling a clock-face pattern. The
cytoplasm contained the usual organelles, similar to those of NS,.
There was only one type of neurosecretory granule in the NS,. In
the cerebtral ganglia, the NS, contained large membrane-bound
granules with a crystalline structure. The NS, of the pleuro-pedal
ganglion contained small electron-dense spherical granules. NS,
were smaller than NS, and NS,. The nucleus had a lace pattern of
heterochromatin. There were fewer cytoplasmic organelles than in
NS| and NS,. The NS, of the cerebral and pleural ganglia con-
tained similar neuro-secretory granules. They were composed of
aggregates of dense osmiophilic globules of various sizes.

M. Tokley, Abalone Industry Association of South Australia Inc.,
10 Alma Court, Flagstaff Hill, South Australia 5159.

The move from open access to limited entry, then to output
controls and minimum size limits changed the way South Austra-
lian abalone divers operate to extract the Total Allowable Catch.
This output/size limit control system used to manage the fishery to
ensure sustainability of the resource, motivated divers to minimize
their efforts and maximize catch efficiencies.

Industry divers restructured the "Le Mans" type race attitude
and method of operating to a more mature and relaxed system with
which to harvest abalone. In turn, the methods, techniques and
periods during which abalone are harvested have also changed.
Abalone divers concentrate on areas where catch per unit effort is
maximized. This reduces the amount of time spent conducting
fishing operations, which in turn, allows juveniles to replace the
larger abalone taken and for stocks to re-aggregate to spawn. The
fact that there is a quota and a minimum size limit at which
abalone can be taken is sufficient to ensure that stocks are not
decimated completely.

Industry believes that indices that can be used to measure the
health of the stocks are CPUE, and the level of stock abundance of
the fishery. Industry acknowledges stocks could be reduced to a
level of eventual collapse, but only where size limits are not im-
posed to ensure next year's breeding stocks are available to help
replenish those harvested. However, industry recognizes that there
are areas in the fishery that were depleted through over exploita-
tion during the open access years, and, up until the introduction of
quotas, prevented divers from taking more than their fair share of
the resource. These areas produce only ten per cent of the amount
taken from the fishery each year, and thus will be left alone to
recruit under natural means.

Industry has embarked on a program to relocate healthy aba-
lone from good grounds to those grounds with low abundance and
stunted abalone in the hope that they will reproduce and provide
valuable breeding stock for future harvestable abalone populations.

540 Ahslnicls. February 2000

4th International Abalone Symposium. Cape Town. South Africa

FIRST CULTURE EXPERIENCES OF HALIOTIS COC-
CINEA CANARIENSIS IN A BIOFILTER SYSTEM. P. H.
Toledo, R. Haroun, H. Fernandez Palacios, M. Izquierdo, and
J. Pena. Institute Canario de Ciencias Marinas (ICCM), Ap. 36
35200, Teide, Las Palmas. Islas Canarias, Spain.

Haliotis coccinea canariensis is an endemic subspecies from
the Canary Islands. Its highly appreciated flavour has led to it
being overfished along the Canarian coasts. The present experi-
ments were aimed to produce cultures of W. coccinea camirieusis
in an integrated biofilter system. Sixty abalone specimens of av-
erage shell length 4.2 cm were carefully collected from rocky
shores by hand and scuba diving. After being sexed and marked,
they were kept under natural light conditions in three indoor cir-
cular tanks of 2000 1 and provided with constant seawater tlux and
aeration. They were fed with four species of algae: Ulva ri)>ida.
Grateloupia cUchotoma. Codiiiiu taylorii. and Cystoseira hiimiUs.
produced in the biofilter system of the aquaculture experimental
plant in the ICCM. Individual size (shell length and width) and
weight were recorded on a monthly basis and spawning was in-
duced in the late spring with U.V. light, seawater. hydrogen per-
oxide and TRIS solution. Abalone grew well with two of the
.selected algal species, namely Ulva rlgiJa and Grateloupia di-
chotoma. although the former was better accepted and more effi-
cient in promoting growth. Induced spawning was successful, and
after two months of feeding with benthic diatoms, settled juveniles
started feeding on macroaljjae.

THE DIGESTIBILITY OF RAW, AUTOCLAVED AND
PHYTASE TREATED LEGUMES IN GREENLIP ABA-
LONE, HAUTOIS LAEVIGATA. M. E. Vandepeer, P. W.
Hone, R. J. van Barneveld, and J. N. Havenhand, SA Research
and Development Iiislilule Aquatic Sciences Centre. PO Bt)x 120,
Henley Beach. South Australia. 5022.

In this study we determined the apparent digestibility of nutri-
ents in field peas, I'aba beans, yellow lupins and vetch in the
greenlip abalone. Halinlis laevigata, and assessed whether auto-
claving or the addition of phytase improveil the digestibility of
nutrients in these legumes. Fifteen isonilrogcnous diets, consisting
of one of three different treatments of the following legumes: field
peas [Pisiiin sativum): yellow lupins (Liipiniis liiieus): faba beans
{Vicia faha) and vetch (Vicia saliva), were formulated. The three
treatments of each legume were raw. raw plus the en/ymc phvlase.
and autoclaved. De-fatted soytlour served as a control. All le-
gumes (whole seed) were ground in a hammer mill and included as
the sole protein source of each diet. Each diet was fed lo four
different replicate tanks of 80-100 juvenile greenlip abalone. Hali-
otis laevigata (shell length 40-60 mm. 70 g wet weight). Faeces
were collected each ilay by setllement and the apparent digestibil-

ity of gross energy, protein, amino acids and phosphorous in each
diet was calculated using chromic oxide as the inert indicator (0.5
%). With respect to N digestibility for the untreated legumes, lupin
> soyflour = beans > peas = vetch. Gross energy and dry matter
digestibility had similar patterns, with lupin = soyflour > beans >
peas = vetch. In general, autoclaving had a negative effect on
digestibility, significantly decreasing the digestibility of all amino
acids and protein from all legumes. Gross energy digestibility
decreased for both soytlour and lupins, but increased for peas and
vetch after autoclaving. The gross energy digestibility of beans
was unchanged. The addition of phytase significantly increased the
digestibility of phosphorous from the lupin diet only (84—91%).
Strangely, the digestibility of phosphorous from pea diet actually
decreased with the addition of phytase (94—87 %). In addition to its
effect on phosphorous digestibility, increases in dry matter, nitro-
gen and the digestibility of some amino acids were observed with
the addition of phytase.

THE EFFECT OF STARVATION ON GRAZING RATES OF
HALIOTIS FULGENS POSTLARVAE. L. A. Velez Espino, R.
Searcy Bernal, and C. Anguiano Beltran, Institute de Investiga-
ciones Oceanologicas. Aparlado Postal 453. Ensenada 22860. Baja
California, Mexico.

Grazing rates of Haliotis fulgens postlarvae (30. 45, and 60
days old) after different periods of starvation (0. 1, 2, 3, and 4
days) were estimated by the digital analysis of video-recorded
images of grazed areas. Trials were conducted in 10ml plastic
dishes colonized by the cultured diatom Naviciila iiuerta at stan-
dard densities (ca. 250-500 cells/mm" ). A significant increase in
grazing rates at longer starvation periods was detected. However,
for 45 and 60 day-old postlarvae, this increase occurred mostly
during the first two days of starvation. The maximum grazing rates
for 30, 45, and 60 day-old starved abalones were 3 861, 6 986, and
10 643 cells/postlar\;i/hour. respecti\'el> . which are similar to rates
observed in parallel trials at much higher biofilm densities (ca.
4,000 cells/mm-).

TETRAPLOID INDUCTION IN THE PACIFIC ABALONE
HALIOTIS DISCUS HANNAI INO WM H 6-DMAP AND CB.
(;. Zhany;, Z. VVanj;, \'. Chang, J. Song, J. Ding, Dalian Fish-
eries University, Key Laboratory of Mariculture Ecology. Ministry
of Agriculture. Dalian. LN, 1 16023, PRC, S. Zhao and X. Guo,
Haskin Shellfish Research Laboratory, Rutgers University, 6959
Miller Avenue, Port Norris, NJ, 08349, USA.

Triploid shellfish are useful in aquaculture becau.se of their
sterility, superior growth, improxement meat quality and some-
times increased disease resistance. The best way to produce trip-

4th International Abalone Symposium. Cape Town. Soutln Africa

Abstracts. February 2000 541

loids is through diploid x tetraploid mating. The Pacific abalone.
Haliotis discus hannai Ino, is a major aquaculture species in
China. We studied tetraploid induction in this species by inhibiting
the release of polar body I (PBl) with 6-dimethylaminopurine
(6-DMAP) and cytochalasin B (CB). Gametes were obtained by
inducing abalone to spawn with UV radiated seawater and artifi-
cially fertilized. Zygotes were treated with 6-DMAP and CB to
block the release of PBl and incubated at 22 °C. The 6-DMAP
treatments, which were applied from 6min post-fertilization (PF)
for 16-I8min at concentrations of 175(aM and 225|j.M. produced
20% and 22.5% of tetraploids, respectively, as determined by chro-

mosome counts at trochophore stage (12-14 hours PF). Significant
numbers of aneuploids (8.0^7.6%) were also observed. The ma-
jority of treated eggs (87.1-91.0%) survived to post-veliger stage
(56-58 hours PF). Two CB treatments (0.8 mg/L) were applied to
zygotes at 8 min PF lasting for 20 and 30 min. The short and long
CB treatments produced 32.9% and 24.75% tetraploids respec-
tively at trochophore stage, and 35.9% and 29.1% of tetraploid at
post-veliger stage, respectively, as checked by FCM. Survival to
post-veliger stage was 55.6% and 50.0% from the short and long
CB treatments. The result suggests that CB is slightly more effi-
cient than 6-DMAP for tetraploid induction in the Pacific abalone.

Joiinuil of Shellfish Rcscanh. Vol. 19. No. 1. 543-562. 2000.

ABSTRACTS OF PAPERS

Presented at the NAFO-ICES-PICES Symposium on Pandalid Shrimp Fisheries

'SCIENCE AND MANAGEMENT AT THE MILLENIUM"

Halifax. Nova Scotia
September 8-10, 1999

543

Symposium on Pandalid Shrimp Fisheries, Hahfux. Nova Scotia Ahstraas. September 8-10, 1999 545

CONTENTS

Bo Bergstrom

The biology of Pandalus 549

Gunnar Stefdnsson

Assessment methods and utilization of shrimp stocks — from simple-minded approaches through resignation to

multispecies and simulation methods 549

Paul J. Anderson

Pandalid shrimp as indicators of marine ecological regime shift 549

R. Ian Perry and J. A. Boutillier

Spatial scales of shrimp {Pandalus jordani) aggregrations, environmental influences, and consequences

for management 549

Don G. Parsons

Forecasting fishery performance for northern shrimp {Pandalus borealis) in NAFO Divisions 2HJ 550

Peter A. Koeller

Relative importance of environmental and ecological factors to the management of the northern shrimp {Pandalus

borealis) fishery on the Scotian Shelf 550

Anne Richards

Physical and biological factors influencing recruitment of northern shrimp Pandalus borealis in the Gulf of Maine — 550

Rene O. Ramseier and C. Garrity

How does the particle organic carbon sedimentation within the seasonal sea-ice regime influence the distribution of

northern shrimp (Pandalus borealis)''! 550

George R. Lilly and D. G. Parsons

Was the increase in shrimp biomass on the Northeast Newfoundland Shelf a consequence of a release in

predation pressure? 551

Hege 0. Hansen and M. Aschan

Growth performance, size and age at maturity of shrimp Pandalus borealis in the Svalbard area related to

environmental parameters 551

Manfred Stein

Hydrographic conditions off East Greenland - their potential effect on the distribution of shrimp {Pandalus borealis) .. 551

Michaela Aschan

Spatial variability in length frequency distribution and growth of shrimp {Pandalus borealis Kroyer 1984) in the

Barents Sea 552

Boris I. Berenboim, A. V. Dolgov, V. A. Korzhev and N. A. Yaragina

Cod impact on the stock dynamics of shrimp Pandalus borealis in the Barents Sea and its application in

multispecies models 55/

Alexey Buyanovsky

Biology and distribution of Pandalus hypsinotus (Brandt) in the northern part of the Sea of Japan 552

Stephen H. Clark, V. Silva, E. Holmes and J. B. O'Gorman

Observations on the biology and distribution of northern shrimp, Pandalus borealis. in the Gulf of Maine, from

research vessel surveys 55 j

Per Kanneworff and D. M. Carlsson

Occurrence of various species taken as by-catch in stratified-random trawl surveys for shrimp {Pandalus borealis) in

NAFO Subareas 0+1 , 1988-98 553

Peter Koeller, R. Mohn and M. Etter

Density dependent sex-reversal in pink shrimp, Pandalus borealis. on the Scotian Shelf 553

Bradley G. Stevens and P. J. Anderson

An association between the anemone, Cribrinopsis fenialdi. and the shrimps of the families Pandalidiae

and Hippolytidae 553

Takashi Minami

Predator-prey relationship and trophic levels of the pink shrimp, Pandalus eons, in the Yamato Bank, the Sea

ceo

of Japan -^-^

Soren A. Pedersen

Hydrographical and biological processes of importance in determining recruitment variability of northern shrimp in
West Greenland waters

546 Abstracts. September 8-10. 1999 Symposium on Pandalid Shrimp Fisheries, Halifax. Nova Scotia

Boris G. Ivanov

Pandalid shrimps of the Boreal area: history of fisheries and research with special reference to Russia 554

Robert Mohn

Data-poor stock assessment methods and their application to shrimp stocks 554

Steve Cadrin

Assessment of Pandalus borecdis stocks in the Northwest Atlantic: challenges with catch and catchability 554

Geoff T. Evans, D. C. Orr, D. G. Parsons and P. J. Veitch

A non-parametric method for estimating biomass from trawl surveys, with Monto Carlo confidence intervals 555

AlfHarbitz

Use of subjective prediction in optimal stratified sampling with application to shrimp surveys in the Barents Sea 555

Dan Carhson, O. Fobner, P. Kaniieworff M. Kingsley and M. Pennington

A trawl survey for Pandalus borealis in West Greenland 555

Stephen H. Clark, S. Cadrin, D. Schick, P. Diodati, M. Armstrong and D. McCarron

The Gulf of Maine northern shrimp fishery — a review of the record 556

John Clark, G. Bishop and T. Koeneman

Estimation of harvest rates in the spot shrimp pot fishery in Southeast Alaska using pre- and post-fishery stock

assessment surveys 556

Robert W. Hannah

By-catch reduction in an ocean shrimp iPainhiliis jordani) trawl from a simple modification to the trawl footrope 556

Louise Savard

Variations in the growth pattern of northern shrimp {Pandalus borealis) in the Gulf of St. Lawrence 556

Jean-Marie Sevigny, L. Savard and D. G. Parsons

Genetic characterization of the northern shrimp Pandalus borealis, in the Northwest Atlantic 557

Stein Tveite

Fixed stations survey for shrimp abundance indices. 15 years of investigations in the Norwegian Deeps

and Skagerrak 557

Hugues Bouchard, J. Lambert and L. Savard

Catching juvenile northern shrimp (Pandalus borealis) in the St. Lawrence estuary with a rigid frame trawl 557

Dan M. Carlsson

A new interpretation of age-at-length for shrimp (Pandalus borealis) in Davis Strait and inshore West

Greenland waters 557

Charmaine M. Gallagher, R. Hannah and G. Sylvia

Biological and economic yield-per-recruit: alternative strategies for managing Pacific Ocean shrimp {Pandalus

jordani) 558

Carsten Hvingel and Michael C. S. Kingsley

The uncertainty of an assessment procedure for the West Greenland stock of Pandalus borealis 558

Unnur Skiiladdttir

Age determination of northern shrimp. Pandalus bmcalis. in Icelandic waters using the deviation method in

conjunction with the method of Macdonald and Pitcher 558

P. Koeller Don G. Parsons, L. Savard and C. Fu Invited Paper

The Tralfic Light: a colourful but ugly approach to precautionary shrimp stock management 559

Rick Harbo, L. Convey, J. Boutillier and I). Hay

Pacific coast shrimp trawl fisheries: new management and assessment co-management programs 559

Caihong Fu, T. J. Quinn and G. H. Kruse

Analyses of harvest strategies for pandalid shrimp populations 559

Gretchen H. Bishop, T. M. Koeneman and C. A. Botelho

Development of a management and stock assessment program for the pot shrimp fishery for Pandalus platyceros in

southeastern Alaska 559

Jennifer A. Bond and J. A. Boutillier

Fixed escapement: an alternati\e to quota management in a shrimp fishery 560

Roger Larsen

Modern trawling and by-catch reducing devices in the North Atlantic shrimp fisheries 560

Symposium on Pandalid Shrimp Fisheries. Halifax. Nova Scotia Abstracts. September 8-10. 1999 547

John Angel

Management of the Canadian shrimp fishery 560

Daniel F. Schick and M. Brown

Gear testing in the northern shrimp fishery in the Gulf of Maine to improve size selectivity, reduce by-catch and

decrease production loss 561

Roland Hurtubise

Trends in processing and marketing in the Gulf of Maine shrimp fishery 561

Petur Bjarneson

The Icelandic shrimp industry 56 1

Vicki H. Kutzikowski. R. Hannah, G. Sylvia and M. T. Morrissey Poster

Finfish by-catch effects on the quality of ocean shrimp. Pandcilus jordani 561

Jason Clarke and W. E. L. Clayton

Monitoring the shrimp trawl fishery in British Columbia 562

Symposium on Pandalid Shrimp Fislieries. Halifax. Nova Scotia

Ahstracls. September 8-10. 1999 549

THE BIOLOGY OF PANDALUS. Bo Bergstrom. The Royal
Swedish Academy. Kristineberg Marine Research Station. S-450
34 Fiskebiickskil. Sweden.

The literature on the biology of 15 described species of the
shrimp genus Pandalus. reported from the Pacific and Atlantic
Oceans in the Northern Hemisphere is reviewed. Information on
taxonomy, external morphology as well as species descriptions,
keys to adult stages and systems for classification and species
determination of larvae is treated. Present knowledge on geo-
graphic distributions, depth distributions and salinity, temperature,
and habitat preferences on the continental shelves and slopes are
also accounted for and discussed. Various aspects of the life his-
tories of these species are described. The treatise on reproduction
biology includes a discussion of evolutionary maintenance of
protandric hermaphroditism, which is the dominating mode of re-
production in the genus, as well as an account of the present
knowledge of the mechanism of sex change in these shrimp. Meth-
ods used to describe reproductive cycles as well as results are
described and exemplified. Temperature effects on the duration of
different phases of the reproductive cycles represented in the genus
are discussed. Detailed accounts of adult behaviour in connection
with mating, oviposition and hatching are presented. A section on
Pandalus larvae includes accounts and discussions of behaviour,
nutrition, growth and survival. Adult behaviour is treated both on
individual and population levels, reports on .schooling, migrations
and food and feeding are reviewed and discussed. Predators, dis-
eases and parasites on Pandalus are accounted for as well as some
information on competitors. Methods for estimating growth and
age and factors affecting growth are described and reviewed.
Population dynamics, including population discrimination, abun-
dance and standing stock estimation, parent stock-recruitment re-
lationships and mortality factors, is discussed and illustrated by
examples. Notes on the fishery on Pandalus, which from an eco-
nomical point of view is surpassed only by the fishery for shrimp
in the family Penaediae, and the potential for aquaculture con-
cludes the review.

ASSESSMENT METHODS AND UTILIZATION OF
SHRIMP STOCKS - FROM SIMPLE-MINDED AP-
PROACHES THROUGH RESIGNATION TO MULTISPE-
CIES AND SIMULATION METHODS. Gunnar Stefansson,

Marine Research Institute. Skulagata 4. P.O. Box 1390. 121-
Reykjavik. Iceland.

Shrimp stock assessment methods have varied from simple
bulk models through VPA approaches. The assumptions of such
models typically fail miserably when shrimp stocks are considered.
Recent years have seen some new methods for estimating popu-
lation abundance and possible methods for utilizing resources dif-
ficult to assess. Such methods range from simulation of simple
harvest control rules through very complex assessment models.
Although these techniques still have a way to go. they represent
promising ways forward.

PANDALID SHRIMP AS INDICATORS OF MARINE ECO-
LOGICAL REGIME SHIFT. Paul J. Anderson, National Ma-
rine Fisheries Service, Alaska Fisheries Science Center, Kodiak
Laboratory, P. O. Box 1638. Kodiak. AK 99615-1638.

Pandalid shrimp are central components of the cold-regime
boreal marine ecosystem in the Gulf of Alaska. Declines in abun-
dance of several Pandalid species occurred quickly following wa-
ter column warming due to an abrupt climate change after 1977.
Shrimp trawl surveys conducted from 1953-1999 are used to de-
scribe how shrimp composition in catches changed relative to en-
vironmental parameters. Proportion of shrimp in survey catches
was found to be negatively correlated with water column tempera-
ture. Pandalid shrimp species which occupied inshore and typically
shallower water declined to near functional extinction, while off-
shore and deep water shrimp species have maintained low popu-
lation levels. Possible mechanisms responsible for the chronic de-
cline of several taxa of Pandalid shrimp and other crustaceans and
replacement by other species are discussed. Abrupt climate change
has an immediate effect on lower trophic levels of boreal marine
ecosystems and rapid pandalid shrimp population changes are one
of the first indicators that a community regime shift is underway.

SPATIAL SCALES OF SHRIMP (PANDALUS JORDAN!)
AGGREGRATIONS, ENVIRONMENTAL INFLUENCES,
AND CONSEQUENCES FOR MANAGEMENT. R. Ian Perry

and J. A. Boutillier, Pacific Biological Station. Fisheries and
Oceans Canada. Nanaimo. B.C.. Canada V9R 5K6.

Medium scale spatial patterns of smooth pink shrimp (Pan-
dalus jordani) off the west coast of British Columbia exhibit pe-
riods of aggregation, and periods of dispersal. This affects catch
rates by commercial vessels and affects the variance of survey
estimates of abundance. Survey procedures for shrimp in this area
have attempted to compensate for such changes in spatial pattern
by using sector and spline interpolators over the area surveyed to
estimate total abundance. Short-term availability of shrimp to com-
mercial fishing gears is known to be influenced by environmental
conditions such as tides, winds, and the amount of sunshine. In this
study, we use commercial and survey data to identify spatial scales
and patterns of shrimp aggregations and dispersal as influenced by
environmental conditions and interannual variations in the abun-
dance of shrimp. The goal is to improve understanding of the
processes affecting the spatial distribution of shrimp so as to im-
prove survey abundance estimates and management advice.

550 Ahsiracls. September 8-10, 1999

Symposium on Pandalid Shrimp Fisheries. Halifax, Nova Scotia

FORECASTING FISHERY PERFORMANCE FOR NORTH-
ERN SHRIMP (PANDALUS BOREALIS) IN NAFO DIVI-
SIONS 2HJ. Don G. Parsons, Northwest Atlantic Fisheries Cen-
tre, P.O. Box 5667, St. John's, Newfoundland, Canada AlC 5X1.

The physical environment is believed to have a major influence
on northern shrimp (Pandatus borealis) populations. Decades ago,
sea water temperatures from Boothbay Harbor were used to predict
shrimp landings two years later in the Gulf of Maine. Recent,
unpublished studies in eastern Canada showed that commercial
catch rates for shrimp were negatively correlated with temperature
(lagged several years) and that nitrate levels in surface layers
might be useful to predict shrimp abundance.

In this paper, I use time-series analysis to construct predictive
models for standardized catch rates (an abundance index) in a
shrimp fishing area off the Labrador coast. Environmental data are
incorporated as input series in transfer functions. Results support
the hypothesis that cold conditions are favourable for shrimp at
early life-history stages (larvae and juveniles). Model results fit the
observed values well in most cases and catch rate predictions for
several years are provided.

RELATIVE IMPORTANCE OF ENVIRONMENTAL AND
ECOLOGICAL FACTORS TO THE MANAGEMENT OF
THE NORTHERN SHRIMP (PANDALUS BOREALIS) FISH-
ERY ON THE SCOTIAN SHELF. Peter A. Koeller, Depart
ment of Fisheries and Oceans. Bedford Institute of Oceanography.
Dartmouth, Nova Scotia, Canada B2Y 4A2.

The biology of P. borealis on the Scotian Shelf Is discussed in
relation to the Gulf of Maine and Newfoundland Shelf stocks. The
Scotian Shelf as a whole is an area of transition for P. borealis with
a change in oceanographic characteristics in the mid shelf area that
has resulted in populations with characteristics of both more south-
ern and more northern stocks. The relatively large, commercially
important population on the eastern Scotian Shelf is restricted to
small areas of suitable habitat despite favorable temperatures over
a wide area. On the southern and western Scotian Shelf a small
population inhabits relatively small areas of marginally suitable
temperatures, despite large areas of suitable habitat. Commercially
important concentrations in this area appear only rarely after tem-
peratures decrease to more favorable levels. Growth rates and age
at first maturity on the Scotian Shelf are intermediate between the
Gulf of Maine and the Newfoundland shelf, and migrations include
inshore movements of ovigerous females in winter as in the Gulf
of Maine, as well as localized ontogenetic migrations. While
shrimp populations on the Scotian Shelf are influenced by water
temperatures and habitat availability, prcdation pressure is also a
significant determinant of abundance. The implications for the
management of shrimp fisheries in the area are discussed.

PHYSICAL AND BIOLOGICAL FACTORS INFLUENCING
RECRUITMENT OF NORTHERN SHRIMP PANDALUS
BOREALIS IN THE GULF OF MAINE. Anne Richards, Na

tional Marine Fisheries Service, 166 Water St., Woods Hole. MA
02543, USA.

The Pandalus borealis population in the Gulf of Maine is at the
southernmost limit of the species' distribution in the northwest
Atlantic. Previous studies have shown that recruitment is related to
both spawning biomass and spring surface temperatures (corre-
sponding to the period of planktonic larval development). The
purpose of this study was to extend these analyses by examining
the influence of additional environmental factors (freshwater run-
off, wind velocity and direction) and predation on recruitment.
Runoff and wind patterns strongly affect circulation within the
western Gulf of Maine and thus may affect recruitment through
effects on retention and/or transport of larvae. Major perturbations
in the predator community in the Gulf of Maine have occurred in
recent decades due to overfishing, thus predation pressure on
shrimp may have varied over time. Data on runoff and wind pat-
terns were available from long term monitoring programs of the
U.S. Geological Survey and NCAA's National Climatic Data Cen-
ter. An extensive food habits database developed by the Northeast
Fisheries Science Center (NEFSC. Woods Hole. MA) was used to
identify primary predators of shrimp in the western Gulf of Maine.
Aggregate abundance indices for the predators were developed
from multispecies trawl surveys conducted by the NEFSC during
1968-1998. Shrimp recruitment was modelled as a function of
shrimp spawning biomass, the environmental factors, and predator
abundance at biologically appropriate lags.

HOW DOES THE PARTICULATE ORGANIC CARBON
SEDIMENTATION WITHIN THE SEASONAL SEA-ICE
REGIME INFLUENCE THE DISTRIBUTION OF NORTH-
ERN SHRIMP {PANDALUS BOREALIS)? Rene O. Ramseier
and C. Garrity, Microwave Group-Ottawa River, Inc.

Based on sediment trap data collected at a depth of 500 m
below the ocean surface, as well as the //; situ sea-ice regime, we
have constructed a sedimentation model to map the amount and
distribution of particulate organic carbon (POC) for the Greenland
Sea (Ramseier et al. 1999). The derived model is based on ice
regimes defined by: ( I ) ice concentration. (2) duration of ice cover
and (3) distance from an ice edge, all relative to a trap location. In
the case of POC the sedimentation can be determined using a mean
annual ice concentration. For a severe ice year, the area of seasonal
ice cover provides 899f- of the POC sedimentation. In a light ice
year the amount of POC is slightly reduced to 87%. To better
understand the distribution of the POC sedimentation the model
tlivides the seasonal Ice cover into three distinct sub-regions, col-

Symposium on Pandalid Shrimp Fislieries. Halifax, Nova Scotia

Abstracts. September 8-10. 1999 551

lectively named the Biological Marginal Ice Zone (BMIZ). The
Biological Marginal Ice Zone does not include all the seasonal ice
cover extent, as would a Marginal Ice Zone. There is a centrally
located sub-region within the BMIZ. where the sedimentation is
non-linear resulting in a band of localized high sedimentation. This
results in an elevated export of biologically produced particles to
the deeper ocean. It is this result of localized sedimentation that is
likely to affect the distribution of shrimp. As an initial test we have
selected the Northern Shrimp Fishery area between 49°N and
60°N. Labrador Sea. Two data sets provided by Don Parsons
(DFO). (1) commercial catches for 1989 and (2) research catches
for Oct.-Dec. 1997, were analyzed in relation to the POC distri-
bution based on mean annual seasonal sea-ice cover extent. Bin-
ning the commercial data according to POC, results in a correlation
coefficient for a linear regression between catch per hour and POC
of r^ = 0.926. Similarly, binning the commercial data according to
depth, results in a correlation coefficient of r^ = 0.995. The re-
search data on the other hand was binned according to (1 ) POC, (2)
depth and (3) temperature with linear regression between total
catch and POC. The correlation coefficients r are (I) 0.535, (2)
0.897, and (3) 0.954 respectively. Analysis of the linear equations
for the commercial fisheries catch per hour data, based on POC
and depth binning, yield catch rates of 57.4% for POC. while depth
accounts for 42.6%. For the research fisheries the relative contri-
butions of the three variables in percent for the weight/shrimp
versus POC give (1) 36.2, (2) 33.1, and (3) 30.7 respectively. The
overall conclusion is that POC plays an important role as a food
supply, and its distribution provides locations where to look for
shrimp. Bibliography: Ramseier. R.O.. C. Garrity, E. Bauerfeind,
and R. Peinert. Sea-ice impact on long term particle flux in the
Greenland Sea"s Is Odden-Nordbukta region. 1985-1996. / Geo-
phys. Res., 104:5329-5343, 1999.

WAS THE INCREASE IN SHRIMP BIOMASS ON THE
NORTHEAST NEWFOUNDLAND SHELF A CONSE-
QUENCE OF A RELEASE IN PREDATION PRESSURE?
George R. Lilly and D. G. Parsons, Science Branch, Department
of Fisheries and Oceans. P. O. Box 5667. St. John's. Newfound-
land. Canada AlC 5X1.

During the late- 1980s and early- 1990s the relative importance
of groundfish and northern shrimp iPaiulalus borealis) was re-
versed on the Northeast Newfoundland Shelf off southern Labra-
dor and northeastern Newfoundland. The biomass of cod ( Gadus
morhiia) and other groundfish decreased greatly whereas the bio-
mass of northern shrimp increased both within the same area and
in areas to the north. It has been suggested that the increase in
shrimp was due to decreased predation. Temporal trends and
biomass indices for shrimp and its predators were examined to

determine if the timing of the changes is consistent with the above
hypothesis. In addition, temporal changes in the quantity of shrimp
consumed by the cod were determined from the catches of cod
during autumn bottom-trawl surveys (1978-1996), analysis of the
stomach contents of cod sampled during those surveys and the
application of a consumption model for cod based on studies of
gastric evacuation rate. Changes in consumption of shrimp by the
cod were compared with changes in the shrimp population, with
emphasis on determining whether the increase in recruitment to the
shrimp population was coincident with a reduction in removal of
young shrimp by the cod stock.

GROWTH PERFORMANCE, SIZE AND AGE AT MATU-
RITY OF SHRIMP PANDALUS BOREALIS IN THE SVAL-
BARD AREA RELATED TO ENVIRONMENTAL PARAM-
ETERS. Hege 0. Hansen and M. Aschan, Norwegian Institute of
Fisheries and Aquaculture Ltd., (Fiskeriforskning), N-9291
Troms0 Norway.

The study area was the shelf slope northwest of Svalbard (north
of 75°00'N) within a depth range of 200-600 m. Seven Subareas
were considered for modal analyses (MIX 2.3) of Pandalus bo-
realis length frequencies from 1992 to 1998. The L50 (carapace
length where 50%' are mature females) was calculated from a line
fitted to the length frequency of each area. Differences in L50.
growth and age at sex reversal were then related to environmental
factors including temperature, stock density and fishing activity.
The data show that the life history of shrimp in this area varies not
only geographically, but also over time. Between six and nine year
classes were identified in each area and age at sex change varied
between five and seven years. Shrimp in the northern areas grew
faster than in the south. Difference in growth and age at sex change
were mainly explained by temperature variations. Areas dominated
by cold polar water, north and south of Svalbard, and areas where
Atlantic and polar water alternate and cause variation in the envi-
ronmental conditions, show a slower growth and a higher age at
sex change. The implications to stock assessments of this plasticity
in the shrimp life history is discussed.

HYDROGRAPHIC CONDITIONS OFF EAST GREEN-
LAND - THEIR POTENTIAL EFFECT ON THE DISTRIBU-
TION OF SHRIMP (PANDALUS BOREALIS). Manfred Stein,

Institut fur Seefischerei. Palmaille 9, D-22767 Hamburg, Ger-
many.

Hydrographic conditions in the major shrimp catching areas
north of 65''N off East Greenland were examined to determine

552 Ahstnias. September 8-10. 1999

Symposium on Pandalid Shrimp Fisheries. Halifax. Nova Scotia

possible changes in the physical environment which might explain
the southward shift of Shrimp aggregations observed from 199,^
onwards. Based on the historic mean summer (JAS) hydrographic
conditions in the area of the Denmark Strait, the paper outlines the
regional distribution of Polar and Atlantic water masses on the
Greenland-Iceland Ridge. It is shown that topographic features
have a steering influence on the flow of these waters, and that they
might be responsible for the entrainment of major shrimp aggre-
gations. A salient topographic feature is the deep Kangerdlugsuak
Fjord which is carved into the East Greenland Shelf. Just southwest
of the Dohrn Bank. Until 1992. the northern shrimp aggregations
were confined to this fjord region. Recent hydrographic data as
sampled during the German bottom trawl surveys off East Green-
land, give a potential explanation for the observed southward shift
of shrimp iPandahis horealis) distribution from 1992 onwards. It
is hypothesised that an increased advection of warm Atlantic water
masses as observed during September 1993 and thereafter, led to
a southward displacement of the shrimp concentrations, and hence
to a southward shift of the catching areas. These "warm water
conditions" are maintained through to the present. Sea surface
anomaly data from the TOPEX/POSEIDON Satellite, show that
the variability of sea surface elevation, which is mostly due to
changes in the heat content in the upper water column of the area
under investigation is in the order of about ten to twenty days.

COD IMPACT ON THE STOCK DYNAMICS OF SHRIMP
PANDALUS BOREALIS IN THE BARENTS SEA AND ITS
APPLICATION IN MULTISPECIES MODELS. Boris I. Be-
renboim, A. V. Dolgov, V. A. Korzhev, and N. A.Yaragina,

Knipovich Polar Research Institute of Marine Fisheries and
Oceanography (PINRO). Knipovich Street, 6 Murmansk 183763,
Russia.

Cod predation is considered to be one of the most important
factors influencing shrimp population dynamics. Historical cod
feeding studies indicate that in the years with high shrimp biomass
their frequency of occurrence in cod stomachs increases. Quanti-
tative cod feeding analyses from the joint Russian-Norwegian da-
tabase indicate that shrimp became most important for cod feeding
during spring-summer. Fish at age 3-6 had the maximum influence
on shrimp stocks. In 1982-1997 shrimp biomass dynamics was
correlated with cod abundance indices. Patterns of cod predation
impact are used in calculating shrimp stock dynamics using mul-
ti.species VPA and production models.

SPATIAL VARIABILITY IN LENGTH FREQUENCY DIS-
TRIBUTION AND GROWTH OF SHRIMP (PANDALUS BO-
REALIS KR0YER 1984) IN THE BARENTS SEA. Michaela
Aschan, Norwegian Institute of Fisheries and Aquaculture Ltd..
(Fiskeriforskning) N-9005 Tromso. Norway.

The length frequency distributions (LFDs) of shrimp (PaiuUitus
horealis. Kroyer 1984) in the Barents Sea varies both spatially and
temporally. Sur\ey stations were defined in four groups using
correlation and cluster analysis. The groups were defined accord-
ing to LFDs with many small shrimp comprising the first group
and stations with the largest shrimp comprising the last. Distribu-
tion was depth dependent for the first three groups, while the
largest shrimp occurred in all depths - their distribution may be
determined by strong currents. Grov\th performance in any area
requires a broad LFD including all length groups and presents a
special challenge in sampling design. Cohort analysis of LFDs for
1992 to 1998 show thai shrimp in the southern Barents Sea grow
faster than in the north. On the basis of similar grow th performance
14 sub-areas were defined, lurlhcr population analysis for each
sub-area is difficult due to gralc selectivity differences, especially
in catch data. Only high resolution data should be selected when
preparing input data for further analysis. Summation of the number
of individuals by year class and area can then be used to run
production models or multispecies virtual population analysis for
the whole Barents Sea.

BIOLOGY AND DISTRIBUTION OF PANDALUS HYPSl-
NOTUS (BRANDT) IN THE NORTHERN PART OF THE
SEA OF JAPAN. Alexey Buyanovsky, VNIRO, V. Krasnosel-

skaya. 17 Moscow 107120. Russia.

Seasonal observations of the Painhiliis Inpsinotiis population in
the Tatar Strait (46-49''N) were made in 1996-1998. Data include
samples from 504 sites. Females release larvae in June. Oocytes
develop from August to December. Fertilisation and spawning
occur in winter and females bear the eggs for six months. How-
ever, females with outer eggs were present in samples through the
whole year. Five age groups were distinguished from the samples
which were collected in autumn 1998. Carapace lengths in the first
group varied from 16 to 27 mm; second = 29-44 mm; third =
29-44 mm; fourth = 37—48 mm; fifth = 44-52 mm. The two first
groups included 97'7f males, the third I009r transitionals. and the
last 2 groups comprised 85% females w ith head row . The probable
age of the first group is 2-H. Males change their sex during the fifth
year. The most significant growth occurs after the summer moult
while the autumn and winter moults are not followed by growth.
Analysis of latitudinal distribution allowed to distinguish three
local populations in the eastern pari of the strait. Dynamics of both
densities and si/e structines of these populations indicate that they
develop rather independently and large-scale migrations between
Ihem do not occur. Stock managcmeni should be based on quan-
lilali\e estinialions ol each local population.

S>mposium on Pandulid Shrimp Fisheries. Halifax, Nova Scotia

Abstracts. September 8-10. 1999 553

OBSERVATIONS ON THE BIOLOGY AND DISTRIBU-
TION OF NORTHERN SHRIMP. PANDALUS BOREALIS.
IN THE GULF OF MAINE. FROM RESEARCH VESSEL
SURVEYS. Stephen H. Clark, V. Silva, E. Holmes, and J. B.
O'Gorman. Northeast Fisheries Science Center. National Marine
Fisheries Service. Woods Hole. MA. USA.

Research vessel survey data collected since 1974 on northern
shrimp (Pandahis borealis) in the Gulf of Maine provide useful
insights on the biology of this species and on factors affecting its
distribution. Length-frequency analysis confirms the basic patterns
of growth and sex reversal determined by previous authors and
also indicates that changes in demographic parameters occur at
different population levels and under different environmental con-
ditions. Spatial and temporal distribution patterns are strongly de-
pendent upon temperature, depth, and substrate conditions, and
different size/year classes tend to occupy different habitats.

OCCURRENCE OF VARIOUS SPECIES TAKEN AS BY-
CATCH IN STRATIFIED-RANDOM TRAWL SURVEYS
FOR SHRIMP {PANDALUS BOREALIS) IN NAFO SUBAR-
EAS 0+1, 1988-98. Per Kanneworff and D. M. Carlsson, Green-
land Institute of Natural Resources, Box 2151, DK-1016 Copen-
hagen K. Denmark.

Random-stratified trawl surveys for assessing the stock of
shrimp (Pandalus borealis) have been carried out annually in the
period 1988-98 in the shrimp distribution areas in NAFO Subarea
I and a part of Div. OA. By-catch has been recorded since 1991 by
weight and number of species. A listing of all recorded by-catch
species (or groups of species as determined on board) is given
together with their prevalence. A preliminary analysis of annual
and spatial variations of shrimp and selected by-catch fish species
based on calculated densities is also presented.

including temperature, are also involved. We concluded that males
have a growth related physiological threshold at which they are
obligated to enter the transitional phase and that final mati'.ration to
the female is associated with a separate size related threshold.
Transition is a growth phase that is extended in areas of early male
maturation until the female threshold is attained. In warmer areas
where the difference between male and female thresholds are great
the benefits of protandry are decreased and the population may
regress to the dioecious state, resulting in more primary females.

AN ASSOCIATION BETWEEN THE ANEMONE, CRI-
BRINOPSIS FERNALDL AND THE SHRIMPS OF THE
FAMILIES PANDALIDIAE AND HIPPOLYTIDAE. Bradley

G. Stevens and P. J. Anderson, National Marine Fisheries Ser-
vice, Alaska Fisheries Science Center. Kodiak Laboratory, P.O.
Box 1638, Kodiak, AK 99615, USA.

A previously unrecognized association between the pink sea
anemone Cribriiuipsis fenialdi and several species of Caridean
shrimp was observed and documented from a submersible at
depths of 75-150 m in Kodiak. Alaska. Shrimp were aggregated in
a radial pattern around anemones, beneath or just beyond the ten-
tacle canopy. Species collected with a suction sampler included
Eitahts suckleyi. Spirontocaris sp.. Lebbeus graiuliinaiuis, L.
groenkmdiciis, and Paiidahts tridens, but not P. borealis or P.
goniunts, although they were probably also present. Numbers of
shrimp per anemone increased with depth from 61 to 115 m, and
more shrimp were observed on silty-sand than on sandy-gravel
substrates. While associations between actinians and Hippolytid
shrimp are common in tropical waters, this association is unusual
because of its northern geographic location and the involvement of
Pandalid shrimp.

DENSITY DEPENDENT SEX-REVERSAL IN PINK
SHRIMP, PANDALUS BOREALIS, ON THE SCOTIAN
SHELF. Peter Koeller, R. Mohn. and M. Etter, Bedford Institute
of Oceanography. P.O. Box 1006. Dartmouth. Nova Scotia.
Canada B2Y 4A2.

On the Scotian Shelf, transition of Pandalus borealis from male
to female occurred at different sizes and ages, and could not be
related to a minimum size, age, or number of instars. Our data do
not show the positive relationship between density of older fe-
males and size at sex transition predicted by sex allocation theory.
Size at transition was inversely related to density, which was at-
tributed to density dependent growth affecting all stages. Density
appears to become increasingly important as a factor determining
growth as densities increase, while at low densities, other factors.

PREDATOR-PREY RELATIONSHIP AND TROPHIC LEV-
ELS OF THE PINK SHRIMP, PANDALUS EOUS, IN THE
YAMATO BANK, THE SEA OF JAPAN. Takashi Minami.

Japan Sea National Fisheries Research Institute. Suido-Cho
1-5939-22. Niigata. Japan 951-8121.

Pink shrimp Pandalus eons is an important commercial species
in Japan and an important member of the deep-water ecosystem in
the edge of continental shelf and isolated offshore banks such as
the Yamato Bank (shallowest depth; 246 ni) in the central part of
the Sea of Japan. In the ecosystem, pink shrimp function as both
predator and prey. An understanding of predator-prey relationships
are essential in elucidating the position of the pink shrimp within
food webs and the ecosystem. Diet composition of the pink shrimp
were investigated and stomachs of the potential predators of the
pink shrimp such as skate, sculpins, flatfish, cod. walleye pollock.

554 Ahsimcts. September 8-10. 1999

Symposium on Pandalid Shrimp Fisheries, Halifax, Nova Scotia

snailfish, lumpsucker, eelpouts collected in the Yamato Bank were
analysed. Pink shrimp prey primarily consisted of small crusta-
ceans groups such as Gammaridea, Euphausia. and Harpacticoida.
Other common prey were Bivalvia, Caridea, and Polychaeta. Little
dietary variation was seen with respect to predator size or depth.
Dominant predators for pink shrimp were skate {Bathyraja
smirnovi). eelpouts {Lycodes tanakai). Allolepis hollandi.
Petroschmidlia toyumaensis. sculpin {Maracocottus gihher). and
cod (Gadiis macrocephalus). There was some evidence of canni-
balism in pink shrimp. Trophic levels were estimated by means of
stable isotope ratios for pink shrimp and other benthic animals
collected in the Yamato Bank.

tional Pandalid Shrimp Symposium (Kodiak. Alaska, 1979) while
that in Russia was poorly documented. In the North Atlantic
USSR/Russia began to fish for P. borealis off West Greenland in
1974 but the introduction of the 200-mile zone in 1977 resulted in
the migration of Soviet boats to the Barents Sea. By 1978 shrimp
catches had reached 18,000 tons. In the Pacific Russia started
shrimping (P. hypsinotus. P. borealis) in the Tartar Strait in 1979,
off northeast Sakhalin in 1995, off south-west Kamchatka in 1996.
After a 20-year hiatus shrimping also recommenced in the western
Bering Sea. The historical fishery for shrimp was conducted in the
Gulf of Alaska (P. borealis) and the Anadyr Gulf [P. i;oiiiiiri(s).

HYDROGRAPHICAL AND BIOLOGICAL PROCESSES OF
IMPORTANCE IN DETERMINING RECRUITMENT
VARIABILITY OF NORTHERN SHRIMP IN WEST
GREENLAND WATERS. Soren A. Pedersen, Greenland Insti-
tute of Natural Resources, C/O Danish Institute for Fisheries Re-
search, Dept. of Marine Ecology, Kavalergaarden 6. 2920 Char-
lotlenlund. Denmark.

The poster presents preliminary results obtained from ongoing
studies of recruitment processes of northern shrimp {Pandahts bo-
realis) in West Greenland waters. Information on distribution and
lipid composition of Pandahts shrimp larvae in relation to hydrog-
raphy and potential food resources is presented. Ongoing studies
with the objectives of 1 ) identifying the effect of hydrographic
frontal regimes on larval and juvenile shrimp condition and sur-
vival potential and 2) investigating the potential use of tracer lipids
to establish food web relationships are outlined and discussed.

DATA-POOR STOCK ASSESSMENT METHODS AND
THEIR APPLICATION TO SHRIMP STOCKS. Robert
Mohn, Marine Fish Division, Department of Fisheries and Oceans,
P. O. Box 1006, Dartmouth. Nova Scotia, Canada B2Y 4A2.

Many stocks, especially invertebrates, do not have enough data
to support traditional assessment methods. These stocks are can-
didates for so-called data poor methods. Recently, 1996-1998.
ICES convened a Study Group (Study Group on the Assessment of
Other Fish and Shellfish Species) to investigate data poor methods
in the context of evaluating unassessed European stocks. The re-
ports of this Study Group provide a catalogue of methods, ap-
proaches and software. A summary of their work, and that of other
fora, is presented with emphasis on data needs, utility of output
and applicability to shrimp stock data. The methods range from
simple production models to heavily parameterized stock synthe-
sis. Determination of the precautionary reference points is in-
cluded. Some of the methods are applied to Scotian Shelf shrimp
data as test cases.

PANDALID SHRIMPS OF THE BOREAL AREA: HISTORY
OF FISHERIES AND RESEARCH WITH SPECIAL REFER-
ENCE TO RUSSIA. Boris G. Ivanov. Russian Research Institute
of Fisheries and Oceanography (VNIRO) 17, V.-Krasnoselskaya,
Moscow 107140, Russia.

The history of research and management of Pandalid shrimp
fisheries arc described with special reference to Russia. All com-
mercial pandalid species were described between 1814-1902.
Hjort and Petersen discovered commercial densities of Pandahis
borealis in Norwegian tjords in the laic 19th century. A. Berkeley
(1929.1930) discovered protandry in pandalids. By 19,Vt-I941. P.
borealis life history had been studied mainly in southern areas and
it was concluded that the species has a similar lite cycle every-
where. Rasmussen ( 1953) broke this assumption and demonstrated
great variability in growth and maturation depending on local en-
virt)nniental conditions. Horsted and Smidt (1956) and Allen
(1959) studied life history in the most and least severe areas. In
Europe and North America the fishery for pandalids began in the
late 19th century. The histories of the fisheries in European.
American, and .lapancse waters was described at the first Intcrna-

ASSESSMENT OF PANDALUS BOREALIS STOCKS IN
THE NORTHWEST ATLANTIC: CHALLENGES WITH
CATCH AND CATCHABILITY. Steve Cadrin. National Ma
rine Fisheries Service, 166 Water St., Woods Hole, MA 02543-
1026, USA.

Lacking precise information on age and growth, some stock
assessments of northern shrimp. PmuUdus borealis. in the North-
west Atlantic have been based on models that integrate catch and
stock size indices. The most commonly used models have been
production models, such as ASPIC, and stage-ba.sed methods, such
as the Collie-Sissenwine model. Both model types can provide
estimates of uncertainty for stochastic risk assessment of manage-
ment options. However, the absolute magnitude of stock size from
either model can be misleading, especially when models are over-
parameterized, and calchability of surveys or fishing effort is
poorly estimated. Simulations indicate that production models re-
quire a time scries with a wide range of stock levels to provide
reliable estimates of absolute stock size. Stage-based estimates of
survey catchability are sensitive to natural mortalit). rclati\e sur-

Symposium on Pandalid Shrimp Fislieries. Halifax. Nova Scotia

Abstracts. September 8-10. 1999 555

vey selectivity of recruits, and the relative weight of observation
errors. In assessments where estimates of catchability are judged to
be unreliable, the results are often still useful for providing insights
on trends in relative stock size and fishing mortality.

A NON-PARAMETRIC METHOD FOR ESTIMATING BIO-
MASS FROM TRAWL SURVEYS, WITH MONTE CARLO
CONFIDENCE INTERVALS. Geoff T. Evans. D. C. Orr,
D. G. Parsons, and P. J. Veitch. Northwest Atlantic Fisheries
Centre. P. O. Box 5667. St. John's, Newfoundland. Canada AlC
5X1.

The probability distribution for biomass of many marine spe-
cies varies in space, partly as a function of bottom depth. We
describe a non-parametric method for using trawl survey data to
estimate the probability distribution at any point in the survey
region whose bottom depth is known. Integrating the expected
value of the distribution over the region provides an estimate of the
biomass in the region. Repeated resampling from the estimated
distributions at the survey points enables us to compute a Monte
Carlo confidence interval for the biomass. When we apply these
methods to northern shrimp (Pandalus borealis) in NAFO Divi-
sions 2HJ. we obtain confidence intervals that are narrower than
those computed using methods based on random-stratitled sam-
pling and an assumed Gaussian distribution.

USE OF SUBJECTIVE PREDICTION IN OPTIMAL
STRATIFIED SAMPLING WITH APPLICATION TO
SHRIMP SURVEYS IN THE BARENTS SEA. Alf Harbitz,

Norwegian Institute of Fisheries. And Aquaculture Ltd.. N-9291
Tromso, Norway.

We consider stratified sampling and the task of applying sub-
jective knowledge in predicting the number of trawl samples per
stratum that minimises the cv of the abundance estimator. The
constraint is a given vessel time available. It is assumed that the
strata biomass means, arbitrarily scaled, are the only unknown
parameters needed to find the optimal solution. The concept of a
subjective prediction distribution of the unknown stratum means is
introduced. The distribution is person-dependent and is determined
based on intervals [LU] for the minimum and maximum subjec-
tively predicted biomass values compared with the true measured
values found after the predictions. The approach assumes a con-
stant subjective confidence level defined as the probability of cov-
ering the true value in a random interval. A pilot subjective pre-
diction experiment was conducted during the 1998 shrimp survey
in the Barents Sea. Based on 62 [LU] predictions of shrimp bio-
mass in the next trawl haul combined with the true biomass. the
subjective prediction distribution for the cruise leader was esti-
mated. The distribution was applied to her stratum predictions for

the next survey. 10,000 random predictions of true strata means
were simulated from the distribution. For each simulation cv-
values of the abundance estimator were estimated based on relative
strata means predicted from historical data as well as the subjective
predictions. A significant cv-reduction was obtained based on a
combination of subjective prediction and historical data, compared
to the use of historical data alone.

A TRAWL SURVEY FOR PANDALUS BOREALIS IN WEST
GREENLAND. Dan Carlsson. O. Folmer. P. Kanneworff, M.
Kingsley, and M. Pennington, Pinngortitaleriffik Greenland In-
stitute of Natural Resources, Nuuk, Greenland.

Stocks of northern pink shrimp Pandalus borealis have been
assessed using CPUE data. Since 1988, a stratified random re-
search trawl survey has also provided estimates of stock abun-
dance, and on biological parameters of the resource. The survey
covers the West Greenland fishing grounds between 150 m and
600 m deep, and occupies a research trawler for about 60 days.
Stratification is based on depth, but strata are fairiy small. Stations
are allocated proportional to stratum area. Over time, the survey
has changed. It has been extended into southern fishing grounds as
the fishery developed there. Two-stage sampling was introduced to
reduce the largest stratum standard errors, and a spline method was
investigated for interpolation of additional stations. Gear changes
have included replacing 44-mm stretch mesh in the liner with
20-mm since 1993, and using a trawleye to determine start position
since 1997. Biomass estimates have had error CVs of the order of
15-30%, and have not been correlated with CPUE. In 1997 a
group comprising science management, biologists, fishery advis-
ers, and external survey expertise reviewed the survey design. The
review suggested: abandoning 2-stage sampling; shortening the
tows and increasing their number, and allocating more stations to
the highest-yield group of strata. Making more tows would im-
prove information about biological parameters, and shorter might
improve biomass estimation. Catches in a small sample of Vz-h
tows in 1998 were no more variable than in 1-h tows, although an
experimental survey in that year showed no correlation between
contiguous 1-h tows and could not predict the effect of shorter
tows. Changes suggested to the analytical methods included pool-
ing the small design strata into 4 large groups based on depth, log
transforming the data to reduce its skewdness, and smoothing the
results to reduce year-to-year variation. Analysis suggests that the
biomass has not varied much over the survey series. To implement
some of these suggestions, a progressive shift toward '/2-h tows has
begun, and it is envisaged that some '/2-h tows will be experimen-
tally carried out as pairs of 15-min tows. The proportion of stations
allocated to high-density strata has been increased. Buffered sam-
pling has been used to control station placement, and to prevent
stations from clustering within strata. A proportion of stations,
randomly selected, has been fixed from year to year.

556 Abstracts. September 8-10. 1999

Symposium on Pandalid Shrimp Fisheries. Halifax. Nova Scotia

THE GULF OF M.\INE NORTHERN SHRIMP FISH-
ERY—A REMEW OF THE RECORD. Stephen H. Clark. S.
Cadrin, D. Schick, P. Diodati. iM. Armstrong, and D. McCar-

ron, Northeast Fisheries Science Center. National Marine Fisher-
ies Ser\ice. Woods Hole. MA. USA.

The Gulf of Maine northern shrimp fishen. has been a dynamic
one. with landings varying greatly in response to resource and
market conditions. A directed winter fishery' developed in coastal
waters in the late- 1930s, which expanded to an offshore year round
fishery in the late- 1 960s when landings peaked at over 12.000 mt
in 1969. Landings subsequent!) declined to ven. low lexels as
recruitment deteriorated and the stock collapsed, precipitating clo-
sure of the fishen. in 1978. The resource reco\ered under restric-
tive management and was relatively stable at low to moderate
levels of exploitation into the 1990s, with 3—1 strong year classes
recruiting to the fishery. In the mid-1990s, landings and fishing
mortalitx increased sharpl\ and abundance and recruitment ha\e
again declined. Environmental conditions have clearlv plaved an
important role in affecting survival and abundance, but fishing
mortalitv has been the overriding factor since the late- 1970s. Stock
assessments have consistentlv shown that poor recruitment is more
likely at low levels of spaw ning stock biomass ow ing to reduced
total egg production. Under normal environmental conditions sus-
tainable yields from the Gulf of Maine northern shrimp stock are
probably about 3000-4000 mt per vear.

ESTIMATION OF HARVEST RATES IN THE SPOT
SHRIMP POT FISHERY IN SOUTHEAST ALASK.A USING
PRE- AND POST-FISHERY STOCK ASSESSMENT SUR-
VEYS. John Clark. G. Bishop, and T. Koeneman, Alaska De-
partment of Fish and Game. P. O. Box 240020. Douglas. .^K
99824. USA.

The pot shrimp fisherv in Southeast .Alaska harvested an annual
average of 355 metric tons of shrimp since the 1990/91 season
with an average ex-vessel value of almost 2 million dollars (U. S.).
Spot shrimp (Paiidaliis platyceros) comprise over 959}- of the
landed weight. Information on abundance, si/e and sex composi-
tion, distribution of spot shrimp populations and evaluation of the
cumulative impact of fishing effort on the abundance and biology
of this resource is essential to achieving an optimum sustainable
harvest. StiK-k assessment surveys were conducted in limited areas
two weeks prior to the fishery in 1997 and 1998 to collect this
Information. In February. 1999. a post-season survey was con-
ducted approximately 4 months after the area was closed to evalu-
ate the effects of the commercial fisherj' on the abundance and
composition of the st(Kks and to determine if a harvest rate could
be reliabh estimated lor spot shrimp stocks in two areas. A simple
ratio estimator is used to estimate the harvest rate on spot shrimp
in two adjacent areas. A more robust and informative abundance
estimator based on the relative estimated abundance and si/e
distribution of spot shrimp in the areas and selectivity of the 1 3/4

and I 1/8 inch mesh pots is developed and applied to the 1997 and
1998/99 survey data. Confidence limits are estimated using a boot-
strap approach for a random stratified sampling plan. Dockside
samples, survey size distributions and residuals from the analysis
are examined for evidence of hiah incidental mortalitv.

BY-CATCH REDUCTION IN AN OCEAN SHRIMP (PAN-
DALIS JORDASI) TIUWVL FROM A SIMPLE MODIFICA-
TION TO THE TR.AWL FOOTROPE. Robert W . Hannah.

Oregon Department offish and Wildlife. 2040 SE .Marine Science
Drive. Newpon. OR 97365. USA.

Two commonly used groundline designs in the ocean shrimp
( Pandahis jordani) trawl fisherv' were fished side by side from a
double-rigged vessel to compare catch rates of shnmp and bv catch.
The designs compared were a traditional "tickler chain" ground-
line, which is shorter than, and runs below and in front of the
fishing line of the trawl, and a ladder chain with a short roller
section, set to run under and slightly behind the fishing line. Both
nets were measured using a SIMR.\D ITl trawl monitoring system
to control for differences in net spread and rise. The ladder/roller
groundline caught iA% fewer slender sole (Eopsetta exilis). 61%
fewer greenstriped rockfish iSebasles el(mi;cttiis) and 33% fewer
small rocktlsh (<8 cm total length) than the tickler chain ground-
line. However, only the difference in slender sole was statistically
significant (P < 0.05) for these sample sizes in an .ANOVA. After
allow ing for a wider net spread w ith the ladder/roller gear, catches
of shrimp and other fish species were comparable for both gears.
These results suggest that ocean shrimp traw Is can be rigged to fish
efficiently for shrimp and marketable fish with a groundline that
runs behind the fishing line of the trawl. A test of an ocean shrimp
trawl using dropper chains to maintain a constant height of the
footrope above bottom, with no groundline at all. is recommended.

\ ARIATIONS IN THE GROWTH PATTERN OF NORTH-
ERN SHRIMP (PAyOALLS BOREALIS) IN THE GULF OF
ST. L.A\>RENCE. Louise Savard. Maurice Lamontagne Insti-
tute. Depanment of Fisheries and Oceans. P. O. Box 1 (X)0. Mont-
Joli. Quebec, Canada G5H 3Z4.

Northern shrimp tPaiulaliis horealis) are found throughout the
Estuarv and the Northern Gulf of St. Lawrence in concentrations
that sustain a commercial fishery that has landed more than 20.000
tons annually, for the last 3 years. The Estuarv and the Gulf of
St. Lawrence form a closed area which is div ided into four fishery
management units located from the west to the east. Bottom trawl
research surveys have been conducted in these management units
in August-September each year since 1990. Carapace length fre-
quency distributions obtained from the surveys indicate that the
mean size of the last mode of males, the length at sex reversal and
the mean si/e of females have varied in two ways since 1990. The
LFDs show a size gradient from east to west that was persistent

Symposium on Pandalid Shrimp Fisheries. Hahfax. Nova Scotia

Abslnicts. September S- 10, 1999 357

from year to year. Howe\'er. similar size variations between years
were also observed within each area. The size gradient indicates
that the areas have a specific influence on growth but the similarity
of the year to year variations suggests a synchronism in the events
that cause the length variations. Hypothesis explaining these re-
sults as well as the impact of such variations on resource conser-
vation and fishery management are discussed.

GENETIC CHARACTERIZ.4TION OF THE NORTHERN
SHRIMP PANDALUS BOREALIS, IN THE NORTHWEST
ATLANTIC. Jean-Marie Sevigny. L. Savard. and D. G. Par-
sons, Ministere des Peches et des Oceans, Institut Maurice-
Lamontagne. Mont-Joli. Quebec. Canada G5\\ 3Z4.

Genetic variability of the northern shrimp, Paiulahts horealis.
in the Northwest Atlantic was studied at nine enzymatic loci.
Samples were collected in seven regions of the Saguenay Fjord,
the Estuary and the Gulf of St. Lawrence and in two regions off the
Labrador-Newfoundland coast. Males, primiparous and multipa-
rous females were sampled in each region in order to determine if
gene frequencies within regions are temporally stable. Genetic
distances between regions are low indicating that the level of gene
flow may be high in this species. A cluster analysis of genetic
distance did not reveal organization on the geographic scale of
sampling. Furthermore, the interpretation of the population genetic
structure of the northern shrimp is complicated by the fact that
significant differences in allelic frequencies were observed among
the maturity stages within some regions. This observation suggests
that differences observed on the geographic scale between some of
the studied regions may not be stable through time. Various hy-
potheses to explain these results are discussed.

FIXED STATIONS SURVEY FOR SHRIMP ABUNDANCE
INDICES, 15 YEARS OF INVESTIGATIONS IN THE NOR-
WEGIAN DEEPS AND SKAGERRAK. Stein Tveite, Institute
of Marine Research. Flodevigen Marine Research Station. N-4817
His, Norway.

Since 1984 about 100 bottom trawl stations at fixed positions
have been completed yeariy in October-November. The catch of
0-group Panclalus horealis give an indication of year class
strength, however, the catch of I-group gives a more reliable es-
timate compared to analytical methods and CPUE statistics. In this
paper comparisons are made between variations in shrimp abun-
dance and environmental factors such as tluxes of Atlantic water
into the area, other hydrographic variations and abundance of fish
species.

CATCHING JUVENILE NORTHERN SHRIMP (PAN-
DALUS BOREALIS) IN THE ST. LAWRENCE ESTUARY
WITH A RIGID FRAME TRAWL. Hugues Bouchard, J.
Lambert, and L. Savard, Maurice Lamontagne Institute, Depart-
ment of Fisheries and Oceans, 850 route de la mer, P. O. Box
1000, Mont-Joli. P.Q.. Canada G5H 3Z4.

Data for stock status assessment of northern shrimp {PwiJaliis
horealis) is traditionally obtained from commercial sampling and
research surveys using a conventional bottom trawl at depths
where small shrimp are rare. To make up for the lack of knowledge
on the relative abundance of juvenile shrimp, a rigid frame trawl.
15 feet wide and 7 feet high, mounted on skates was developed at
Maurice Lamontagne Institute with financial support from the
shrimp fishing industry. The trawl was designed to be easily ma-
nipulated on a rear trawler of 65 feet in order to sample several
stations in one day. The catch in the trawl should reliably represent
the proportions of the different size-classes of shrimp present on
the bottom sampled. The trawl is 67 feet in length. A liner of 9
mm-stretched mesh was added to the last 51 feet to retain small
shrimp. During the research survey, the trawl was towed at about
2 knots for 20 min at depths varying from 50 to 260 m. The poster
presented at the symposium deals with 3 main points: a) design of
the rigid frame trawl with dimensions, b) method of fishing with
the trawl, and c) handling the catch.

A NEW INTERPRETATION OF AGE-AT-LENGTH FOR
SHRIMP (PANDALUS BOREALIS) IN DAVIS STRAIT AND
INSHORE WEST GREENLAND WATERS. Dan M. Carls-
son, Greenland Institute for Natural Resources. P.O. Box 2151.
DK-016 Copenhagen K.. Denmark.

Since the introduction of annual stratified-random trawl sur-
veys in 1988 interpretation of age at length for the shrimp stock in
West Greenland waters has been based on the age-length structure
established by Savard et al. (1994) for shrimp in the Davis Strait
using modal analysis of samples from 1982 to 1987. Survey
samples from the inshore Disko area have shown similar modes as
found in the offshore surveys from 1988 to 1997. and shrimp in the
two areas have been considered to belong to the same stock and
have been assessed as such. The progression from 1996 to 1997 of
a distinct and significant mode of males in survey samples from
the Disko area indicated that the old interpretation is not applicable
in this area, even though reasonable results have been obtained
with its use in recent years. Therefore, survey samples from both
the offshore areas and the Disko area were reanalysed by modal
analysis, and a new age-at-length structure derived, indicating that
shrimp in both areas change sex from males to females at age 6
rather than at age 7. The new interpretation also shows distinct van
Bertalanffy growth.

558 Abstracts. September 8-10, 1999

Symposium on Pandalid Shrimp Fisheries, Halifax, Nova Scotia

BIOLOGICAL AND ECONOMIC YIELD-PER-RECRUIT:
ALTERNATIVE STRATEGIES FOR MANAGING PACIFIC
OCEAN SHRIMP (PANDALUS JORDAN!). Charmaine M.
Gallagher, R. Hannah, and G. Sylvia, Oregon State University,
Corvailis, Oregon, USA.

Selecting "optimal' strategies for managing Pacific Ocean
shrimp is challenging due to uncertain and variable natural mor-
tality, recruitment, and growth. Although there are no explicit
objectives for managing Oregon's shrimp fishery, managers have
developed measures they believe will prevent long-term biological
damage to the stock, protect age-1 shrimp from overharvesting and
sustain long term fishery benefits. Developing harvest strategies
such as mesh size and season dates are complicated by economic
factors including differences in output prices as a function of
shrimp size. To evaluate the potential importance of market price
on shrimp management, this research uses equilibrium yield per
recruit analysis to compare biological and revenue yield. The
analysis is conducted over a range of natural mortality rates, for
multiple age-at-entry dates, and fishing mortality rates. Growth is
determined using length at age samples from the commercial
catch. Results indicate that higher rates of natural mortality shift
the biological and economic optimum toward younger shrimp and
lower fishing mortality rates. Incorporating output prices may shift
the optimum toward an older age of entry and a lower fishing
mortality. For all analyses, the revenue generated at biological
yield optimums is compared to revenue from maximizing eco-
nomic yields. Future research will build on this analysis by includ-
ing selectivity at length, variable recruitment, harvester and pro-
cessor costs, and product quality.

THE UNCERTAINTY OF AN ASSESSMENT PROCEDURE
FOR THE WEST GREENLAND STOCK OF PANDALUS
BOREALIS. Carsten Hvingel and Michael C. S. Kingsley, Pin-
ngortitaleritfik. Greenland Natural Resources Institute, P. O. Box
570, DK-3900 Nuuk, Greenland.

Fishery resources are often asses.sed by standardizing catch
performances of fleets over time, to create series of CPUE indices
that are not affected by the way the fleets have changed, but which
instead represent indices of biomass. Simple population dynamics
models can be fitted to these scries of yearly estimates of biomass
index, along with the yearly landings. The shrimp resource off
West Greenland was assessed by fitting separate models lo four
different fleets operating over different periods between 1976 and
19SS. The four CPUE series were united by weighted least
squares, and the licet catch series were summed. A logistic model
of population dynamics was then fitted lo this data and a standard
trawl survey series. It was ditficull lo know how reliable this
complex threc-siagc process was, and a jack-knife procedure of
leaving out one year's dala at a time was adopted lo find out. First
we Jack-knifed the entire procedure, leaving out, for each year in
turn, all Ihe dala from commercial CPUF and survevs from all

stages of the process. The results were: small scatter (CV 9.8%) in
optimal fishing mortality, but larger and off-centre scatter in sus-
tainable catch, ranging from 8.7% lower to 52.0%' higher than the
value based on all the data. Omission of the first year's data
created an outlier with a 47.3% lower sustainable fishing mortality.
The components of this variation were investigated by using all
years' data as input to the standardized CPUE series, and then
jack-knifing only the input to the logistic population model. The
optimal fishing mortality varied about as much as before (CV
7.7%), but the sustainable catch varied much less and more sym-
metrically (from 8.5% lower to -i-9.2% higher). Much of the varia-
tion in estimated sustainable catch therefore appears due to uncer-
tainty in the standardized CPUE series. However, omitting the first
year's data from the input to the logistic population modeling
phase continued to give an outlying point, with an MSYF that was
46.8% lower. The parameter estimates of the fitted population
model therefore appear sensitive to characteristics of the first
year's data.

AGE DETERMINATION OF NORTHERN SHRIMP, PAN-
DALUS BOREALIS, IN ICELANDIC WATERS USING THE
DEVIATION METHOD IN CONJUNCTION WITH THE
METHOD OF MACDONALD AND PITCHER. Unnur Skii-
ladottir. Marine Research Institute, Skulagata 4, P. O. Box 1390,
121 Reykjavik, Iceland.

Aging of shrimp was carried out for 10 consecutive years in
two very different areas in Icelandic waters - the Arnartjordur
fjord, and the offshore area north of Iceland. In the Arnartjordur,
it was not possible to interpret length frequency distributions of
individual years with the technique of Macdonald and Pitcher i.e.,
Mix. Recruitment failed completely twice during the period so 2
year-classes were missing altogether in the series. It was therefore
necessary to look at a series of years using deviations from the
mean length frequency distribution of the 10 year series. By fol-
lowing the positive deviations, one age class could e.g., be fol-
lowed for 7 years as a positive deviation. The mean lengths as-
.sessed from the deviations were then used as inputs for the mix
method to calculate Ihe proportions, new mean lengths for each
age class for a given number of age classes. The offshore popu-
lation was very different from ihe secluded fjord populalion. The
former area had to he divided into many smaller areas in order to
inlcrprel age classes properly. Deviations calculated Doni a com-
bined length frequency distribution accentuated peaks lor each
small area but combinalion before aging was not useful in this
regard. Eventually mix was applied lo length frequency distribu-
tions of e\ery small area and the results combined and weighted by
nominal catch of each small area to an overall catch number for the
whole area per year. Up to 8 year-classes were detected.

Symposium on Pandalid Shrimp Fisheries. Halifax. Nova Scotia

Abslracts. September 8-10. 1999 559

THE TRAFFIC LIGHT: A COLOURFUL BUT UGLY AP-
PROACH TO PRECAUTIONARY SHRIMP STOCK MAN-
AGEMENT. P. Koeller Don G. Parsons, L. Savard, and C. Fu,

Northwest Atlantic Fisheries Centre. P. O, Box 5667. St. John's.
Newfoundland. Canada AlC 5X1.

At a recent meeting of the NAFO Scientific Council partici-
pants considered three example stocks for the application of pre-
cautionary methods, including one "data poor" stock i.e. shrimp
{P. borealis) on Flemish Cap (NAFO Division 3M). For stocks
such as 3M shrimp for which quantitative reference points, targets
or limits could not be defined. Scientific Council endorsed the
interim use of stock specific checklists which include multiple,
qualitative indicators of resource status. This method, which is
similar to the matrices used in Environmental Impact Assessment,
uses the "Traffic Light" analogy because assessment results are
categorized as "green." "yellow." or "red." corresponding to fa-
vorable, uncertain, or unfavorable stock conditions. Recent stock
assessments for Atlantic Canadian shrimp stocks, including the
Scotian Shelf, the Gulf of St. Lawrence and the Labrador-
Newfoundland Shelf, were conducted using the Traffic Light/
checklist approach and results were viewed positively by scientists
(some), fisheries managers and industry. A major drawback of the
method in its current form is that it does not link assessments to
TACs or other management controls. Modeling results suggest that
"Traffic Light" results could be linked to simple harvest control
rules in a way that is consistent with shrimp stock dynamics and
management requirements, creating an integrated management
framework.

PACIFIC COAST SHRIMP TRAWL FISHERIES: NEW
MANAGEMENT AND ASSESSMENT CO-MANAGEMENT
PROGRAMS. Rick Harbo. L. Convey, J. Boutillier. and D.
Hay, Department of Fisheries and Oceans. Operations Branch,
Fisheries Management. Pacific Region. Nanaimo, B.C.. Canada.
The diverse and complex Pacific shrimp trawl fishery takes
place along the British Columbia coastline, in a number of small
inshore areas and large offshore grounds. The fleet of 248 licences
is a mix of beam and otter trawls. There are seven Pandalid species
harvested commercially and fisheries vary in complexity from
single to multiple species fisheries with a variety of markets, in-
cluding machine-peeled, hand-peeled, frozen-at-sea, fresh, and
live shrimp. Landings peaked at over 7300 tons, with annual
landed values reaching $13.6 million. Landings have declined
since 1996. to annual levels ranging from 2000 to 3000 tons at $5
to $7 million, due to low stock levels in offshore areas and more
restrictive, precautionary management practices. Fishery manage-
ment has developed rapidly from passive management at relatively
low levels of efforts to a complex suite of management programs
starting in 1997. including time and area closures, catch ceilings
(arbitrary precautionary limits, historically based or forecasted)
and quotas assigned to more than 30 new shrimp management

areas. In-season area-swept trawl surveys using commercial and
research vessels have led to fishery independent biomass indices
and exploitation rates of 25-33% have been set initially. A long-
term collaborative management and assessment program is being
developed with stakeholders. It will include logbooks, catch moni-
toring, biological sampling, and fishery independent surveys. At-
sea observers on board commercial vessels, research cruises and
plant sampling have been undertaken to determine the catch com-
position and develop preliminary estimates of by-catch, with an
emphasis on eulachon and halibut. There have been efforts in the
fleet to develop gear improvements and a code of responsible
fishing practices.

ANALYSES OF HARVEST STRATEGIES FOR PANDALID
SHRIMP POPULATIONS. Caihong Fu, T.J. Quinn, and
G. H. Kruse, School of Fisheries and Ocean Sciences, University
of Alaska Fairbanks, 1 1 120 Glacier Highway, Juneau AK 99801-
8677, USA.

Pandalid shrimp species have unique life history features, for
instance sex change, which could have great effects on population
dynamics. These populations are also highly variable in annual
recruitment, seasonal growth and natural mortality, which have
profound influence on their dynamics. Our research was aimed at
evaluating harvest strategies with these features and variations
explicitly incorporated for achieving better management. In this
paper, population dynamics were simulated over a 50-year time-
frame. A constant harvest rate of 0.3 was imposed when the popu-
lation level was above its threshold, and 0.0 when below the
threshold. The following alternative harvest policies were evalu-
ated: fishing right after hatching, fishing at the end of the growing
season but before spawning, fishing right after spawning, reducing
(or increasing) mesh size to increase (or reduce) catch of young
shrimp, and imposing an area closure to protect ovigerous females.
The policies were evaluated based on the fishing effort corre-
sponding to the catch quota, probability of population going below
threshold level over the 50 years, and annual recruitment success.
The effectiveness of these policies was contingent upon seasonal
and annual variations in growth and natural mortality. Recommen-
dations are made on harvest policies corresponded to situations of
various growth and natural mortality variations.

DEVELOPMENT OF A MANAGEMENT AND STOCK AS-
SESSMENT PROGRAM FOR THE POT SHRIMP FISHERY
FOR PANDALUS PLATYCEROS IN SOUTHEASTERN
ALASKA. Gretchen H. Bishop, T. M. Koeneman, and C. A.
Botelho, Alaska Department of Fish and Game. Commercial Fish-
eries Management, and Development. P.O. Box 240020, Douglas,
Alaska 99826-0020, USA.

The spot shrimp fishery in southeast Alaska is the last viable
pot shrimp fishery in the state. Pandaliis phayceros comprise 95%
of the landed weight: the remainder is primarily P. hypsinotus. A

560 Abstracts. September 8-10. 1999

Symposium on Pandalid Shrimp Fisheries, Halifax. Nova Scotia

10-fold increase in participating vessels since 1960 has heightened
concern for conservation and led to increasing restrictions, includ-
ing limited entry in 1996. Vessel configuration is a function of
market but appears to be evolving from small vessels with a lim-
ited fresh local market to larger catcher-processors, which sell
frozen whole shrimp, primarily to the Japanese market. The season
has shortened from year-round in 1981 to the current season which
begins on October I and closes on February 28 or when the upper
end of the guideline harvest level is reached, and opens again in
districts with quota left on May 1 . A mesh size of ! 3/4-in. and two
categories of pot configuration, "large' and "smaH' with associated
pot limits of 100 or 140 were defined in 1997. Guideline harvest
levels (GHL's) for all districts were implemented in 1995 based
upon long-term average catches. More active management, with
in-season monitoring of catch began at this time. With increasing
effort in this fishery we recognized a need to move towards a
harvest rate management strategy, thus we began stock assessment
of a limited area southwest of Prince of Wales Island (district 3) in
1997. We have established a survey protocol and collected base-
line data on catch rate index of abundance of P. pkityceros for
1997, and before and after the fishery in 1998/99. There is no
consistent trend in shrimp carapace length, number per pot, or size
at 50 percent female (L50) between 1997 and 1998. Pre and post
fishery length frequency and catch per pot data are modeled using
change in ratio techniques to attempt to estimate harvest rate, as
described in a separate paper. We examined length frequency and
L50 data for 1997 and 1998 and used Arcview CIS to map harvest
information from 1960-1997 in order to detect evidence of serial
depletion. There was evidence of reduced harvest in 1997 and a
significant decrease in average carapace length in 1998 for some
sub-districts of district 1.

FIXED ESCAPEMENT: AN ALTERNATIVE TO QUOTA
MANAGEMENT IN A SHRIMP FISHERY, .lennifer A. Bond
and J. A. Boutillier, Department of Fisheries and Oceans, Pacific
Biological Station, Hammond Bay Road, Nanaimo. B.C.. Canada
V9R 5K6.

Growth and recruitment overfishing in the spot prawn {Pun-
dalus j>kilyccros) trap fishery in British Columbia is managed
using si/.e limits and a fixed escapement system. The fixed escape-
ment is implemented using an index of the number of spawners/
trap caught in the connncrciai fishery. The development and ap-
plication of this system is reviewed, including a description of the
theoretical basis for this method of managemenl. problems with
implcmcmalion. and Ihc unplcniciilatioii procedure. The si/e limits
were introduced In 1985 and Implemented in 1988. Industry has
recognised the benefits of the si/e limits and has recently sug-
gested increases to take advantage of price dilTerenlials. The fixed
escapement system was initially based on empirical sur\ev
data. To evaluate the efficacy of this management system a num-
ber of experimental management areas were developed. This

paper reviews the present state of progress in the development of
a more model-based rationale for this system using data gathered
from one of these experimental management areas. Howe Sound.
Howe Sound has been closely monitored as an experimental prawn
management area since 1985. Data available for the area includes
catch records from the commercial fishery, biological sampling
and catch composition monitoring of the fishery, and detailed in-
formation from pre- and post-fishery research surveys earned out
every year.

MODERN TRAWLING AND BY-CATCH REDUCING DE-
VICES IN THE NORTH ATLANTIC SHRIMP FISHERIES.
Roger Larsen, Norwegian College of Fisheries Science, Univer-
sity of Tromso. 9037 Troms0. Norway.

This presentation deals with by-catch related problems in trawl-
ing for Pandalus borealis. including recent developments in fish
excluder devices. New techniques in sorting grates, and double and
triple trawl methods are also covered.

MANAGEMENT OF THE CANADIAN SHRIMP FISHERY.
John Angel, Canadian Association of Prawn Producers. P.O. Box
ICl, Head of St. Margaret's Bay. Nova Scotia, Canada BOJ IRO.
The Canadian northern shrimp fleet has been cited for its re-
sponsible fishing practices and for its success in resource conser-
vation and sustainable economic development. The northern
shrimp fishery has been managed conservatively since its incep-
tion with sustainable development being the guiding principle.
Quotas increased slowly in the early years of the fishery and ex-
ploitation rates remain low. Canada implements an elaborate man-
agement scheme for northern shrimp with light controls on quotas,
vessels, gear, and landings, including 100% observer coverage in
the offshore factory free/er fleet. The offshore fishery is rights-
based with license ht)lders held to strict Enterprise .MIocations per
company. The inshore quota is flshed competitixels among par-
ticipating vessels with quota being assigned on a coastal commu-
nity basis. Penalties and license sanctions are severe for \ iolalors.
Great strides have been made in technological de\clopmenl in-
cluding gear selectivity, bycalch control, size selection, etc. The
fishery is based primarily on a single species. Pandalus horcalis
(northern pink shrimp) and takes place off eastern Canada from
49 N to approxiniately 63 N. A second species. Painhdiis iiioii-
tiif'iii (striped shrimp), is commercially less important and is fished
in limited quantities. The commercial fishery began in 1978 and
dc\ eloped slowly until 1986 v\hen catches began increasing. Since
I99(i. quo(as have increased rapidly from some 36.000 mt in 1996
to approximately 90. 000 mt in 1999. Scientists helie\e the shrimp
resource to be comprised of a single stock or stock complex al-
though there are differences in rates of growth and maturation
across the geographic range of the species. These difterences pro-

Symposium on Pandalid Shrimp Fisheries. Halifax. No\a Scotia

Abstracts. September 8-10. 1999 561

vide the basis for delineating assessment and management units.
Current stock status, especially in the southern areas, is very
favourable with high biomass of male and female components. The
offshore fishery is prosecuted by thirteen offshore factory freezer
trawlers. All of the offshore catch is processed and frozen on board
as either cooked or raw product. The inshore fishery is prosecuted
by some Trf^Q vessels landing fresh product to shore plants for
cooking and peeling. Major markets are Asia, Europe and the U.S.

GEAR TESTING IN THE NORTHERN SHRIMP FISHERY
IN THE GULF OF MAINE TO IMPROVE SIZE SELECTIV-
ITY, REDUCE BY-CATCH AND DECREASE PRODUC-
TION LOSS. Daniel F. Schick and M. Brown, Dept. Marine
Resources. McKown Point. W. Boothbuy Hbr.. ME 04-575. USA.
The Nordmore grate has greatly reduced finfish by-catch ex-
cept for finfish of a similar size to shrimp. Also, the grate appeals
to have shifted the size selection for northern shrimp downward
somewhat. Two studies of size selection for northern shrimp and
finfish were conducted comparing small diamond mesh with l-.^/4
inch diamond mesh (commercial minimum mesh) in the cod end
with and without the Nordmore grate and with square mesh of
three sizes in the cod end behind the Nordmore grate. The square
mesh was tested using both knotted and knotless twine. Initial tests
with a double Nordmore grate showed reasonable release of small
shrimp with a bar spacing of 1" in the first grate and 7/16" in the
second grate. Trials with bar spaces of 1/4" (6.4 mm), 5/16" (7.9
mm). 3/8" (9.6 mm), 7/16" (11.1 mm) and 1/2" (12.7 mm) in the
second grate showed 1/2" provided the best escapement of small
shrimp, but allowed too many larger shrimp to escape. The 7/16"
bar spacing gave the best combination of retention of larger shrimp
and release of small shrimp. Small finfish did not readily escape
through the bars of the second grate. Cod end strengtheners of 6"
diamond and 6" square mesh placed outside the shrimp cod end
revealed no shift in the size selectivity for shrimp. Thus strength-
eners of sufficient mesh size to prevent masking of cod end mesh
may be permitted to prevent the loss of shrimp production through
cod end splitting on haulback.

TRENDS IN PROCESSING AND MARKETING IN THE
GULF OF MAINE SHRIMP FISHERY. Roland Hurtubise,

Tang of the Sea. Inc., Standish, ME, USA.

This paper reviews the history of the Gulf of Maine shrimp
fishery; with particular reference to trends in processing and mar-
keting, based on the author's 39 years of involvement with north-
ern shrimp. Operations have been greatly affected by biological
factors including unpredictable trends in abundance and seasonal
changes in product quality. Industry representatives played a major
role in developing the management program, and in recent years
industry involvement has remained strong. Final comments center

around the importance of maintaining continued production of
Gulf of Maine shrimp and the author's perspectives on options for
achievinc this goal.

THE ICELANDIC SHRIMP INDUSTRY. Petur Bjarneson,

Fisheries Association of Iceland. Reyjavik, Iceland.

This talk deals with the development of the Icelandic shrimp
industry, including a history of its expansion and the current state
of processing and marketing. The industry developed slowly from
1935 to 1967 and then grew quickly during a period of decreasing
prices. Out of necessity Nordic countries, including Iceland, began
to cooperate in marketing activities and have a long history of
experience in this area. Current challenges include decreasing local
catches in a situation where catches are increasing elsewhere (e.g.,
Canada) and world trade of raw materials has reached unprec-
edented levels.

FINFISH BY-CATCH EFFECTS ON THE QUALITY OF
OCEAN SHRIMP, PANDALUS JORDANI. Vicki H. Kutz-
ikowski. R. Hannah, G. Sylvia, and M. T. Morrissey, Hatfield
Marine Science Center, Oregon State University, 2040 Marine
Science Dr.. Newport, Oregon 97365, USA.

By-catch reduction devices (BRD's) have been used voluntar-
ily in the ocean shrimp, Pandalus jordani, fishery off the Pacific
Northwest United States to reduce by-catch and the associated
labor and time costs. It has been hypothesized that by-catch may
affect the quality of the shrimp by causing breakage. In this study,
the effect of finfish bycatch on the quality of shrimp was evalu-
ated. A double-rigged commercial shrimp vessel was chartered for
test fishing. One net employed a Nordmore grate BRD and the
other served as a control. By-catch was measured from each net.
The shrimp catch was kept separate by side of gear and day of
fishing and sampled throughout processing. Shrimp samples were
collected at seven stages from the deck to finished product and
evaluated for percent breakage by weight. At greater than 50%
by-catch, there was a small (1-7%), but statistically significant (P
< O.I) decrease in breakage of .shrimp in the excluder net for
samples taken before placement in the hold, after being iced and
raked, before being cooked and after the second shaker. After this
point the differences became non-significant. The mean percent
breakage increased from a mean 2.4% breakage before placement
in the hold to a mean 1 8.0% breakage in the final product. Other
factors such as the molt condition, count per pound, carapace
length, phosphate treatment, moisture and microculture were ex-
amined and found to not influence breakage. Based on these find-
ings, it is concluded that finfish by-catch does contribute to the
breakage of ocean shrimp, however the impact is small relative to
breakage caused by other handling procedures.

562 Abstracts. September 8-10. 1999

Symposium on Pandalid Shrimp Fisheries. Halifax. Nova Scotia

MONITORING THE SHRIMP TRAWL FISHERY IN BRIT-
ISH COLUMBIA. Jason Clarke and W. E. L. Clayton, Archi-
pelago Marine Research Ltd., 200-525 Head St.. Victoria BC V9A
5S1. Canada.

In 1997. the Department of Fisheries and Oceans. Canada
(DFO) implemented a new management regime for the shrimp
trawl fishery in British Columbia in response to concerns of in-
creased fishing pressure and lack of fishery data. The BC coast was
divided into Shrimp Management Areas (SMA). and catch ceilings
for these areas were established. To support this change, shrimp

license holders were required to fund a data collection program
that included; ( 1 ) a hail reporting system to monitor vessel activity,
(2) a logbook catch monitoring system to monitor catch by area
and species, and (.■!) an offshore observer by-catch monitoring
program to collect catch composition information for shrimp
trawls. Since 1997. Archipelago Marine Research Ltd.. an inde-
pendent service agency, has been providing these data collection
services on behalf of the Pacific Coast Shrimpers" Cooperative
Association. The information collected is forwarded to DFO for
use in the management of the shrimp trawl fishery.

J

.Iminuil of Shellfish Research. Vol. 19. No. 1. .'563-584. 2000.

ABSTRACTS OF TECHNICAL PAPERS

Presented at the 20th Annual Meeting

MILFORD AQUACULTURE SEMINAR

Milford. Connecticut
February, 2000

563

Milford AqiiacLillure Seminar, Miltbrd. Connecticut Abstnicrs. February 2000 565

CONTENTS

Walter J. Blogoslawski

Overview. 20"' Milford Aquaculture Seminar 567

Craig L. Appleyard and Joseph T. DeAlteris

Maximizing efficiency of liard clam, Merccnaria mercenaria. culture in an experimental-scale upweller 567

Bethann Balazsi and Gary Wikfors

Experimental evidence for phytic acid-phosphorus use by pure cultures of marine microalgae 567

Brian F. Beat

The importance of temporal and spatial replication of field experiments: effects of seagrass cover on the growth and

survival of cultured juveniles of the soft-shell clam, Mya arenaria, and hard clam, Mercenaria mercenaria 568

Diane J. Broiisseau, Margot Gallowitsch and Lenka Hurton

Prey size and species selection by the Asiatic shore crab, Hemigrapsus sanguineus (deHaan) feeding on bivalve prey. . 568
Maronda V. Brown, Linda Strausbaugh and Sheila Stiles

Methodology for the generation of molecular tags in Placnpecten magellanicus (sea scallop) and Argopecten

irradians (bay scallop) 569

John J. Curtis and Sherry Lonergan

Educational partnerships: its value to the future of aquaculture 569

Russell P. Davis

James River market sized oysters have their late summer survival rates doubled by marl treatment of their water 569

Eric Edwards

Developments cultivating bivalve molluscs in Europe 570

Susan Ford, Zhe Xu and Gregory DeBrosse

Acquisition and prevention of MSX and Denno in a hatchery and land-based nursery: A DNA assay investigation 571

Susan Ford, Roxantia Smolowitz and Marnita Chintala

The question of temperature and Perkinsus marimis (Denno) activity in the northeastern United States 57 1

Ronald Goldberg, Jose Pereira, Paul Clark, Bernard Faber, David Porter, Lorenz Rinek, Barbara Ranter, Eric Kanter and
Walter Lord

Population enhancement efforts for the bay scallop, Argopecten irradians. in the Niantic River estuary,

Connecticut, USA 572

Ximing Guo, Susan Ford, Gregory DeBrosse and Roxanna Smolowitz

Breeding for a superior eastern oyster for the northeastern region 572

J. Michael Hickey

State implementation of the NSSP interim control plan for Vibrio paraluwmolyticus 573

Porter Hoagland and Haiike L. Kite-Powell

The economics of blue mussel grow-out: aquaculture at an offshore site 573

Richard C. Karney, John C. Blake and Thomas E. Berry

Shellfish production in the Blake Floating Hatchery and in modified tidal upweller nurseries in 1999 574

Gordon King

Operations at Taylor Seafood 575

Richard Langan

Submerged longline culture of blue mussels (Mxtilus edulis) at an open ocean site in the Gulf of Maine 575

Ken Leonard lU, Josefa Dougal, Marta Gomez-Chiarri and Arthur Ganz

Detecting the presence of Perkinsus marinus and Haplosporidium nelsoni in the oyster, Crassostrea virginica. in

Rhode Island waters: A survey update 575

Dale F. Leavitt, William Burt and Charles Koines

Federal crop insurance becomes available for quahog farmers 575

Chang Chun-Mean Lin, Sineenat Siri, Sheila Stiles and Thomas Chen

Production of transgenic mollusks and crustaceans 576

Clyde L. MacKenzie, Jr.

A review of current clam culture in New England 576

Mark S. Miller and Joseph K. Buttner

Symbiotic relationship of the sea scallop and red hake as a possible management tool 577

Brandy M. Moran, Clifford A. Goudey and Jessica Rabe

The culture of haddock, Melanogrammus aeglefinus, using a recirculating system in an urban setting 577

566 Ahstracls. February 2000 Milford Aquaculture Seminar. Milford. Connecticut

Dean M. Perry, Laurel Ramseyer and Joseph E. Goncalo

Growth of juvenile tautog fed commercial diets in a cross-over experiment 578

Dean M. Perry, David A. Nelson and Joseph E. Goncalo

Growing rotifers on single and mixed alga! strains to be used as a first feed for larval tautog 578

Enrico Picozza, Joseph Crivello, Maronda V. Brown, Linda Strausbaiigh and Sheila Stiles

Status report for the characterization of the bay scallop. Aii><ipecten inadians, genome 578

Steven Pitchford, Richard Robohm, Sharon MacLean and Laurel Ramseyer

Observations on mycobacteriosis in the tautog (Taiitoga nnitis) 579

Leonora Porter, Eugene Zamojcin, Joe DeCrescenzo, Inke Sunila, John Volk, and John Karolus

The presence of Vibrio parahaemolyticus in Crassostrea virginica at specific locations along the Connecticut and

Long Island Shore — participation in the Vibrio parahaemolyticus ISSC — FDA survey for June 1999 to June 2000 579

Julia Rankin, Michael Wilcox and Donald Harris

Exploring diversity through aquaculture 580

Michael A. Rice, April Valliere, Mark Gibson, and Arthur Ganz

Ecological significance of the Providence River quahogs: Population filtration 580

Spencer Russell, Soledad Penna and Richard French

Comparative evaluation of the multiplex PCR with conventional detection methods for Haplosporidium nelsoni

(MSX) Haplosporidium coshde (SSO). and Perkinsus marinas (Dermo) in the eastern oyster, Crassostrea virginica ... 580
Spencer Russell, Kristen Hobble, Tom Barrage, Claudia Koerting, Sylvain De Guise, Salvatore Frasca Jr. and
Richard A. French

Identification of a protozoan parasite in the American lobster, Homarus americaniis. from Long Island Sound 581

Barry C. Smith and Mark Dixon

Living contaminants in microalgal feed production tanks — what do we do now? 581

Roxanna Smolowitz, Ernest Marks, Chris Brothers, Dale Leavitt and Bruce Lancaster

Recent results from field and laboratory studies of QPX 582

Bethany A. Starr

Effects of various microalgal diets on the growth and survival of larvae of sea scallops. Placopecten magellcmicus 582

Sheila Stiles, Tasha Robinson and Joseph Choromanski

Observations on growth and survival of juvenile bay scallops (Argopecten irradians) from genetic lines under

different density and holding conditions 582

Inke Sunila, Joseph DeCrescenzo, John Karolus and John Volk

Principal diseases of Connecticut's oysters 583

Karin A. Tammi, Wayne H. Turner, Luning Sun and Michael A. Rice

Establishment of Rhode Island's first conmiercial shellfish hatchery 583

James C. Widman, Jr.

An inexpensive digital temperature sensor for data acquisition use in aquaculture 584

Gary H. Wikfors, Jennifer H. Alix, Sara Barcia, Julie Cullum, Sandra E. Shumway and Roxanna M. Smolowitz

Responses of bay scallops, at several life-history stages, to cultures of potentially-harmful marine microalgae 584

Loy Wilkinson

Process design for Artenna culture at Coastal BioMarine 584

Milford AquiiL-ultiiie Seminar. Milt'oid. Connecticut

Abstnicts. February 2000 567

OVERVIEW. 20^" MILFORD AQUACULTURE SEMINAR.
Walter J. Blogoslawski, U.S. Department of Commerce. National
Oceanic & Atmospheric Administration. National Marine Fisher-
ies Service, Northeast Fisheries Science Center. Milford Labora-
tory. 212 Rogers Ave.. Milford. CT 06460.

The 20''' Anniversary Meeting of the Milford Aquaculture
Seminar attracted 39 speakers who gave 42 presentations covering
such topics as bivalve culture in Europe and New England, clam
culture. Vibrio paraliaemolyticus surveillance on shellfish beds,
oyster and lobster diseases, transgenic species and genetic modi-
fication to prevent disease, harmful algal blooms, high school
aquaculture curricula, blue mussel cultivation in the Northeast and
Pacific Northwest, designs for recirculating fish culture systems,
and diseases of cultured tautog. Over 160 attendees from the US.
Canada and Great Britain met during the two-day conference to
discuss recent problems of the aquaculture industry and suggest
potential solutions. The cooperative spirit shared by the partici-
pants, many of whom compete against each other in a difficult
business, was evident in the roundtable discussion.

Staff from 41 different aquaculture companies, scientists from
1 1 universities, teachers from two local high schools with aqua-
culture-vocational programs, and staff from 7 marine laboratories
attended the seminar. Also, the Director of the National Marine
Fisheries Service met many of the attendees and presented a lun-
cheon address.

The meeting was sponsored by the US Department of Com-
merce NMFS Milford Laboratory, Milford. CT and abstract print-
ing was courtesy of the US Department of Agriculture. Northeast-
em Regional Aquaculture Center. N. Dartmouth. MA. Their sup-
port is greatly appreciated.

MAXIMIZING EFFICIENCY OF HARD CLAM, MERCE-
NARIA MERCENARIA, CULTURE IN AN EXPERIMEN-
TAL-SCALE UPWELLER. Craig L. Appleyard and Joseph T.
DeAlteris. Department of Fisheries, Animal and Veterinary Sci-
ence. University of Rhode Island. Kingston. Rhode Island 02881.
Upwellers have proved to be extremely effective as bivalve
nursery units and their use is steadily increasing in North America.
The re-analysis of previous work by others suggested an asymp-
totic relationship between growth {9c volume increase per 30 days)
and chlorophyll-a effective flow rate (chlorophyll-a flux per unit
biomass. jji.g/min kg"'). The purpose of the study was to develop
a relationship between flow rate, stocking density and growth in
order to determine the flow rate and density that optimizes growth.
Furthermore, the study was designed to investigate other signifi-
cant environmental parameters influencing bivalve growth in an
experimental-scale upweller system. Hard clams, Meivenaria iner-
cenaria. were grown from ~2 mm (longest axis) to -13 mm in a
forced flow floating upweller from June 21 to August 19. 1999 in

Point Judith Pond. Wakefield. Rhode Island. The flow rate and
stocking density were varied in order to produce a chlorophyll-a
effective flow rate range of 360 to 1500 (j.g/min per liter of clam
volume, and growth and environmental parameters were measured
semi- weekly. During the initial experimental period (June 21 to
24) an asymptotic relationship was observed between growth (%/
day) and chlorophyll-a effective flow rate. The a.symptotic rela-
tionship did not hold for the remainder of the experiment because
ambient environmental conditions appeared to limit growth. Spe-
cifically, growth was linearly correlated with morning-dissolved
oxygen. The relationship is strongest for the upper third of the
fastest growing animals because the removal of the slower grow-
ing animals eliminates the effect of flow rate on growth.

EXPERIMENTAL EVIDENCE FOR PHYTIC ACID-
PHOSPHORUS USE BY PURE CULTURES OF MARINE
MICROALGAE. Bethann Balazsi, Natural Science Division,
LIU. Southampton. NY 11968: Gary H. Wikfors, USDOC.
NCAA. National Marine Fisheries Service. Northeast Fisheries
Science Center. Milford Laboratory, Milford. CT 06460.

Phytic acid, or phytin. is a cyclic organic molecule containing
six phosphorus atoms. Approximately half of the phosphorus in
agricultural livestock feeds, e.g., com and soybeans, is in the form
of phytic acid. Most livestock animals are unable to digest phytic
acid; therefore, essentially all phytic acid consumed is released as
waste that may find its way into coastal waters. Some soil fungi
and bacteria produce a phytase enzyme that breaks down phytic
acid, thereby releasing the phosphorus in the oxidized form of
phosphate ions. The capability of marine and estuarine microalgae
to obtain phosphorus from phytic acid directly, without fungal or
bacterial decomposition, has not been investigated systematically.
although some reports have suggested that harmful dinoflagellates
may be selectively fertilized by phytic acid in coastal waters pol-
luted with livestock waste. In this study, we tested the capacity of
eight bacteria-free microalgal strains, from several major algal
classes, to grow on phosphorus from phytic acid.

Culture media (E medium basal) were prepared containing iso-
molar phosphorus (P) as either phosphate, phytic acid, or a 50:50
mix of the two, or with no added phosphorus source. Each of these
media then was treated with commercially-available phytase en-
zyme (BASF Corp.) or left untreated, resulting in eight different
medium treatments. Triplicate. 10-ml test tubes of each medium
were inoculated with algal strains that had been grown for one
subculture in medium with no added phosphate to deplete any
cellular P reserves. Algae tested were: dinoflagellates, Pruiocen-
tniin inicans (CCMP1589) and P. ininimiiin (EXUV); diatoms,
Chaetoceros neogracile (Chaet-B 1 and Amphora cojfeaeformis ( A-
ORA). prymnesiophytes: Isochrysis sp. (T-ISO) and Pavlova gy-
rans (#93), and green algae; Tetraselmis chid (PLY429) and Du-

568 Ahstracrs. February 2000

Milford Aquaculture Seminar. Milford. Connecticut

luiliellci tertiolecta (DE). Culture growth was monitored with
thrice-weekly spectrophotometer readings, and final cell densities
were determined by cell counts after 40 days.

Growth curves plotted from spectrophotometer readings
showed at least some initial growth of all strains (with one excep-
tion) in all media, including media with no added P. This growth
indicates that seawater used in the media contained some available
P, or possibly that stored P in inoculum cells was sufficient to
support limited cell division. This initial growth made maximal
algal division rates unreliable for comparing experimental treat-
ments; therefore, results were analyzed in terms of final population
densities. P. inicans did not grow in any medium, possibly because
of lethal phosphorus starvation of the inoculum culture, and was
eliminated from results analysis. Of the seven remaining algal
strains, only two showed clear evidence of phytic acid-P use with-
out phytase enzyme: T-ISO and A-ORA: neither of these is a
dinoflagellate. Interestingly, it was not clear from statistical analy-
sis if phytase enzyme effectively released P from phytic acid in a
form usable by the algae, because addition of phytase enzyme
alone (without phytic acid) consistently stimulated algal growth
equal to the phytic acid -I- phytase treatment. The commercial
phytase product is extracted from microbial biomass. and may
contain phosphorus itself. These results indicate that microalgae
other than dinotlagellates can possess the capacity to obtain phos-
phorus directly from phytic acid. This finding has application in
management of coastal water quality, as well as in aquaculture.
where phytic acid in cultured fish waste can be expected to support
a more diverse algal assemblage than previously suggested.

Effects of submerged aquatic vegetation, such as eelgrass,
Zostera marina, on the survival and growth of infaunal bivalves
provides an interesting example for culturists to consider. The
current paradigm states that grass beds provide spatial refuges for
clams and quahogs. The mechanism for this protection has been
shown experimentally to be related to the presence of the upright
blades which affects visual predators, and. more importantly, the
presence of below-ground roots and rhizomes which reduce the
mobility and effectiveness of both infaunal and epifaunal preda-
tors.

From 1990-1999. five independent 3- to 6-month field tests
using hatchery-reared juveniles oi Mya arenaria (SL = 5 to 15
mm) were conducted on a tidal fiat in Cutler. Maine to examine the
influence of the presence or absence of eelgrass on clam growth
and survival. From 1980-1981. similar field tests using cultured
juveniles of Mercenaria mercenaria (SL = 8 to 15 mm) were
conducted in two shallow subtidal regions of eastern North Caro-
lina. Results from both geographic locations and across time gen-
erally discount and challenge the current paradigm. Sur\ ival rates
of M. arenaria were significantly reduced in eelgrass beds com-
pared to adjacent, unvegetated areas primarily due to increased
predation by crabs and other crustacean predators. Similarly, grass
beds in North Carolina afforded M. mercenaria no refuge from
predators. Growth rates of M. arenaria were significantly de-
pressed inside vs. outside eelgrass beds. Growth rates of M. mer-
cenaria either showed no difference between habitats or were
higher in grass beds. These studies indicate the importance of
replicating field experiments temporally and spatially. Both public
and private aquaculture programs should exercise caution about
placing small bivalve seed into or near grass beds as they are
habitats where crabs seek refuge from their predators (gulls, large
fish, and other crustaceans).

THE IMPORTANCE OF TEMPORAL AND SPATIAL REP-
LICATION OF FIELD EXPERIMENTS: EFFECTS OF SEA-
GRASS COVER ON THE GROWTH AND SURVIVAL OF
CULTURED JUVENILES OF THE SOFT-SHELL CLAM,
MYA ARENARIA. AND HARD CLAM, MERCENARIA MER-
CENARIA. Brian F. Beal, University of Maine at Machias. 9
O'Brien Avenue. Machias. ME 04654.

Field experiments in marine soft-bottom habitats that are de-
signed to test specific hypotheses about mechanisms regulating
growth. sur\ ival. or fecundity of infaunal, hatchery-reared clams
and quahogs are difficult to conduct due to logistical problems
imposed by the nature of soft sediments.

These constraints mean that field trials typically suffer from
poor replication (both spatially and temporally). Generalizations
about processes that regulate these populations rarely can be made.
For field culturists or communities seeking practical information
concerning seed size, planting dates and locations, or what types of
predator deterrent devices to use. the scientific literature provides
conflictinij lessons that relate directly to this inability to generalize.

PREY SIZE AND SPECIES SELECTION BY THE ASIATIC
SHORE CRAB. HEMIGRAPSUS SANGUINEUS (DeHAAN)
FEEDING ON BIVALVE PREY. Diane J. Brousseau. Margot
Gallnwitsch, and Lenka Hurton. Fairfield University. Fairfield.
CT 06430.

Prey selection by the Asiatic shore crab (Hcnnnrapsiis san-
s^iiinen.s I was investigated in relation to bivalve prey of different
sizes and species. In the laboratory, four size classes of male crabs
(Small. 10-15 mm CW; Medium. 15-20 mm CW: Large. 20-25
mm CW; X large, >25 min CW) and three size classes of female
crabs (Small. 10-15 mm CW; Medium. 15-20 mm CW; Large,
20-25 mm CW) were offered three bivalve species. Mylihis edidis.
Mya arenaria and Cras.sostrea virginica. Ninety-eight percent of
both the male and female crabs tested ate bivalve prey. Male crabs
in all size classes selected larger M. edidis than similarly-sized
female crabs, w hereas medium and large male crabs selected larger

Milt'ord Aqiiacultiire Seminar. Miltord, Connecticut

Abstracts. February 2000 569

C. virginica than female crabs but only the largest male crabs
selected larger M. arenaria than the females. Maximum prey sizes
eaten by the Asiatic shore crab were M. arenaria. 20-25 mm SL:
M. edulis. 15-20 mm SL and C. virginica. 10-15 mm SL. Both
male and female crabs ate significantly more M. arenaria than
either M. edulis or C. virginica (59% M. arenaria vs. 40% M.
edulis. P = .001; 83% M. arenaria vs. 22% C. virginica. P —
.0001) indicating a strong preference for soft shell clams. Sexual
differences in predation patterns reported here are likely due to
differences in the cheliped morphology of male and female crabs.

Community

METHODOLOGY FOR THE GENERATION OF MOLECU-
LAR TAGS IN PLACOPECTEN MAGELLANICUS (SEA
SCALLOP) AND ARGOPECTEN IRRADIANS (BAY SCAL-
LOP). Maronda V. Brown and Linda Strausbaugh. University
of Connecticut, Department of Molecular & Cell Biology. Storrs,
CT 06269: Sheila Stiles. USDOC. NCAA, National Marine Fish-
eries Service, Northeast Fisheries Science Center, Milford Labo-
ratory, Milford, CT 06460.

Using various molecular genetic techniques that include restric-
tion fragment length polymorphisms (RFLPs). Randomly Ampli-
fied Polymorphic DNAs (RAPDs), and Polymerase Chain Reac-
tion (PCR). we have begun to examine regions associated with
molecular markers in the sea scallop. Placopecten magellanicus.
P. niagellaniciis samples were obtained from several regions off
the Northeast Atlantic coast at multiple sites around Georges Bank
and from the southeastern Canadian waters. We analyzed samples
from both inside & outside of the United States governmental
restricted area. Preliminary screening of a Placopecten magellani-
cus genomic library seems to reveal positive clones using primers
made to both Drosophila melanogaster (fruit tly) histone H2A-
H2B and to the Pisaster ochraceus (sea star) H3 histone gene.
Preliminary comparisons were made with the bay scallop Ar-
gopecten irradians. Further analyses should result in the identifi-
cation of a series of polymorphic regions in both Placopecten
magellanicus and Argopecten irradians as well as provide basic
molecular biology characterization of the highly conserved histone
gene family in mollusks.

Industry-

Educational and
Professional
Technology

Figure \. STAR diagram.

Education -i- Partnerships -l- Aquaculture
Success

High School Student

Education is defined as the development of a person by fostering
to varying degrees the growth of expansion of knowledge, wis-
dom, desirable qualities of mind or character, physical health, or
general competence especially by a course of formal study or
instruction.

Partnership, by definition, is an alliance of at least two parties
with a common interest usually leading to a mutual benefit and
accomplishment.

Aquaculture. a rapidly growing global industry whose own
success will be dependent on the numbers of young, creative and
committed students who we can encourage to pursue it as a life-
long career.

The Bridgeport Regional Vocational Aquaculture School, in its
short history, realized from the beginning the value of partner-
ships. Traditional education will always be the foundation, but like
any structure, success is measured by the extent of additions to that
foundation. Early on, the administration and staff of the Bridgeport
Regional Vocational Aquaculture School committed themselves to
not only providing the solid academic foundations but also seeking
multiple opportunities for the students to apply their hard-earned
knowledge to real-life and meaningful collaborative projects. It
worked! And it continues to work! More students are scoring
higher on standardized academic tests and there continues to be a
significant yearly increase in the numbers of students who pursue
related post-secondary education.

Through the dedication of staff and a commitment to introduce
students to the value of their own commitment to learning and
involvement, they partnered with their students and a STAR (Fig.
I ) was bom. As a result, the aquaculture industry and the students
who choose to attend the Bridgeport Regional Vocational Aqua-
culture School will continue to benefit from the school's commit-
ment to this simple mathematical equation.

EDUCATIONAL PARTNERSHIPS: ITS VALUE TO THE
FUTURE OF AQUACULTURE. John J. Curtis and Sherry
Lonergan. Bridgeport Regional Vocational Aquaculture School.
60 St. Stephens Road, Bridgeport, CT 06605.

A simple mathemafical equation that has been the driving force
of the Bridgeport Regional Vocational Aquaculture School since
its inception in the 1988-89 academic year is:

JAMES RIVER MARKET SIZED OYSTERS HAVE THEIR
LATE SUMMER SURVIVAL RATES DOUBLED BY MARL
TREATMENT OF THEIR WATER. Russell P. Davis, Sweet-
Water Oysters. 1521 Quail Ft. Rd.,Va. Beach. VA 23454.

James River oysters, market sized at about 250 per bushel, are
not normally expected to survive another summer. The cau.se of
death is usually attributed to either MSX or Dermo.

570 Abstracts. February 2000

Milford Aquaculture Seminar. Milford. Connecticut

This experiment appears to reproduce conditions under which
oysters thrived abnormally well. Prior to this experiment it appears
that no one successfully intervened late in the disease process. The
closest research is particularly relevant in that iron is proven to be
a factor in the Dermo disease process.

A lot of those older oysters were given water that was run
through a mesh bag of fossil shell hash. Twice the proportion of
oysters survived in the treated water as did in the untreated water.

Approximately, 20.8% of the no-marl oysters survived and
41.7% of the marled oysters survived. Given the sample size and
the binomial nature of the survival statistic there is a 1:16 chance
that the marl treatment made no difference.

The shell hash was dissolved by the passing water indicating
calcium carbonate under-saturation. The existence of instances
of calcium carbonate under-saturation appears controversial. The
accumulation of iron sulfide in the troughs was surprising
and inspired additional inquiry into the geochemistry of iron sul-
fide. Apparently, extremes in .sediment carbonaie/sulfide ratios do
occur.

When iron sulfide is resuspended and oxidized, the resulting
burst of sulfuric acid can produce calcium carbonate undersatura-
tion particularly at the sediment/water boundary layer. This pro-
cess could explain some disappearance of shell hash and cultch.
Additional sediment chemistry experiments shed light upon the
geochemical mechanisms behind the abrupt discontinuity in Vir-
ginia between prehistoric sediments that are dominantly oxic/
carbonate and historic sediments that are dominantly anoxic/
sulfide.

These sediment chemistry experiments explored extremes in
carbonate/sulfide ratios and suggest that a restoration to prehistoric
levels of estuarine productivity is too conservative a goal - Sun-
light-limited high goals for shellfish productivity may be easier to
achieve than more modest goals provided that "whole-river hus-
bandry'" is allowed.

DEVELOPMENTS CULTIVATING BIVALVE MOLLUSCS
IN EUROPE. Eric Edwards, Shellfish Association of Great Brit-
ain. Fishmongers' Hall. London Bridge, l.ondon, EC4R 9EL. L'K.

Increasing t|uantilics of bivalve molluscs are now being pro-
duced in Huropcan waters by various cultivation techniques. Eu-
ropean production of bivalves is now around S2.S.()00 metric
tonnes a year. Mussels and Pacific oysters represent Just over 90'7r
of this total. The lead countries arc: France, Spain. Holland and
Italy.

Clams arc another important species, especially in soulhcrn
Europe, where a number of different types are cultivated or har-
vested from wild resources. Exciting developments are also taking
place in the culture of scallops, abalone and cxolic clams. The

2000s will see increased production of these molluscs using hatch-
eries and technology adopted from other countries. Historical
records show that bivalve molluscs like oysters and mussels have
been cultivated in Europe for generations using traditional culti-
vation methods. But the last 100 years have seen big changes in
many of these shellfisheries caused by natural and economic prob-
lems.

For example, France was once a major producer of the Euro-
pean flat oyster {Ostrea edulis) selling over 500 million a year
between 1877 to 1887. But massive mortalities of these native
oysters encouraged the French growers to bring in Portuguese
cupped oysters. In the I960"s. a viral infection hit the "Ports', then
the parasite Bniuimia infected the natives and since the 1970s, the
French have cultivated the Pacific oyster. As a result, their oyster
industry is now dominated by Crassoslrea ^igas. at an annual
production level of 150,000 tonnes. Similar changes have also
taken place in the UK. Holland and Ireland where oyster produc-
tion has also dramatically declined.

As a result, mussels have steadily taken over as Europe's pre-
mier bivalve species. Once the convenience food of the working
classes, mussels have now become a favourite seafood in most
European countries, especially France, Belgium and Spain. Two
species are grown: the northern blue mussel (Mytilns edulis) and
the Mediterranean species (Myti/us galloprovincialis). Culture
methods range from the mechanized bottom system used in the
Netherlands to rope culture using rafts or buoys in Spain and Italy.
These two methods have also been used successfully, although on
a more limited scale, in Ireland, UK, Sweden and Greece.

As well as various natural diseases. Europe's bi\alve industry
has suffered from the effects of pollution, mainly from coastal
sewage discharges and organo-tin (TBT) antifoulant paints used on
ship's hull and fish cages. Agricultural run off is also a new prob-
lem as are toxic algal blooms. The need to supply safe shellfish has
prompted the European Commission to formulate and introduce
Shellfish Hygiene Directives which classify har\esting areas and
have set bacterial and algal toxin standards for waters and shellfish
flesh. The use of approved depuration plants — mainly using ultra-
violet light or chlorinalion to sterilise seawater — has helped to
reduce public health outbreaks in many countries. TBT paints,
which caused malformations in Pacific oysters ani.1 killed bixalve
larvae, have since been controlled by national legislation in many
European countries.

There is a growing market for bi\al\e molluscs in Europe and
in general the demand is increasing. Exports between Member
Stales are also growing in imporlance, as more consumers perceive
shellfish as attracli\e, healthy and differenl. There are opportuni-
ties to be had in both niche markets or the bulk Tuarkel. Added-
value products are gaining in popularity, both in terms of improv-
ing ihc produce presentation, convenience and for extending shelf
life. Europe's huge bi\al\e production and the traditional expertise
of shellfish producers, along with new technologies, will ensure
the continued success of the culli\alion industrv in the 200()s.

Milford Aquacullure Seminar. Milford. Connecticut

Abstracts. February 2000 571

ACQUISITION AND PREVENTION OF MSX AND DERMO
IN A HATCHERY AND LAND-BASED NURSERY: A DNA
ASSAY INVESTIGATION. Susan Ford. Zhe Xu, and Gregory

DeBrosse. Haskin Shellfish Research Laboratory. Institute of Ma-
rine and Coastal Science, Rutgers University, 6959 Miller Avenue,
Port Norris, NJ 08349.

Over the past decade, the two major pathogens of eastern oys-
ters [Haplosporidiiim nelsnni [MSX] and Perkinsus marinus
[Dermo] have become epizootic in many areas of the northeastern
United States where oyster culture depends on hatchery-produced
seed. Questions often arise as to whether larval or juvenile stages
become infected in the hatchery or nursery phase of production. To
help answer these questions, we utilized both traditional and mo-
lecular diagnostic methods to detect the pathogens in larval and
juvenile oysters reared at our Cape Shore hatchery/nursery on the
shore of lower Delaware Bay where both parasites are enzootic. In
1995, we found very low levels of P. marinus in juveniles held for
7 weeks in a nursery upweller system receiving raw bay water.
Detection was made using the whole-body parasite burden assay
and Ray's Fluid Thioglycollate Medium (RFTM). In 1998, we
analyzed juveniles in the same upweller system for both parasites.
The body burden assay was used for P. marinus, whereas H.
nelsoni was diagnosed with both traditional tissue-section histol-
ogy and pathogen-specific primer/PCR technology. Oysters resi-
dent in the system for 7-8 weeks were infected with both parasites,
In 1999, we expanded our investigation to include oysters in the
hatchery, as well as in the nursery, and we employed both mo-
lecular and classical methods for detection of both parasites. Three
spawnings were made at different times during the summer. From
each spawning, samples of eyed larvae and 1-mm spat were col-
lected from the hatchery, where water was filtered to 1 p. and
UV-treated. Juveniles were taken 5 weeks after deployment in the
nursery.

One aliquot of the first spawning remained in the filtered/UV-
treated hatchery water for an additional 10 weeks and was com-
pared with animals kept in the raw-water nursery upwellers during
the same period. Neither pathogen was detected, using the mo-
lecular assays, in eyed larvae, 1-mm spat, or spat held in the
hatchery for an additional 5 weeks. A positive response to H.
nelsoni was detected after 10 weeks in the hatchery, although none
was found for P. marinus. The PCR assay detected H. nelsoni in
juveniles held in the nursery, although traditional histology failed
to find infections. Unexpectedly, the PCR assay for P. marinus
failed to tlnd infections in the nursery system, whereas the whole-
body RFTM method detected small numbers of parasites. It is clear
that oysters in a nursery system receiving raw water pumped from
an area enzootic for P. marinus and H. nelsoni are highly likely to
become infected by both parasites, although infections may be
very light. Filtration to 1 |jl and treatment with UV appear to
protect larval and early spat stages from both pathogens, although
there was some evidence that juveniles held for a prolonged period

in the hatchery did become infected with H. nelsoni. Whether this
was because of infective particles passing through the system or
accidental contamination of the sample at some stage of processing
is presently unknown.

THE QUESTION OF TEMPERATURE AND PERKINSUS
MARINUS (DERMO) ACTIVITY IN THE NORTHEAST-
ERN UNITED STATES. Susan Ford, Haskin Shellfish Research
Laboratory, Institute of Marine and Coastal Science, Rutgers Uni-
versity, 6959 Miller Avenue, Port Norris, NJ 08349; Roxanna
Smolowitz, Marine Biological Laboratory, Woods Hole, MA.
02543; and Marnita Chintala. Atlantic Ecology Division, U.S.
EPA, Narragansett, RI 02882.

During the early 1990s, epizootic outbreaks of Denno disease,
caused by the water-borne parasite Perkinsus marinus. occurred
over an area from Delaware Bay, NJ to Cape Cod, MA, areas
previously free of the disease. The apparent northward movement
of P. marinus coincided with a pronounced warming trend, par-
ticulariy during the winter. Knowledge that the parasite is most
active at high temperatures led to the hypothesis that the warmer
water temperatures had created a more favorable environment for
P. marinus in the northeastern US, which allowed it to proliferate
and spread from localized foci in infected oysters historically in-
troduced from the south. An alternative hypothesis was that a
low-temperature tolerant strain of P. marinus had developed and
invaded northeastern oyster growing areas.

Surprisingly, anecdotal reports from oyster growers indicated
that the disease was causing few deleterious effects in the North-
east. It was thought that the lower temperatures and shorter grow-
ing seasons might prevent P. marinus from reaching harmful lev-
els. To investigate this possibility and to document the relationship
between temperature cycles and Dermo disease developinent in the
Northeast, we monitored oyster stocks between Delaware and
Cape Cod Bays over a two-year period from 1996 to 1998. We
regulariy measured P. marinus prevalence and intensity; oyster
growth, condition index and mortality; and the presence of other
disease agents. Results showed that in contrast to the prevailing
reports, P. marinus behaved in its new range very much as it does
in southern areas where it has been enzootic for decades. Seasonal
cycles were similar, as was the 2-3 year progression to a full
epizootic. The pathogen reduced oyster meat condition, but gen-
erally not until oysters had been under disease pressure for two
years. Mortality was clearly associated with elevated infection
levels, but did not become important until the second year ot
exposure. Cumulative mortality over the two years of the present
study was at least as great as in more southern locations. The most
obvious reason for these observations is that temperatures in most
of the growing areas examined readily became warm enough to
sustain high P. marinus proliferation, and winters were not cold
enough to limit disease cycles.

572 Abstracts, February 2000

Milt'ord Aquaculture Seminar. Milford. Connecticut

Tine distribution and abundance of P. maiinits seems to have
stabilized after its initial incursion into the Northeast. Its presence
has not diminished in areas where it has become well established
and it has not intensified in areas that experienced little or no
infection pressure during the early 1990s. This second observation
does not mean that these areas are likely to remain free of Dermo
disease. There are no obvious environmental limits (i.e.. tempera-
ture or salinity) to the parasite in many of these areas - only a
(cuiTent) lack of infective elements. There is no reason that epi-
zootics should not occur if more infective particles are introduced
into these or adjacent sites, either naturally or through the impor-
tation of infected oysters.

Based on an in vitro assay of P. marinus isolates originating
from Massachusetts to South Carolina, there appear to be physi-
ological responses (growth, metabolic activity, or both) to tem-
perature that vary along a latitudinal cline; however, there was no
consistent evidence in this assay, or in comparison of in vivo
proliferation in field samples, that a low-temperature tolerant strain
off. nuniniis was present in the Northea.st. Nevertheless, given the
current abundance of the parasite in this region, it would be unwise
to dismiss the possibility that selection for low-temperature toler-
ance is underway.

POPULATION ENHANCEMENT EFFORTS FOR THE
BAY SCALLOP. ARGOPECTEN IRRADIANS, IN THE
NIANTIC RIVER ESTUARY, CONNECTICUT, USA.
Ronald Goldberg, Jose Pereira, and Paul Clark, USDOC,

NCAA, National Marine Fisheries Service, Northeast Fisheries
Science Center, Milford Laboratory, Milford, CT 06460; Bernard
Faber, David Porter, Lorenz Rinek, Barbara Kanter, Eric
Kanter, and Walter Lord. Waterford East- Lyme Shellfish Com-
mission. 5 Rope Ferry Road. Waterford CT 06,^85.

The Nianlic River supports presently u small population of bay
scallops, Ariiopccten irradians. that is harvested recreationally.
Numbers of bay scallops have tluctuated greatly with a peak level
estimated as high as 20 million animals in the 194()s (Marshall.
1960). Three potential enhancement strategies were evaluated: 1 )
collection of natural spatfall. 2) direct re-seeding, and ?<) over-
wintering of hatchery-reared stock for creation of spawner sanc-
tuaries. Assessment of natural spatfall in 1997 indicated that peak
spawning occurred in late July and that spat were dispersed widely,
however, too few spat were a\ailablc for cnhancemeni activities.
In direct re-seeding experiments, time of planting and the inferred
pretlalion intensity were major factors affecting survival, while
|ilaiiling density had no significant effect. The Waterford F.asi-
Lyme Shellfish Commission (WF.L.SCO) held 26,000 bay scallops
in suspension culture during the 1998 - 1999 winter, of which. 60
- 80 Vf survived and spawned during the summer of 1999 within

mobile spawner sanctuaries (cages suspended on long-lines). This
effort is being repeated during 1999 - 2000. An annual recreational
harvest survey has been initiated to assess enhancement activities.
The pro-active approach of WELSCO in using aquacultural meth-
ods for enhancement of bay scallop populations is appropriate
when natural recruitment is poor and habitat and environmental
conditions are not limiting.

BREEDING FOR A SUPERIOR EASTERN OYSTER FOR
THE NORTHEASTERN REGION. Ximing Guo. Susan Ford.
and Gregory DeBrosse. Haskin Shellfish Research Laboratory,
Institute of Marine and Coastal Science, Rutgers University, 6959
Miller Avenue, Port Nonis, NJ 08349; Roxanna Smolowitz, Ma-
rine Biological Laboratory, 7 MBL Street, Woods Hole, MA
02543.

The eastern oyster, Crassostrea virginica, supports a major
aquaculture industry in the Northeastern (NE) region. An impor-
tant need of this industry is disease-resistant and faster growing
stocks. The eastern oyster faces three major diseases in the NE
region; Dermo (caused by the parasite Perkinsiis mcirinns). MSX
(caused by the parasite Haplosporidiiiin nclsoni) and JOD (caus-
ative agent unknown). Rutgers University has maintained an oyster
breeding program since the early 1960s, established by Harold
Haskin. Rutgers resistant strains have shown strong resistance to
MSX and recently some resistance to Dermo. They have not been
exposed to JOD and are probably susceptible to this important
disease. Rutgers strains have recently been re-grouped into a mid-
Atlantic strain (DBH) and a Northeastern strain (NEH). The NEH
strains are originated from the NE region and known for their
better growth coinpared to the DBH strains. Research funded by
Sea Grant and the State of New Jersey is underway to enhance
further the disease resistance and growth of the NEH strain.

To obtain JOD-resistance, the NEH strain will be crossed with
a JOD-resistant strain from the FM Flower Oyster Company (NY)
and evaluated for resistance throughout the NE region. To enhance
growth significantly, we plan to de\elop a tetraploid stock of dis-
ease-resistant strains for the production of all-triploid and disease-
resistant oysters. Triploids are organisms \\ ith three sets of chro-
mosomes instead of two sets found in normal diploids. Triploid
shellfish grow significantly faster than normal diploids in almost
all shellfish studied so far. Triploid eastern oysters produced by
chemical uiduclion grow l2-4l'/( faster than diploids, and triploids
produced from tetraploids are expected to grow c\en faster. The
enhanced growth reduces not only production duration, but also
exposure tii diseases. Triploid oysters also have improved meat
quality during the summer season. The combination of disease-
resistant strains and the triploid-lelraploid technology should pro-
duce a superior stock for the NE oyster industry.

Milford Aquaculture Seminar. Mllt'ord. Connecticut

Abstracts. February 2000 573

STATE IMPLEMENTATION OF THE NSSP INTERIM
CONTROL PLAN FOR VIBRIO PARAHAEMOLYTICUS.

J. Michael Hickey. Interstate Shellfish Sanitation Conference. 1 15
Atrium Way, Suite 117. Columbia. SC 29223.

Vibrio parahaemolyticiis (Vp) is a bacterium found commonly
and at seasonally high numbers in coastal waters worldwide. Some
serotypes of this organism cause gastroenteritis in the general
population, not solely in high-risk groups, as is the case for V.
viiliiificKs. Sporadic cases along with occasional outbreaks of gas-
troenteritis have been linked to consumption of raw seafood, in-
cluding molluscan shellfish, and seafood re-contaminated after
cooking. The actual incidence of these illnesses is unknown since,
like many foodbome illnesses, this disease is self-limiting and
those affected may not seek treatment. Also, states vary consider-
ably in their Vp investigations and reporting practices. However,
four outbreaks in 1997 and 1998 resulting in over 700 cases have
been traced to consumption of raw oysters and some additional
seafood products from Texas. New York, and the Pacific North-
west. These issues and the growing evidence of more virulent
strains of Vp involved in many of the cases have initiated a con-
certed effort by the shellfish industry, state and federal regulators
through the ISSC to better understand the organism and limit risk
to shellfish consumers.

The Interstate Shellfish Sanitation Conference (ISSC). in 1998.
adopted an interim control plan for addressing illnesses associated
with Vibrio parahaemolyticiis. This interim control plan was modi-
fied by the ISSC in 1999 to incorporate the use of a gene-probe for
identification of pathogenic strains of Vp. The presentation will
provide an update of recent ISSC actions and include a report
regarding state implementation of the interim control plan for Vp.

THE ECONOMICS OF BLUE MUSSEL GROW-OUT:
AQUACULTURE AT AN OFFSHORE SITE. Porter Hoag-
land and Hauke L. Kite-Powell. Marine Policy Center. Woods
Hole Oceanographic Institution. Woods Hole. MA. 02543.

It is widely believed that the commercialization of ocean mari-
culture is an area of tremendous future economic potential. In the
United States, this perceived potential exists because of an increas-
ing longterm trend in the per capita consumption of seafood, limits
on the output of some important commercial wild harvest stocks,
the availability of offshore locations that both minimize the pos-
sibility of conflicts with other uses and permit operations large
enough to achieve efficient scale economies, and good water qual-
ity, among other reasons. However, several serious scientific ques-
tions, technological problems, and economic and policy issues
must be clarified or resolved before this potential can be realized.
As a problem of economic development with the potential to ben-
efit coastal communities, the central issue is one of reducing risks

arising from all of these areas to levels that improve the likelihood
of investment flows.

With the assistance of Blue-Gold Ltd.. located in New Bedford
and the largest U.S. mussel processor, scientists and engineers at
WHOI are taking an interdisciplinary approach to the development
of a framework for reducing the risks of ocean mariculture opera-
tions, focusing on the production of the blue mus.sel (Mytilus edii-
lis). Suspended ocean culture of mussels is likely to result in a
product of superior taste and quality, free of pearls and grit con-
tamination, and with reduced levels of commensal organisms and
predation. We are combining offshore engineering, biological
studies, and the analysis of project economics to characterize a
technically optimized and commercially feasible submerged sus-
pension structure. Here, we report on the continuing development
of a framework for evaluating the commercial viability of offshore
farming.

We define a full scale offshore mussel farming operation to be
one that fully utilizes the annual capacity of one service vessel
(e.g., a small scalloper of approximately 20 GRT). Such a vessel
requires fixed cost payments on the order of $80,000 per year.
Daily variable costs (fuel and supplies) are $1400. including $800
in crew member wages. We estimate that one vessel is capable of
servicing a field of 300 longlines. We assume that 150 longlines
are harvested once every two years. Each year, about 225 days are
spent maintaining the longlines and 38 days are required for har-
vesting. During years when the longlines are being deployed, an
additional 38 days are required to deploy one-half the field (150
longlines). Larger farms can be scaled as multiples of this basic
farm.

Each longline is designed to support 25 mussel socks or ropes.
Each sock produces, on average, 350 pounds of mussels over a
two-year growout period. A longline costs $400 to deploy and
$250 to maintain each year, not including the costs of running the
service vessel. Each longline is designed to last ten years, at which
time it must be replaced. Additional costs include those associated
with processing (sorting, debearding, and cleaning), transport to
the market, and management costs.

Prospective offshore aquaculture entrepreneurs face a wide va-
riety of environmental, engineering, economic, and regulatory
risks. Many specific risk categories cannot be characterized fully
until offshore aquaculture takes place on a commercial scale. Nev-
ertheless, our model has been developed to handle two main types
of risk: market and production. These risks are simulated using
assumed distributions for the relevant parameters: price and natu-
ral growth.

A model of market price is estimated from the monthly distri-
bution of the value per pound of imported Canadian cultured blue
mussels during 1990-97 (averaging 91(i per pound). We assume
that price is distributed lognormally with a mean of 64c per pound
and a standard deviation of lie per pound. The mean has been
adjusted downward to account for the effect of domestic produc-
tion on market price.

574 Abstracrs. February 2000

Milford Aquaculture Seminar. Milford. Connecticut

Developing a model of production rislc is more problematic in
the absence of a history of offshore production activity. This pa-
rameter can be affected by storm events, predaiion. parasitism.
disease, temperature, and availability of food, among other things.
We assume that production takes an extreme value distribution
with a mode of 380 pounds per sock and a scale of 50 pounds. The
majority of possible values for production from a sock thus range
between 80 and 480 pounds per sock.

We assume a two percent rate of inflation, and we discount
revenues and costs at a rate of 12 percent. The model predicts a
marginally profitable operation with a discounted cash flow of
$0.3 million over a thirteen year period. This result suggests that
full scale mussel farming at an offshore location is commercially
feasible. However, the possibility of losses is not insignificant.
Farm profitability is greatly enhanced if the current market price
for Canadian aquaculture product is used in the model. We expect
to refine the model as we gain more experience with the prototype
longline.

This research has been sponsored with funds from the U.S.
Department of Commerce. NOAA. National Sea Grant College
Program under grant Number NA86RG0075 (Woods Hole
Oceanographic Institution project number RyA-40) and the WHOI
Marine Policy Center.

SHELLFISH PRODUCTION IN THE BLAKE FLOATING
HATCHERY AND IN MODIFIED TIDAL UPWELLER
NURSERIES IN 1999. Richard C. Karney, Martha's Vineyard
Shellfish Group. Inc.. Box 1552. Oak Bluffs, MA 02557; John C.
Blake. Sweet Neck Farm, Box 1468, Edgartown, MA 02539; and
Thomas E. Berry, Martha's Vineyard Shellfish, Box 1660, Edgar-
town, MA 02539.

With funding from the National Fish and Wildlife Foundation
and the Massachusetts Department of Food and Agriculture two
innovative field shellfish culture systems were successfully dem-
onstrated. The floating hatchery/nur.sery and the modified tidal
upweller nursery were both designed by Edgartown aquaculturist.
.lack Blake.

First tested in 1998. the Blake Floating Hatchery/nursery pro-
totype was modified in 1999 with the addition of a 400 watt wind
turbine which reduced the labor of recharging and exchanging the
four 6 volt batteries which powered the system. The prototype was
tested in three culture modes: as a 340 gallon larval tank, as a
nursery capable of holding eight downweller sieves for post set
culture, and as a nursery with eight upv\cllcr silos for rearing
juveniles. All except the flow through larval culture mode proved
successful.

In the first culture trial. 21) million quahog embryos were in-
troduced into the larval lank which received an approximate flow
of 146 gallons of 5 micron bag-filtered seawater per hour. The lank
was fitted with three 5 1 micron exit sieves w ilh a surlace area ol

about 700 sq. inches to prevent loss of larvae. By Day 3. the tank
was contaminated with copepods and the larvae were observed to
be hollow looking and in poor health. The poor condition of the
larvae resulted from either a lack of food due to competition from
the copepods or stress related to impingement on the mesh of the
exit sieves. The copepods were thought to have entered the system
when waves pounded the exit sieves.

In the second culture trial. 10 million 48 hour old oyster larvae
were successfully taken to the eyed stage in a closed, aerated,
larval culture with a daily addition of cultured phytoplankton and
a change of seawater every second day. Approximately 1 .6 million
eyed larvae were set on crushed oyster and poultry shell cultch in
eight downweller sieves. The post set were cultured in the system's
upweller silos which received a maximum flow of about 10 thou-
sand gallons/silo/day and resulted in the production of 130,000
single seed oysters.

Blake's modified tidal upweller was designed to maximize the
flow of water through the unit by eliminating flow constricting
outlet ports. In place of standard upweller silos, Blake's upweller
uses stacked bins to hold the seed. The nursery has 24 support
racks, each of which can hold three stacked bins. On 6 August.
1999 during a time of average tides, the flow rate through the tidal
upweller measured with a Marsh McBirney Paramagnetic Current
Sensor was determined to be about 175 gallons per minute.

Over the course of the 1999 growing season, over 0.5 million
oyster seed and 1.4 million quahog seed were cultured in four
upweller nurseries. The oyster seed (3-7 mm) was set out in
batches throughout the growing season, between 21 June and 8
September. On average, the oyster seed were rinsed clean about
twice a week, sieved to size every week or two. and thinned in half
about every ten days. At the beginning of the culture, the seed was
about 4-5 mm in size, and held at an initial density of about 20.000
per bin. Within about six weeks, the oysters had grown to about 37
mm and were at a density of about 1,400 per bin. When they
reached about 37 mm the oysters were transferred from the up-
weller nurseries into growout cages. Oyster seed survival in the
units was nearly 100%.

Quahog seed, at about 0.75 mm. was introduced into two of the
upwellers on 25 June and 3 July at an initial density of about
180,000 per bin. It was rinsed clean about every two to five days
(more frequently when it was smaller). After about three weeks il
was thinned to a density of about 60,000 per bin. After about a
month, it grew to an average size of about 5.7 mm. Quahog seed
mortality was reported to be about 5-IO''f.

An experiment was conducted to determine if bins built from
ACQ"'' pressure treated lumber would have any negative impacts
on the growth of small seed. Equal numbers of 2-3 mm oyster seed
were placed into upweller bins, one with a pressiue treated frame,
and the other u ith a frame of uiilrealed painted wood. After two
weeks, a sample of seed from each treatment was measured and the
difference in growth was found to be insignificant.

Milford Aqiiacultiire Seminar. Milt'ord. Connecticut

Abstracts. Februai^ 2000 575

OPERATIONS AT TAYLOR SEAFOOD. Gordon King . Tay-
lor Resources Inc.. Slielion. WA 98584.

Taylor United. Inc. inas been growing Mytiliis galloprovinciatis
for approximately ten years. Presently we market about 800.000
pounds per year. All production is hatchery seed based and grown
out on rafts with average spawn to harvest of 16-18 months. The
company also produces about 140.000 gallons of shucked oysters,
one-million dozen single oysters and 3,000.000 pounds of manila
clams. Recently. Taylor Seafood has been planting two million
geoduck seed a year and should start harvesting significant vol-
umes in the next two years. The company has a hatchery in
Quilcene, Washington, a floating upwell nursery in Shelton, Wash-
ington, a leased hatchery in Tillamook, Oregon and a hatchery
nursery in Kona, Hawaii. We also recently started a scallop farm
in Mexico.

These operations will be described in the presentation.

SUBMERGED LONGLINE CULTURE OF BLUE MUSSELS
(MYTILUS EDULIS) AT AN OPEN OCEAN SITE IN THE
GULF OF MAINE. Richard Langan, Jackson Estuarine Labo-
ratory, University of New Hampshire, 85 Adams Point Road,
Durham, NH 03824.

In the northeastern US, competing and often conflicting uses
limit the availability of protected inshore sites for shellfish culture.
Though not totally without conflict, open ocean sites provide
greater opportunity for shellfish culture. However, the greater
depth and rigorous conditions in the open sea require engineering
adaptations of the existing technology. The University of New
Hampshire, in collaboration with the Portsmouth, NH Commercial
Fishermen's Cooperative, has established a pilot-scale submerged
longline culture system in order to demonstrate the feasibility of
culturing mussels in the open ocean environment. The project is
located at a fully exposed site eight kilometers from shore in the
open waters of the Gulf of Maine.

In the spring of 1999, a longline system consisting of large
(3200 kg) concrete anchors spaced 180m apart, 30 mm polysteel
line for anchor and headlines, and both steel and hard plas-
tic submersible buoys for floatation was deployed. The depth
at the site is 52 m. and the horizontal headline is submerged 15 m
below the surface. A standard 12.5 m lobster fishing boat was
outfitted with deck gear needed to tend submerged longlines. In
June 1999. one hundred 12 m mesh socks filled with mussel seed
collected in the summer of 1998 were deployed vertically from the
headline. In October 1999, seed collected in spring of 1999 were
used to fill mesh socks 60 m in length that were attached to the
longline in a looping pattern. Mussel growth and density, and
physical and biological conditions at the site have been monitored
monthly.

Mussel growth for the 1998 year-class of seed has averaged
1.6 mm per week for the period June 1999 to November 1999.

This rate of growth suggests that a period of 15 to 18 months
post-set would be required for mussels to reach market size.
The longline has thus far been unaffected by severe weather and
wave heights in excess of 6 m. Preliminary economic analysis
indicates a favorable outlook for both small and large-scale opera-
tions.

DETECTING THE PRESENCE OF PERKINSUS MARINUS
AND HAPLOSPORIDWM NELSON! IN THE OYSTER,
CRASSOSTREA VIRGINICA, IN RHODE ISLAND WA-
TERS: A SURVEY UPDATE. Ken Leonard HI. Josefa Dou-
gal, and Marta Goniez-Chiarri, Fisheries, Animal and Veterinary
Science, University of Rhode Island. Kingston. RI 02881: Arthur
Ganz. Rhode Island Department of Environmental Management.
Coastal Fisheries Laboratory, Wakefield, RI 02879.

Dernio and MSX. diseases caused by the parasites Perkinsiis
marinus and Haplosporidium nelsoni respectively, have been re-
sponsible for oyster mortalities throughout the east coast of the
United States. We report here the latest results from an oyster
disease survey that began in May 1998. Oysters (30 per site) were
collected from 8-10 locations in Rhode Island, including 2 aqua-
culture sites, in May, August, and November 1998, and February.
August, and November 1999. The prevalence and intensity of
Dermo infections were evaluated using the Ray's Fluid Thiogly-
collate Medium (RFTM) method. MSX infections were evaluated
using histological examination. Perkinsiis marinus and Haplospo-
ridium nelsoni infections were detected in August 1998 and con-
tinued to be present in samples collected in August and November
1999. The highest weighted prevalences of Perkinsus marinus
were observed in August 1998 and 1999 in samples from Har-
rington River. Wickford Cove, and Charlestown Pond, three loca-
tions historically known to support oyster populations. Oyster mor-
talities reported at these locations may be due to high Perkinsus
marinus infections. Some initial and intermediate Haplosporidium
nelsoni infections were observed in oysters from 5 locations. No
advanced infections have been detected so far.

FEDERAL CROP INSURANCE BECOMES AVAILABLE
FOR QUAHOG FARMERS. Dale F. Leavitt and William Burt.

SouthEastern Massachusetts Aquaculture Center. Buzzards Bay.
MA 02532; Charles Koines. Risk Management Agency - USDA,
Ballston Spa. NY 12019.

What do quahogs have in common with soy beans, cranberries,
and corn? They are all agricultural crops that are recognized by the
federal government for inclusion in a federally subsidized crop
insurance program. In a pilot program developed by the United
States Department of Agriculture (USDA) Risk Management Of-
fice, quahog farmers in areas within Massachusetts, Virginia,
South Carolina, and Florida are eligible for crop insurance. The
1999 crop insurance program was initiated in November 1999 with
a cut-off date of 1 December for this year's crop. At this point, the

576 Abstracts. February 2000

Milford Aquaculture Seminar. Milford. Connecticut

crop insurance program covers the grow-out phase of hard clain
culture and the field planted seed quahogs have to be larger than 10
mm to be eligible for insurance. The crop insurance program will
be further developed during the next three years to include the
nursery stage of clam farming and possibly clam hatcheries. An
overview of the quahog crop insurance program will be presented
with insight as to how it was developed by the USDA using field
agents and the industry to set the standards.

PRODUCTION OF TRANSGENIC MOLLUSKS AND
CRUSTACEANS. Chun-Mean Lin. Sineenat Siri. University of

Connecticut Biotechnology Center, Storrs. CT 06269: Sheila
Stiles, USDOC, NCAA, National Marine Fisheries Service,
Northeast Fisheries Science Center. Milford Laboratory, Milford,
CT 06460; and Thomas Chen, University of Connecticut Biotech-
nology Center, Storrs, CT 06269.

It has been demonstrated that introduction of naked DNA into
newly fertilized or unfertilized eggs by microinjection or elec-
troporation reproducibly results in production of transgenic finfish.
However, almost no examples have been shown concerning pro-
duction of tran.sgenic mollusks and crustaceans by direct microin-
jection and electroporation of naked DNA into these animals. Re-
cently, a new gene transfer vector, defective pantropic retroviral
vector, has been developed that contains a glycoprotein (VSV-G
protein) from Vesicular Stomatitis Virus (VSV). We have demon-
strated successful transfer of foreign genes into dwarf surfclams or
medaka by electroporating the newly fertilized eggs or exposing
the developing gonads to these gene transfer vectors. We, there-
fore, believe that the same gene transfer technology may be used
to produce transgenic crustaceans.

Defective pantropic retroviral vector containing a lacZ or neoR
reporter gene was introduced into immature gonads of male and
female crayfish iinmediately post the last larval molt by microsur-
gery. The treated animals were maintained in separate aquariums
with respective untreated females or males for maturation and
spawning, and the newly hatched larvae were collected for rearing.
Leg appendages from the presumptive transgenic animals were
removed for the determination of the presence of the lacZ or neoR
transgene by PCR amplification. Results of PCR amplification
analysis showed that ranging from 30 to 50% of the progeny
produced by the treated males or lemales contained the lacZ or
ncoR transgene. Integration of the transgene into the host genome
was further confirmed by linker mediated PCR of the genomic
DNA isolated from the PCR positive animals. Expression of the
transgencs in the transgenic animals was also detected by the re-
verse transcription/PCR analysis. These results demonstrate con-
clusively that crustaceans can be produced routinely by infecting
the immature gonads with defective pantropic retroviral vectors
carrying desirable transgcnes.

Recently, we have also introduced a common carp B-actin
promoter trout GH cDNA transgene into newly fertilized bay scal-
lop eggs by electroporation. A significant fraction of the hatched
animals are shown to carry the GH transgene and have grown to
adulthood. F, transgenic animals have also been produced by
crossing P, transgenics with non-transgenics. These results
showed that transgenic bay scallops can be produced by electropo-
rating naked DNA into newly fertilized embryos and the transgene
can be transmitted into the subsequent generation. Work is under-
way to characterize the integration and expression of the transgene
and the performance of the transgenic progeny. (This research is
supported by a grant from the Connecticut Sea Grant Program to
Thomas Chen).

A REVIEW OF CURRENT CLAM CULTURE IN NEW
ENGLAND. Clyde L. Mackenzie. Jr., USDOC, NOAA, Na-
tional Marine Fisheries Service, Northeast Fisheries Science Cen-
ter, James J. Howard Marine Sciences Laboratory, Highlands. NJ
07732.

This review includes the culture of softshell clams, Myci
arenaria, in Maine, and northern quahogs, Mercenaria inerce-
naria. in Massachusetts, Rhode Island, and Connecticut. Clam and
quahog culture practices differ among the four states.

Clain culture in Maine consists of spreading hatchery seed and
natural seed on the intertidal public clam beds in several towns in
northeastern Maine. The hatchery seed is produced in the state's
only clam hatchery at Beals Island, while the natural seed is dug
in the high areas of fiats where it would die if left in place and
then transplanted to low areas in the same flats. The stimulus for
constructing the hatchery was light clam sets in Washington
and Lincoln Counties for approximately 20 years. During the
past few years, however, natural setting has increased which may
cause the need for planting hatchery seed on the public beds to
diminish.

In Massachusetts. c|uahog hatchery-grov\oul culture occurs on
public beds and private leases and has been increasing during the
1990's. At least 20 towns purchase quahog .seed to spread on their
beds for public fishermen to harvest after they attain market size,
and about 250 leaseholders in various towns grow quahogs under
plastic screens to avoid predators; leases range from one to ten
acres in size. Compared with wild quahog, hatchery quahogs (no-
tata strain) have relatively brittle shells and gape more in storage.
The seed is purchased from hatcheries in Maine. Massachusetts,
and New .Icrscy. The state also transplants quahogs from tuiccrli-
fied waters to public town waters.

Culture in Rhode Island consists of a state-run program of
transplanting quahogs from uncertified grounds to certified
grounds and then hokling them for as long as a year until they
depurate and also spawn. Only a few acres of bottoin are leased for
shellfish culture.

Miltbrd Aquaciilture Seminar. Milt'ord. Connecticut

Abxtnicts. February 2000 577

In Connecticut, all quahog harvesting tal<es place on private
leases. Quahog culture consists of transplanting stocks from re-
mole beds to beds closer to ports. This enables boats to harvest
quahogs more easily during adverse weather. Quahog abundance
has increased sharply in Connecticut during the 1990's. MacKen-
zie and Pikanowski (1998) believe the cause of this has been
diminished numbers of starfish, a quahog predator, during that
decade. The practice of hatchery-growout culture of quahogs
would be difficult because few intertidal areas are available for
leasing and most beds are too deep. 3-12 m, to grow quahog seed
under screens for predator control.

Average weight of fish increa.sed from 3.4 g on 12 April to 320 g
on 2 December 1999.

Collected data indicate that significant numbers of red hake are
lost when scallops are harvested, a potential bycatch of one fish for
every two scallops. Our data suggest that scallop harvest con-
ducted between June and August would minimize the coincidental
catch of hake. Alternatively, if hake were collected and retained
during the colder part of the year (October to March) when post
capture mortality was minimal (<30%) and fish were most abun-
dant, the potential exists for commercial culture or stock enhance-
ment.

SYMBIOTIC RELATIONSHIP OF THE SEA SCALLOP
AND RED HAKE AS A POSSIBLE MANAGEMENT TOOL.
Mark S. Miller and Joseph K. Buttner, Northeastern Massachu-
setts Aquaculture Center and Department of Biology, Salem State
College. Salem, MA 01970.

The symbiotic relationship between the sea scallop {Pla-
copecten luagellaniciis) and red hake iUmphycis cliiiss) in near
coastal waters of New England was monitored by researchers us-
ing scuba gear. Scallop beds off the coast of Salem, MA were
sampled on a monthly basis as conditions permitted between Feb-
ruary and December 1999. After being brought to the surface,
scallops were measured and opened to extract hake. Collected hake
were counted, measured, and transported to the Cat Cove Marine
Laboratory. Salem State College.

Water depth ranged from 18 to 21 m. and bottom tempera-
ture varied between 2° and 15 °C. All dives were conducted
adjacent to the mouth of Salem Sound along a line running
roughly north and south between the coordinates of 42°32.857' N
X 070°48.757'W and 42".^0.502'N x 070°47.032'W. A total of
2,978 scallops and 1,421 hake were collected or 0.48 hake per
scallop. Percent of scallops with hake varied seasonally, declin-
ing sharply to 0% in July and increasing to 96'7r in mid September
as large numbers of young-of-the-year appeared. Scallop size
remained fairly constant (avg. = 119.4 mm, S.D. = 14.3 mm).
Fish length ranged from 44 to 122 mm. reflecting recruitment
and growth. Individual scallops frequently contained two to
four fish; the greatest incidence occurred in October, when mul-
tiples represented 299f of fish collected. Fish frequently exited
scallops after collection, and 22% of all hake were collected as free
fish.

Hake transported alive to the laboratory were transferred to
1900 L recirculating systems and readily ingested commercial feed
within 24h. Fish fed. survived, and grew despite problems associ-
ated with the newly set up facility. On occasion, water temperature
rose above 25 °C and total ammonia nitrogen exceeded 15 ppm.

THE CULTURE OF HADDOCK, MELANOGRAMMUS AE-
GLEFINUS, USING A RECIRCULATING SYSTEM IN AN
URBAN SETTING. Brandy M. Moran, Clifford A. Goudey,
and Jessica Rabe. Massachusetts Institute of Technology. Sea
Grant College Program. MIT Bldg. E38 - 300, 292 Main Street,
Cambridge, MA 02139.

The techniques of recirculating aquaculture are applicable to an
urban setting because of the characteristically high growing den-
sities and low water usage of aquaculture facilities. In addition,
many coastal urban centers have lost their maritime industrial base
and are seeking commercial activity that can maintain the vitality
of their waterfronts. MIT Sea Grant College Program, recognizing
the potential opportunities that exist for urban aquaculture, has
begun a program of research and outreach aimed at promoting
sustainable economic development of Boston Harbor based on
marine finfish culture. Our Marine Finfish hatchery was dedicated
in May 1998. We are now expanding our facilities to increase our
production potential and explore additional species as candidates
for commercial exploitation.

We have developed techniques for culturing haddock. Melano-
graiiiiiius aei^lefmiis. in a pilot commercial-scale recirculating sys-
tem at the hatchery. Light intensity is maintained at 1500 lux. Eggs
are incubated in an up-welling concept and held at a constant
temperature of 8 °C {+/- 1). Enriched rotifers and Anemia are
initially offered to the larvae at the density of 7 animals/ml on D2
and D15. respectively. No natural feed supplements or algae are
used in the culture methods. Culture temperatures are increased by
1.5 °C over a week each time there is a change to a new feed type.
A dry commercial weaning diet is offered to the larvae on D30.
The amount of live feed offered to the larvae is decreased until
they are completely weaned onto the dry diet. Grading contuiu-
ously occurs during the juvenile stage.

A haddock growth model has been developed and will be tested
against the actual culture of the fish during this 2000 .season. The
model was used to determine the tank sizes for the facility by

578 Ahitracls. February 2000

Milford Aquaculture Seminar. Milford. Connecticut

predicting growth rates, survivorship and acceptable densities
within a recirculating system. Fish from this year's spawn will be
reared for growth trials in the Isles of Shoals Open-Ocean Aqua-
culture test cages starting in May 2001. A control population will
be maintained by MIT to compare the ocean trials with recircu-
lating tank culture at optimal temperatures.

GROWTH OF JUVENILE TAUTOG FED COMMERCIAL
DIETS IN A CROSS-OVER EXPERIMENT. Dean M. Perry,

USDOC, NCAA, National Marine Fisheries Service, North-
east Fisheries Science Center. Milford Laboratory, Milford, CT
06460; Laurel Ramseyer, Wildlife Conservation Society, Bronx.
NY 10460: Joseph Goncalo. 231 Harrison Ave., Milford, CT
06460.

A 2x2 cross-over experimental design was used to evaluate the
growth of tautog. Tautoga onitis. fed two commercial feeds and
reared in twin recirculating systems. An additional objective was
to determine whether a simple 2x2 cross-over experimental design
was appropriate for a short-term feeding trial. Seventy juvenile
(x= 13 g) tautog were assigned randomly to tanks across systems
(n = 3). Tautog were fed either Zeigler High Performance Trout
Grower or BioKyowa C for 28 d, then switched to the alternate diet
for the following 28 d. Diets were fed at a rate of 3% wet body
weight/day (dry matter basis). Daily rations were divided into 4
meals/day. and fed with automatic feeders. Tautog fed BioKyowa
had significantly higher weight gain (0.50 g/d. P < 0.001 ), higher
thermal growth coefficient (1 1.8 x 10""* ), and lower feed conver-
sion ratios (0.97) than fish fed Zeigler (0.28 g/d, 7.58 x lO"-", and
1.5. respectively) during the first 28 d of the experiment. Unbal-
anced carry-over effects resulting from switching the diets at day
28 precluded full use of the 2x2 cross-over model. A switchback
design or a 2x2 design with a wash-out period may be more
appropriate if unbalanced carry-over effects are expected. Since
water quality was not significantly different in tanks of fish as-
signed to the two different feeding regimes, simple randomization
of treatments across recirculating systems would have been suffi-
cient to control for system etfects in this experiment. However,
maintenance of comparable conditions in two separate systems can
never be guaranteed, so further development of cross-over designs
is warranted.

GROWING ROTIFERS ON SINGLE AND MIXED AL-
(JAL STRAINS TO BE USED AS A FIRST FEED FOR
LARVAL TAUTOG. Dean M. Perry and David A. Nelson,

USDOC, NCAA, National Marine Fisheries Service, North-
east Fisheries Science Center, Milford Laboratory, Milford, CT
06460: Joseph E. Goncalo. 231 Harrison Ave., Milford, CT
06460.

This research exaiualcd the population growth ol rotifers [lini-
chioiiiis pluLililis) led lour individual unicellular algal strains; Dii-

nalieUa tertiolecta (DE), Nannochloropsis sp. (UTEX 2341), ho-
chrysis sp. (T-Iso). and Tetra.selmis chiii (Ply 429), or a mixture or
"cocktail" of three algal strains; DE, T-Iso, and Ply 429. An ad-
ditional objective was to determine whether certain algal strains
known to contain high levels of the n-3 and n-6 series of polyun-
saturated fatty acids used for enrichment of rotifers, also promote
high densities of rotifers. The algal cells of the four individual
strains used to feed the rotifers averaged 6 x lO*' cells/ml. Counts
of rotifers grown on these single algal strains were done once a
week for 12 weeks. Rotifer populations fed DE, UTEX 2341,
T-Iso, or Ply 429 increased an average of 29.8X, 18.5X, 19. IX.
and 28. 7X, respectively. The algal cells of the three mixed strains
used to feed the rotifers averaged 5.0, 6.0, and 30.0 x 10* cells/ml
for DE. Ply 429, and T-Iso, respectively. Counts of rotifers grown
on a "cocktail" of these three algal strains were done at 4d and at
7d over a 9-week period. Rotifer populations fed the algal "cock-
tail" increased an average 3.8X at 4d, and 5.4X at 7d. Results of
rotifers fed single strains of algae indicate DE and Ply 429 to be
the best choices to feed rotifers to maximize reproduction. Dii-
luiliella, while promoting good reproduction, is lacking in essential
fatty acids. Termsclinis (Ply 429) is a better algal strain to feed
rotifers, since it contains the n-3 and n-6 polyunsaturated fatty
acids that are necessary for larval marine fish growth and survival.
The algal "cocktail" increased the rotifer population dramatically
within the first 4d, showing continued reproduction to the seventh
day. Although feeding rotifers single algal strains appears to pro-
mote higher rotifer densities than feeding them the mixed algal
strains, initial rotifer stocking density and culture container size
can both be limiting factors that affect population size.

STATUS REPORT FOR THE CHARACTERIZATION OF
THE BAY SCALLOP, ARGOPECTEN IRRADIANS, GE-
NOME. Enrico Picozza and Joseph Crlvello, University of Con-
necticut, Department of Physiology & Neurobiology. Storrs. CT
06269; Maronda V. Brown and Linda Strausbaugh. University
of Connecticut Department ot Molecular & Cell Biology, Storrs,
Connecticut 06269; Sheila Stiles, USDOC. NOAA, National Ma-
rine Fisheries Service. Northeast Fisheries Science Center. Milford
Laboratory. Milford, CT 06460.

The bay scallop {Argopecten iirailicins} is a valuable food
source as well as an important inhabitant of marine estuaries and
ecosystems, yet little is known about its genome. As we are learn-
ing with other organisms, it is important to have a better under-
standing of the genome structure, and more specifically the struc-
ture ol particular genes. This will allow us in the future to select
scallops for various properties such as increased growth or surviv-

Milford Aquaeulture Seminar, Milford. Connecticut

Abstracts. February 2000 579

ability for aquaeulture. or even as environmental indicators of their
micro-environments.

In an attempt to begin understanding the genome of the bay
scallop, we spent time developing various tools critical to this task.
A genomic library was generated to serve as the basic tool for
breaking down the genome into a manageable size. A cDNA li-
brary was also generated that will allow us to examine and under-
stand expressed genes and to generate an Expressed Sequence
Tags (EST) database. The EST database can be used for managing
the functional components of the genome.

Finally in an attempt to begin applying these tools, we are in the
process of cloning the metailothionein (mt) gene, a gene that has
been shown in other organisms to serve as an indicator of animal
e.xposure to various hazardous pollutants. Details will be presented
of the various tools and of the progress being made in the cloning
of the mt aene.

OBSERVATIONS ON MYCOBACTERIOSIS IN THE TAU-
TOG {TAUTOGA ONITIS). Steven Pitchford and Richard
Robohm, USDOC , NOAA, National Marine Fisheries Service.
Northeast Fisheries Science Center, Milford Laboratory. Milford.
CT 06460; Sharon MacLean, USDOC , NOAA. National Marine
Fisheries Service, Northeast Fisheries Science Center, Narragan-
sett Laboratory, Narragansett, RI 02882; Laurel Ramseyer, Wild-
life Conservation Society. Bronx. NY 10460.

During the spring and summer of 1999, mortalities occurred in
1-2 year old juvenile tautog being held for nutritional studies at the
Milford Laboratory. Internal examination of the dead fish revealed
that most contained large numbers of white, round nodules. Nod-
ule imprints and histological sections stained with Kinyoun Acid-
Fast stain revealed the presence of acid-fast, rod-shaped bacteria.
To prevent infection of other tautog at the facility and to determine
the extent of infection, the remaining fish were sacrificed and
examined for the presence of nodules. Thirty six of the 123 fish
examined (29%) had evidence of nodular growth.

Multiple organ involvement of nodule growth was found in
71.43% of the infected fish. Nodules were found primarily in
kidney tissue (85.71%). liver (57.14%) , and spleen (45.71%).
Nodules also were seen to a lesser extent in the heart (20.0% I.
visceral membrane ( 1 \A%) and one instance each in the intestines
and gonads (2.9%). With the exception of a few heavily infected
fish which became lethargic and had swollen abdomens, very few
signs of external pathology were noted.

Slow-growing, acid-fast bacteria were isolated from the nod-
ules using Middlebrook 7H10 Agar media supplemented with
ADC enrichment media. After 2-3 weeks, visible growth of raised.
roughly textured, off-white colonies was observed. Ongoing and
future plans for these isolates call for identification to species level
and pathogenicity testing by challenge in healthy tautog.

Several species of Mycobacteria have been found to cause
diseases in a number of aquatic species including frogs, amphib-

ians, and both freshwater and saltwater fishes. Some of the these
same organisms also have been known to infect humans. This is
the second known occurrence of mycobacteriosis in the tautog or
the wrasse (Lahrulae) family of fishes. Aquaculturalists should be
made aware of the possible infection of their stocks by this patho-
gen.

THE PRESENCE OF VIBRIO PARAHAEMOLYTICUS IN
CRASSOSTREA VIRGINICA AT SPECIFIC LOCATIONS
ALONG THE CONNECTICUT AND LONG ISLAND
SHORE - PARTICIPATION IN THE VIBRIO PARA-
HAEMOLYTICUS ISSC - FDA SURVEY FOR JUNE 1999 TO
JUNE 2000. Leonora Porter and Eugene Zamojcin, State of
New York, Department of Environmental Conservation, 205 North
Belle Mead Rd., East Setaucket, NY 11733; Joe DeCrescenzo,
Inke Sunila, John Volk. and John Karolus, State of Connecticut,
Department of Agriculture. Bureau of Aquaeulture. P.O. Box 97,
Milford. CT 06460.

Vibrio parahaemolyticiis is an enteric pathogen transmitted
to humans primarily through consumption of raw or mishandled
seafood. Like other members of the genus Vibrio, it is a gram
negative, halophilic bacterium that occurs naturally in estu-
arine environments. In the late summer of 1998, an outbreak of
Vibrio parahaemolyticiis was epidemiologically linked to a
shellfish bed on the northern shore of Long Island, NY. Based
on an Interstate Sanitation Shellfish Conference (ISSC) 1998
Interim Control Plan for Vibrio parahaemolyticiis. routine moni-
toring was required at this location and other locations where
outbreaks of Vibrio parahaemolyticiis had occurred. FDA scien-
tists at the laboratory in Dauphin Island, Alabama developed rapid
DNA probe procedures for the detection of Vibrio parahaemolyti-
ciis and for the detection of the human pathogenic form of this
bacterium.

Briefly, the procedure requires oysters to be homogenized with
alkaline peptone water (1:1). Aliquots are then placed on TiN,
agar plates for overnight growth at 35" C. Whatman 541 filters are
applied on the surface of the agar plate for adhesion of the bacterial
growth to the filter. The bacterial colonies are lysed, followed by
DNA splitting. Then the alkaline phosphatase labeled DNA probe
hybridization occurs. After washing, the filters are placed in an
NBT/BCIP solution which will precipitate dark blue dyes in the
presence of the DNA bound alkaline phosphatase. Blue black spots
representing the Vibrio parahaemolyticiis colonies are counted for
a result.

The training of individuals from eight different states in
these procedures resulted in the development of an FDA-ISSC
partially funded national oyster surveillance program. Each state
is testing oysters from two locations along their shore for twelve
months. All testing is performed at the individual state labora-
tories. CT had started testing the first week of June 1999 and
New York started in August 1999. Vibrio parahaemolyticiis

580 Abstracts. February 2000

Milford Aquaculture Seminar. Milford, Connecticut

has been found in CT and NY oysters. Results have shown
greater levels of the bacterium in the summer when the water
temperature is higher. When the water temperature dropped below
16-17 °C, Vibrio parahaemolyticus could not be detected. No
forms of the bacterium pathogenic to humans have been found.
Results from all states are being sent to the FDA Laboratory in
Alabama for analysis.

EXPLORING DIVERSITY THROUGH AQUACULTURE.
Julia Rankin, Michael Wilcox and Donald Harris, AgriScience
& Technology Center. Bloomfield. CT 06002.

Exploring Diversity Through Aquaculture (EDA) involves 40
racially diverse students from 8 districts in the Greater Hartford
area. This number reflects 20% deduction in proposed numbers
due to a 25% reduction in funding. These students have success-
fully completed the first two weeks of this scientifically intensive
four-week residency program which began in July 1999 and will
be completed in late June 2000. The first week utilized the re-
sources of the University of Connecticut, ending with an overnight
trip to coastal Rhode Island. The second week, also in July, ex-
plored the fisheries and aquaculture industries of Maine, beginning
in Eastport, the easternmost point in the continental United States,
and finishing in Bath, in southern Maine. Students used the Uni-
versity of Maine at Machias and Husson College in Bangor as
home bases. The first two weeks were tremendously rewarding, as
students forged lasting friendships across racial and cultural
boundaries while constantly exploring myriad aspects of science,
often in a hands-on environment to which they would not normally
be exposed. The third week of the program will occur later in
2000, with the same group of students exploring warm-water aqua-
culture at Auburn University in Alabama in April and studying the
fisheries of the Gulf of Mexico. The final week of the program will
allow students to study the burgeoning aquaculture history of Ja-
maica. At each location, students have been and will be given the
opportunity to explore the cuisine and culture specific to each
region, through interviews and festivals. Students are also prepar-
ing on-going portfolios featuring CAPT-based activities that focus
on the scientific, sociological, economic and anthropological as-
pects of each activity in the program.

ECOLOGICAL SIGNIFICANCE OF THE PROVIDENCE
RIVER QUAHO(;S: POPULATION FILTRATION. Michael
A. Rice, Department of Fisheries, Animal and Veterinary Science,
University of Rhode Island. Kingston. Rl 02SSi; April Valliere.
Mark (Jibson and .Arthur Ganz, Rhode Island Di\ isiiin of t-ish
and Wildlite. Coastal Fisheries Laboratory, 12.''1 Succotash Rd.,
RR#I. Wakefield. Rl. 02S97.

Filler feeding by populations of bivalves has been suggested as
a means of reducing euirophicaiion in coastal estuaries by exerting

control of phytoplankton populations in the water column. Fre-
quently, large populations of mature shellfish residing behind pol-
lution closure lines in estuaries represent a large filter feeding
biomass. The standing crop of quahogs, Mercenaria mercenaria.
in the Providence River averages 9.1 clams/m" or about 26,400
tonnes, filtering about 1.05 x 10' m"* of water daily or a rate
equivalent to 21% of the rate of water exchange during a tide
cycle. Due to annual temperature effects, population filtration
ranges from 0 in the winter to 2 x 10' mVday in August. The
population of quahogs, however, is composed of mostly older
adults with valve lengths in excess of 60 mm. These large animals
are slow growing, have a low rate of secondary production in
relation to standing crop biomass, and have a neutral nitrogen
balance (organic-N assimilated = NH,-N excreted). These large
adults increase sedimentation through filter feeding, but since they
are neither harvested nor growing they do not directly remove
much nitrogen from the system, although the increased sedimen-
tation rates may result in increased sediment denitrification. Fil-
tration by the .standing crop of quahogs may remove 76.2 tonnes of
organic nitrogen from the estuary annually by depositing it to the
benthos. Harvest of quahogs at MSY can remove 8 tonnes of
organic nitrogen annually. As part of a Narragansett Bay wide
shellfisheries management plan, 10% of the standing crop of qua-
hogs in the Providence River is recommended for relay to man-
agement beds down bay for later harvest. Smaller more rapidly
growing quahogs have the capability of incorporating organic ni-
trogen into growing tissues and, if harvested regularly, provide a
mechanism for direct removal of nitrogen from the estuary. The
removal of quahogs from the dense assemblages in the Providence
River reduces the population filtration by only 10%, but it culls the
population making room for faster growing juveniles and small
adults. This is publication 3785 of the Rhode Island Agricultural
Experiment Station. University of Rhode Island.

COMPARATIVE EVALUATION OF THE MULTIPLEX
PCR WITH CONVENTIONAL DETECTION METHODS
FOR HAPLOSPORIDILM NELSONSI (MSX) HAPLOSPO-
RiniUM COSTALE (SSO), AND PERKINSUS MARINVS
(DERMO) IN THE EASTERN OYSTER. CRASSOSTREA
VIRdlNlCA. Spencer Russell. Soledad Penna and Richard
French, University of Connecticut, Dept. of Pathobiologv. 61
North F.agleville Rd, U-89, Storrs, CT 06269.

Presently, the monitoring of cultured oyster populations for
pathogeirs is intVequent due to the dependence on traditional, time
consuming diagnostic assays. A multiplex polymerase chain reac-
tion (MPCR) has been developed which rapidly detects the proto-

Milford AqiiacLiltiire Seminar. Milford. Connecticut

Abstracts. February 2000 581

zoan parasites. Perkinsus mariniis (Dermo). Haplosporicluiiii nel-
soni (MSX) and Haplosporidium costale (SSO), which infect the
cultured oyster. Crassostrca virginica. Conventional diagnostic
methods (hislopathology and Ray/Mackin fluid thioglycollate as-
say) for H. nelsoni, H. costale and P. marinus respectively were
compared and evaluated with the MPCR. Ninety-one adult oysters
were collected from randomly selected beds in Westport, CT.
(n = 37) and Milford. CT (n = 54) and subjected to all three assays.
The Ray/Mackin assay detected P. marinus infections in 59 of 91
(64%) oysters and MPCR revealed infections in 73 of 91 (80%)
oysters. Histological examination detected 37 of 91 (40%) oysters
infected with Haplosporidium plasmodia. The MPCR was able to
differentiate between the two Haplosporidium plasmodia. detect-
ing 9 of 9 1 ( 1 0% ) oysters infected only with H. nelsoni. 37 of 9 1
(40%) oysters with only H. costale. and 32 of 91 (35%) oysters
with mixed infections of H. iielsoiii and H. costale. These results
indicate the MPCR is a more .sensitive assay for the detection of P.
marinus and is able to detect and differentiate between the two
Haplosporidium species. This would suggest that the MPCR can
be useful at low infection intensity by being able to detect patho-
gens, based on pathogen DNA concentrations as low as lOfg.. for
H. nelsoni and Ipg. for both H. costale and P. marinus.

IDENTIFICATION OF A PROTOZOAN PARASITE IN
THE AMERICAN LOBSTER. HOMARUS AMERICANUS,
FROM LONG ISLAND SOUND. Spencer Russell. Kristen
Hobble. Tom Burrage. Claudia Koerting. Sylvain De Guise,
Salvatore Frasca Jr., and Richard A. French. University of Con-
necticut. Department of Pathobiology. 61 North Eagleville Rd.
U-89. Ston-s. CT 06269.

Mortalities of the American lobster. Homarus americaims. in
Long Island Sound have severely increased and as a result are
critically damaging the regional lobster industry. Necropsies were
performed on 75 individual lobsters collected from six different
locations in Long Island Sound. Gross observations found in 'sick'
lobsters included a pink discoloration to the ventral surface of the
abdomen (tail meat) and lethargic/limp behavior. An associated
coagulopathy of hemocytes is also observed in affected lobsters.
Initial bacteriology findings include isolation of Vibrio spp. and
spirochetes. No Aerococcus have been isolated to date. Histologic
examination has been conducted on various tissues, including
heart, gill, hepatopancreas. antennary glands, intestine, muscle,
exoskeleton. eyes, antennae, and central nervous system. The his-
lopathology is consistent with a systemic inflammatory disease
affecting multiple tissues but primarily the nervous system. Asso-
ciated with lesions is a protozoan parasite morphologically char-
acterized as an amoeba, tentatively Paramoeha sp.

LIVING CONTAMINANTS IN MICROALGAL FEED PRO-
DUCTION TANKS - WHAT DO WE DO NOW? Barry C.
Smith and Mark Dixon. USDOC. NCAA. National Marine Fish-
eries Service. Northeast Fisheries Science Center. Milford Labo-
ratory, Milford, CT 06460.

The Greenhouse for Research on Algal Mass Production Sys-
tems (GRAMPS) at the Milford Laboratory can produce 20.000
liters of dense algal culture per day. This is done by using half of
two 20.000-liter tanks each day and refilling one while the other is
being drained. Backup starter cultures for these tanks can be
housed in up to eight 500-liter cylindrical tubes. Algal production
can be reduced severely if any of these cultures become contami-
nated with unwanted organisms.

Contaminants that invade algal cultures lower the yield of al-
gae, increase the cost of production, and sometimes destroy the
culture. Some contaminants are benign in that they establish a
minor population that has no detectable effect on the algal culture
or on what the culture is used for. Other contaminants, such as
many ciliates. algae of unsuitable nutritional value that out-
compete the desired algae, and algae that are harmful to the ani-
mals to be fed. spell disaster for an algal culture. Routine quality
control measures, such as microscopic observation, should be used
to detect contaminants as early as possible. When a contaminant is
identified in an algal culture, the fate of the culture must be as-
sessed. Will the contaminant dominate or destroy the culture? Is it
harmful to the use of the algae? Can the contaminant be tolerated?
If the decision is made that the contaminant cannot be tolerated,
the source of the contaminant must be located. There are several
possible contamination vectors. Any fiow, act, or event involving
the algal cultures, or even in the facility, could be responsible.
Perhaps the most common source of contamination is the treatment
of the water used to fill the culture. Contaminants can get on an
operators' hands, hoses, and other accessories that may contact,
even briefly, the culture. Even a random splash from a nearby
workstation can inoculate an algal culture with a contaminant.

Once the source of contamination is located, a remedy can be
found and assessed economically. The best way to solve the prob-
lem of contaminants in an algal culture is to prevent them from
entering the system; this sounds easier than it is. One consideration
is the cost of increasing the level of filtration or pasteurization. Is
treatment with ultraviolet light more desirable? Hygiene/
microbiological sanitation is one way to reduce the risk of con-
tamination. Some remedies have been as simple as switching the
order of daily tasks performed by an operator so that possible
contaminants do not contact the person until after the algal cultures
have been cared for. Contaminants may become resident in the
culture vessels or plumbing; once established, the only way to
remove them is with microbiological sanitizing measures and tac-
tics. Every surface must be sanitized in such a way that the system
has been thoroughly cleaned and no surface has been re-
contaminated.

Living contaminants in an algal culture are best managed by an

582 Abstracts. February 2000

Milford Aquaculture Seminar. Milford, Connecticut

hierarchical decision tree that 1 ) assesses the impact of the con-
taminant, 2) locates the source of harmful contaminants, and 3)
evaluates and tests possible control measures to identify proce-
dures that are effective and economical.

RECENT RESULTS FROM FIELD AND LABORATORY
STUDIES OF QPX. Roxanna Smolowitz, Ernest Marks, and
Chris Brothers, Marine Biological Laboratory. Woods Hole. MA
02543; Dale Leavitt and Bruce Lancaster, Woods Hole Oceano-
graphic Institution, Woods Hole, MA 02543.

Field studies of clam disease (QPX) have been ongoing in
Provincetown and Duxbury. MA since October. 1997. Data col-
lected to date show development of visible QPX nodules in 38% of
mixed parentage (wild/notata) hard clams (Mercenaria merce-
iwria) planted in Duxbury, MA in Oct. 1997. However, wild/
notata, 100% notata and 100% wild parentage clams planted and
sampled at the same times listed above from Provincetown, MA
have shown no nodules to date. Clams from Duxbury are signifi-
cantly larger than Provincetown clams, so decreased food quantity/
quality does not appear to be a significant factor in the develop-
ment of the disease. While the percentage of animals grossly posi-
tive in Duxbury was high during the Oct. 1999 collection period,
no mortality was noted. It is expected that mortality will occur
during the spring of 2000.

In the laboratory, raceways were constructed and put into use in
August of 1999. Raceways consisting of a total of 32 individual
containers received hard clams of approximately 30 mm in shell
height thai had been treated in one of five exposure methods (no
exposure, saline only injection. QPX injection, exposure to QPX in
the water column, exposure to QPX infected two year old adults
gathered from Provincetown flats). Cultured QPX was used in the
injection and water column exposures. To date, no significant mor-
tality has occurred in the raceways.

EFFECTS OF VARIOUS MICROALGAL DIETS ON THE
GROWTH AND SURVIVAL OF LARVAE OF SEA SCAL-
LOPS. PLACOPECTEN MAGELIANICVS. Bethany A. Starr.

Beais Island Regional Shellfish Hatchery. P.O. Box S3, Beals, ME
04611.

A series of feeding experiments using single and mixed species
of unicellular microalgae to determine the growth and survival of
Placupecten inagelUiniiiis lar\ ae were conducted at the University
of Maine at Machias Aquaculture Room from late August - No-
vember 1999. The following algal species were used: Isoclirxsis
galhana (Tahitian strain), Chaetoceros iieognicitc. TluilUisiosini
\vi'i.'isflt)i;i;i. RIioiIdiiuiiuis scilina. and Tetraselinis cliiii.

Experiments were conducted at 14 ± 1 °C: algal cultures were
maintained at temperatures between 17-20 "C. The experiment
was a random block design with five treatments of food combi-
nations (n = 4). Experimental units consisted of 40 L aerated krie-

sels. In early trials, larvae were fed at a rate of 20,000 cells/ml for
single algal species, and 10,000 cells/ml/species in mixed algal
combinations. The feeding regime was later adjusted to begin at
5000 cells/ml with trocophores and increased by 5000 cells/ml at
days 6, 11. 16, and 21 until 20,000 cells/ml were attained after
which time this feeding density was maintained. A feeding trial
was concluded when larvae reached the pediveliger stage or died.
Growth and survival rates were determined by subsampling each
kriesel on drain-down days (occurring every 2 — 3 days). Animals
were measured using an ocular micrometer to determine growth
rates.

Preliminary results indicate that sea scallop larvae grew best on
a combination of Isocliiysis gaibana and Tetraselinis chiii. This
may be due to the high fatty acid content of the T. chid. Other
preliminary results indicate that survival rates were stable early in
the larval stage, but decreased as the larvae reached the pediveliger
stage (depending on food treatment). Future research will focus on
identifying the specific fatty acid content of each algal species, and
also the assimilation of these algae by the scallop larvae.

OBSERVATIONS ON GROWTH AND SURVIVAL OF JU-
VENILE BAY SCALLOPS {ARGOPECTEN IRRADIANS)
FROM GENETIC LINES UNDER DIFFERENT DENSITY
AND HOLDING CONDITIONS. Sheila Stiles, Tasha Robin-
son, and Joseph Choromanski, USDOC, NOAA, National Ma-
rine Fisheries Service. Northeast Fisheries Science Center. Milford
Laboratory. Milford, CT 06460.

Hatchery-reared juvenile bay scallops (Argopccten irnulians)
from mass-spawned foundation crosses for genetic selection were
tested under various nursery and holding conditions to maximize
growth and survival for breeding. Lines consisted of scallops of
two initial mean sizes. MS99-2 (12.5mm) and MS99-4 (4 mm).
Scallops from each line were divided into four groups under dif-
ferent holding conditions: laboratory trays at 2 different densities,
a raceway tank, and a suspension dock at approximately 10 feet
under water. The latter two groups were in pearl nets. Scallops
were measured for length and their volume estimated weekly over
a five-week period during the summer.

Overall survi\al was high and growth was significant for most
of the scallops. Best growth tor the smaller-sized scallops (MS99-
4) initially occurred up to 10 mm in the flowing water trays, after
which growth was better in the raceway tanks, and then tnially was
best at the dock site, with a mean size of IS mm after 5 weeks.
Survival was best in the trays and lowest at the dock. A few small
crabs were observed in the pearl net with the scallops which ac-
counted lor the higher mortality there. The larger scallops (MS99-
2) grew better in the raceway tank and the tray with less density,
at the beginning of the experiment. However, by the end of the
study, the scallops from the MS99-2 line at the dock were more
than 2-fold larger (26.7 mm). Mean sizes of scallops held in the
trays at the two densities were not very different for either line. No

Miltbrd Aquaculture Seminar, Milford, Connecticut

Ahslracts. February 2000 583

line-specific performance was apparent. Results generally con-
firmed those of other researchers for the efficient production of
bay scallops under different holding conditions at various nursery
and growth stages.

PRINCIPAL DISEASES OF CONNECTICUT'S OYSTERS.
Inke Sunila, Joseph DeCrescenzo, John Karolus, and John
Volk. State of Connecticut, Department of Agriculture, Bureau of
Aquaculture, P.O. Box 97, Milford, CT 06460.

Oysters are long-lived, sessile animals, which have the ability
to accumulate hundredfolds of micro-organisms and pollutants
while filter feeding. This makes them susceptible to diseases. Sev-
eral factors can induce pathological changes in oysters: infections
caused by \iruses. bacteria and parasites, age, fouling, predation,
siltation, biotoxins, starvation, pollutants, oxygen deficiency and
variations and extremes in temperature and salinity. These factors
may cause pathological changes such as inflammatory responses
(acute or chronic), degenerations (vacuolization, inclusions, ceroi-
dosis). cell and tissue death (necrosis, apoptosis). growth derange-
ments (hyperplasia, metaplasia), hemodynamic and fluid derange-
ments (edema, hemorrhage), and neoplasia (benign or malignant).
The sum of environmental stimuli, together with the genetic make
up of the oysters, will determine their likelihood for disease.

From 1 997 to 2000 we collected 3000 oysters from a hundred
sampling stations in Connecticut, processed them for histology and
diagnosed them for different categories of pathology. Despite the
tradition of transplanting oysters, different areas of CT's oyster
grounds were characterized by different conditions. Fouling or-
ganisms such as Crepidula spp. occurred in the western and central
part of CT and were replaced by limpets in the eastern part. Am-
pharetid worms. Sabelkiria vulgaris, calcareous tubeworms and
different species of Bryozoa were present in the central and west-
em part of CT. Parasitic infestation by trematodes and Turbellaria
were prominent in the eastern part. Neinatopsis ostrearum was
present at high prevalence from New Haven to Westbrook. Poly-
dora websteri occurred in seed beds in the Housatonic. Quinnipiac
and Thames Rivers and also in small rivers between Guilford and
Westbrook. Cliona spp. and ciliates were present in all sampling
stations. MSX-disease (caused by Haplospohdiiim nelscmi) oc-
curred at epizootic prevalence causing high morlaHties in western
and central CT. Infected animals had chronic hyaline hemocyte
inflammatory responses. Dermo-disease (caused by Perkinsus
mariiuts) established high prevalence on the entire coastline. In-
fected specimens were likely to have ceroidosis. Ulcers were found
in the intestine and stomach epithelia in oysters from Bridgeport.
Norwalk and the Housatonic River. Xenomas in the gills and viral
gametocyte hypertrophy occurred at low prevalence in all sam-
pling areas. Both benign and malignant tumors were detected. A
large pericardial tumor, described as a vesiculo-epithelial polyp,
was found in one oyster in Groton. Several cases of enteric ad-
enocarcinoma /;; situ were detected. Tumors were composed of

cystic glands with cellular debris and mucus inside the lumens.
Epithelia forming the glands were basophilic, thickened and with
increased cellularity and mitotic figures. According to published
reports, similar lesions have been induced in the laboratory by
exposing oysters to sediment from Black Rock Harbor, Bridgeport.
In conclusion, disease resistance to MSX is developing in the
oyster population. The overall health of the Connecticut oyster can
be considered good.

ESTABLISHMENT OF RHODE ISLAND'S FIRST COM-
MERCIAL SHELLFISH HATCHERY. Karin A. Tammi,
Wayne H. Turner, and Luning Sun, Hope Shellfish Company.
Post Office Box 4, Portsmouth, Rhode Island 02871 1; Michael A.
Rice. Fisheries. Animal and Veterinary Science Department. Uni-
versity of Rhode Island. Kingston. Rhode Island 0281 1,

Hope Shellfish Company (HSC) recently completed permitting
to become the first commercial shellfish hatchery in the history of
Rhode Island. This project has been four years in the making and
will be operational in the spring of 2000. The principals of the
HSC have invested a considerable amount of time, patience and
energy securing the financing and permits. Traversing the recent
aquaculture regulations promulgated by the state's Department of
Environmental Management and the Coastal Resource Manage-
ment Council were not easy tasks. In order to acquire the proper
approvals. Hope Shellfish Company networked with more than 25
regulatory representatives from federal, state and local agencies.
Explaining the aquaculture project to regulatory agencies required
preparation of detailed and lengthy applications with the total
amount of documentation easily exceeding 500 pages.

By combining years of experience in shellfish restoration and
commercial culturing. the principals anticipate producing over 40
million large-size shellfish seed annually when the facility is fully
operational. HSC will begin producing bay scallops. Argopecten
inadians. eastern oysters. Crassostrea virgiiiica. quahogs. Merce-
naria mercenaria. and soft-shell clams. Mya arenaria. It was es-
timated that this project could generate enough "raw product" to
allow 500 fishermen to earn a competitive day's pay year-round
and could support jobs in value-added industries such as process-
ing, marketing, and distribution and will have a positive influence
on recreational shellfishing and tourism in Rhode Island. This
facility offers tremendous opportunities to aquaculturists. shell-
fishermen, researchers, educators, and resource managers in the
State of Rhode Island and beyond. The benefits of this project are
numerous, yet none more significant than the overall economic and
cultural benefits to the citizens in this region. This project has the
ability to elevate the status of aquaculture in the State as well as to
educate students and researchers from the region's schools and
universities.

584 Ahstnuts. February 2000

Milford Aquaculture Seminar, Milford, Connecticut

AN INEXPENSIVE DIGITAL TEMPERATURE SENSOR
FOR DATA ACQUISITION USE IN AQUACULTURE.
James C. Widman, Jr., USDOC, NOAA, National Marine Fish-
eries Service, Northeast Fisheries Science Center, Milford Labo-
ratory. Milford, CT 06460.

A few parts, a personal computer, and some mechanical ability
can yield an inexpensive digital thermometer which can be used
for monitoring air and water temperatures in an aquaculture facil-
ity. Digital temperature sen.sors (DS1820, DS18B20) and COMM
port adapters (DS9097U) are available from Dallas Semiconductor
at http://www.dalsemi.com. The temperature sensors have an ac-
curacy of ± 0.5 °C from -10 to 85 °C and a full range of -55 to
125 °C. By using category-5 cable, sensors can be placed at a
distance of up to 300 meters from a personal computer. After
soldering the cable to the sensor, a small piece of vinyl tubing is
placed over the wire and attached to the body of the sensor with
underwater epoxy or silicone. To ensure no electrical leakage, the
entire surface of the sensor is coated. This adds slightly to the
thermal mass and increases response time, but temperatures gen-
erally change slowly in an aquaculture facility. These sensors have
been used for over a year in a submerged marine environment.

mollusks themselves have received less attention. As part of a
larger study designed to investigate the role of grazing in the
bloom dynamics of microalgae for which there is some evidence of
grazing suppression, we conducted experimental exposures of bay
scallops. Argopecteii inadians. at several life-history stages (em-
bryos, larvae, post-set, and juveniles) to a number of cultured
microalgal strains. Microalgae investigated included; 1 ) di-
noflagellates, two strains of Prorocentnim minimum. Gyrodinium
aureoluin. and Gymnodiniiim splendens; 2) a raphidophyte, Het-
erosigma carterae; and Prymnesiophytes, two strains of Prymne-
siiim pannim and one of P. patellifenim. Scallop response vari-
ables measured included survival, growth, development, feeding
behavior, and histopathology. Effects ranging from subtle and sub-
lethal to acute toxicity were observed. The most dramatic, lethal
effects were seen with a new strain of Prorocentnim minimum,
collected by Dr. Patricia Gilbert from a 1998 bloom in the Chop-
tank River, MD, and with a new strain of Prymnesium parvum.
isolated by Dr. Robert Guillard from Boothbay Harbor, ME. In
addition to limiting the harvest of molluscan shellfish for human
consumption, harmful algal blooms have the potential to affect the
population biology of molluscs themselves.

RESPONSES OF BAY SCALLOPS, AT SEVERAL LIFE-
HISTORY STAGES, TO CULTURES OF POTENTIALLY-
HARMFUL MARINE MICROALGAE. Gary H. Wikfors,
Jennifer H. Alix, Sara Barcia, and Julie Cullum, USDOC.

NOAA, National Marine Fisheries Service, Northeast Fisheries
Science Center, Milford Laboratory, Milford, CT 06460; Sandra
E. Shumway, Southampton College, LIU, Southampton, NY
11968; Roxanna M. Smolowitz. Marine Biological Laboratory.
Woods Hole. MA 02543.

Widespread use of the term "Harmful Algal Bloom" begs the
question: Harmful to whom? Molluscan shellfish have been rec-
ognized as vectors of microalgal toxins to human consumers for
millennia, but detrimental effects of some microalgae upon the

PROCESS DESIGN FOR ARTEMIA CULTURE AT
COASTAL BIOMARINE. Loy Wilkinson. Coastal BioMarine,
250 Northrup St., Bridgewater. CT 06752.

A method for the design of a closed cycle system for the in-
tensive production of Anemia from algae is described. The process
for the intensive production of Anemia is an adaptation of the one
practiced at the Laboratory of Aquaculture and Artemia Reference
Center. University of Ghent and described in the Manual of the
Production of and Use of Live Food for Aquaculture. The overall
material balances are presented as well as the design method for
individual equipment in the process. Emphasis is placed on the
treatment of the water to remove dissolved organics and ammonia.
Theoretical and in-practice parameters for effective removal of
these contaminants by means of a trickle bed filter are discussed.

JoKi-mil of Shellfish Research. Vol. \9. No. 1. 585-668. 2000.

ABSTRACTS OF TECHNICAL PAPERS

Presented at the 92nd Annual Meeting

NATIONAL SHELLFISHERIES ASSOCIATION

Seattle, Washington
March 19-23. 2000

585

National Shellfisheries Association. Seattle. Washington Ahslracrs. 2000 Annual Meeting. March 19-23, 2000 587

CONTENTS
ENVIRONMENTAL AND ANTHROPOGENIC INFLUENCES ON SHELLFISH

Fu-Lin E. Chit, Phillippe Soudant and Robert C. Hale

PCB assimilation in oysters {Crassostreci virgiiiica): an implication for reproductive impairment 597

Gaskov Clerge. Mahendra H. Kothary, Marianne D. Miliotis, Darcy E. Hanes, Seynabou Fall. Jeffrey W. Bier,
Dhirendra B. Shah, B. D. Tall, Broderick Eribo, Jerome F. La Peyre and Mohamed Faisal

Adherence and invasion mechanisms of Vibrio vulnificus with oyster and fish cultured cells 597

Luis A. Cruz-Rodriguez, Fu-Lin E. Chu and Philippe Soudant

Stress protein (HSP70) response in oysters Crassostrea virginica exposed to various stress agents 598

Susan E. Ford, Roxanna Snwlowitz and Marnita M. Chintala

Temperature and range extension by Pcikinsus nuiriuus 598

Fred J. Genthner, William S. Fisher, Aswani K. Volety, Ben D. Tall, Sherill K. Curtis and Susan A. McCarthy

Responses of oysters and their hemocytes to clinical and environmental isolates of Vibrio parahaemolyticus 598

Amro M. Hamdoun, Daniel Cheney, Ralph Elston, Brian McDonald and Gary N. Cherr

Summer stress protein responses of cultured Pacific oysters: does chronic stress reduce tolerance? 599

Allison C. Luengen, Carolyn S. Friedman and A. R. Flegal

Immune responses of two species of mussels (Mytilus cctlifornianus and Myriliis galloprovincialis/trossuliis hybrid) to

pollutants in San Francisco Bay. CA 599

L. M. Oliver, W. S. Fisher, A. K. Volety and Z. Malaeb

Relationships between oyster (Crassostrea virginica) defense measurements and tissue contaminants 599

Aswani K. Volety and William S. Fisher

In vitro killing of Perkinsus marinus by hemocytes of oysters 600

Inge Werner

Stress proteins as biomarkers in estuarine shellfish species 600

FEEDING AND NUTRITION

Peter G. Beninger and Suzanne C. Diifour

Evolutionary and functional trajectories of the bivalve gill abfrontal surface: lessons from contemporary cilia and

mucocyte distributions 600

Eleanor Bochenek, Eric Powell, John Klinck and Eileen Hofmann

A biochemically-based model of the growth and development of Pacific oyster Crassostrea gigas larvae 601

Martha G. S. Brillant and Bruce A. MacDonald

Using Chlorellu to study postingestive selection in bivalves 601

Peter J. Cranford, Shelley L. Armsworthy, Michael J. White and Timothy G. Milligan

Natural diet effects on food utilization by sea scallops and blue mussels 601

C. L. Demetropoulos and C. J. Langdon

Enhanced production of Pacific dulse [Pabnaria mollis) for co-culture with red abalone [Haliolis rnfescens) in a

land-based system 602

Catherine M. Gatenby, Daniel A. Kreeger, Vanessa A. Jones, David M. Orcutt, Bruce C. Parker and Richard J. Neves

Nutritional status of four algal diets for the captive care of freshwater mussels 602

J. M. Hall. R. J. Thompson and C. C. Parrish

Changes in the tluidity and fatty acid composition of cell membranes froin the .sea scallop (Placopeclen

magellcmiciis) during short-term cold acclimation 602

Daniel A. Kreeger, Roger /. E. Newell and Shou-Chung Huang

Natural sources of nutrition for the mussel Geukensia demissa 603

C. J. Langdon, C. Seguineau, B. Ponce, J. Moal and J. F. Saniain

Riboflavin supplements for larval and adult Pacific oysters (Crassostrea gigas) delivered by lipid spray beads 603

Lisa M. Milke and J. Evan Ward

Pallial cavity residence time in two species of bivalved mollusks: Mytihis ecliilis and Crassostrea virginica 603

Carter R. Newell, Cynthia Pilskaln, Shawn Robinson and Bruce MacDonald

Particle tlux and consumption by mussels at Roque Island. Maine: the importance of marine snow 604

Eric Powell, John Klinck, Eileen Hofmann and Eleanor Bochenek

Food quality and feeding strategies in hatchery rearing of Pacific oyster Crassostrea gigas larvae:

a modeling approach 604

Gunlher Rosen, Chris J. Langdon and Ford Evans

The nutritional value of Palmaria mollis cultured under different light intensities and water exchange rates for

juvenile red abalone Haliotis rnfescens 604

588 Absiructs. 2000 Annual Meeting. March 19-23. 2000 National Shellfisheries Association, Seattle. Washington

J. F. Samain, C. Quere, J. R. Le Coz, C. Seguineau, P. Sotidaiit, J. Moal, P. Sorgeloos, M. Caers, C. Van Ryckeghem,
O. Garcia, J. Espinosa, Y. Marty, M. Mathieu and C. Berthelin

Gigas, nutrition and gametogenesis; presentation and first results of the European project Giganuga 605

Philippe Soiidant, Fu-Lin Chu and Jean-Francois Samain

Lipid requirements in some economically important marine bivalves 605

Kevin R. Stuart, Arnold G. Eversole and David E. Brune

Algal uptake rate of freshwater mussels 605

S. C. Feindel, R. J. Thompson and C. C. Parrish

Changes in the fatty acid composition of the flagellate Pavlova pingnis ICCM0459) during culture 606

J. Evan Ward, Jeffrey S. Levinton, Sandra E. Shiiinway and Terri L. Cucci

Influence of diet quality on pre-ingestive feeding strategies of bivalves: connecting paliial cavity function to

ecosystem processes 606

Gary H. Wikfors

Aquacultural feeding standards for molluscan shellfish seed: a first cut 606

FORUM: BIOLOGICAL AND TECHNOLOGICAL TRANSFERS IN SHELLFISH

Joth Davis

Biological and technological transfers in shellfish aquaculture 607

FORUM: THE APPROPRIATENESS OF CULTURING BIVALVES FOR FOOD, PROFIT, RESOURCE
RESTORATION, HABITAT AND WATER QUALITY MITIGATION

William F. Dewey and Daniel P. Cheney

Forum on the appropriateness of cuituring bivalves for food, profit, resource restoration, habitat and water quality

mitigation 607

FUNCTIONAL ROLE OF BIVALVES IN MARINE ENVIRONMENTS
Loren D. Caen, Mark W. Luckenbach and Denise Breitburg

Oyster reefs as essential fish habitat for finfish and decapod crustaceans: a comparison from natural and

developing reefs 608

Richard Dame, David Biishek, Dennis Allen, Alan Lewitus, Eric Koepfler, Leah Gregory and Don Edwards

Bivalves or nekton? Is that the question? 608

Brett R. Dumbauld, Steven P. Ferraro and Faith A. Cole

Oyster aquaculture and benthic invertebrate communities in West Coast estuaries: an update 608

A. J. Erskine, David Biishek, Richard Dame, Nancy Hadley and Loren Coen

Juvenile oyster growth and carrying capacity of intertidal creeks in North Inlet. SC 609

Ray Grizzle and Mike Castagna

Natural intertidal oyster reefs in Florida: can they teach us anything about constructed/restored reefs? 609

Michael L. Judge, Loren D. Coen and Kamille Hammerstrom

The ecological implications of high density hard clam (Mcrccnaria mcrcciuiria) mariculture on tidal

creek environments 609

Mark Luckenbach, Francis O'Beirn, Juliana Harding, Roger Mann and Janet Nestlerode

Temporal patterns of fish and decapod iilili/alion of oyster reefs: comparison across an estuarine gradient 610

Carter R. Newell and John E. Richardson

Grazing of natural particulates by blue iiuisscls on rafts: simulations using Flow-.^D 610

Roger I. E. Newell and JeffC. Cornwell

Role of suspension leediiig bi\al\es in mediating estuarine nutrient cycling 610

Bradley ./. Peterson and Kenneth L. Heck, Jr.

Inlerrelalionships between seagrasses ami benlhic suspension feeders 610

Martin H. Posey, Troy I). Alphin, Christopher M. Powell and John M. Rhoads

Use of oyster reefs by mobile fauna: consequences lor adjacent saiultlat habitats 611

Linda Righetti

Nitrogen excretion by (he Pacific oyster. Crassosiica ifii^as: a contributor to estuarine nutrient cycling in Tomales

Bay. CA 611

National Shellfisheries Association. Seattle, Washington Abstracts. 2000 Annual Meeting, March 19-23, 2000 589

Ray Seed

Mussels: space monopolisers or ecosystem-engineers? 611

Janet K. Thompson

Two stories of phytoplankton control by bivalves in San Francisco Bay: the importance of spatial and temporal

distribution of bivalves 612

GENETICS AND BREEDING

Standish K. Allen, Jr.

Research and development on suminoegaki, Crassostrea ariakensis, for aquaculture in Virginia, and other activities

with non-natives 612

Pierre Boudry, Bertrand Collet, Florence Cornette, Veronique Hervouet and Frangoise Bonhomme

Microsatellite markers as a tool to study reproductive success in the Pacific oyster, Crassostrea gigas (Thunberg),

crossed under controlled hatchery conditions 612

Ben Bowen

Bead-based genomics technologies at Lynx: applications for Pacific oyster breeding 613

John T. Buchanan, Amy D. Nickens, Terrence R. Tiersch and Richard K. Cooper

Transfection of Eastern oyster embryos 613

Christopher V. Davis

Estimation of nanow-sense heritability for larval and juvenile growth traits in selected and unselected sub-lines of

Eastern oysters 613

Daniel DenDanto, Bonnie L. Brown, Chris Davis and Irving Kornfield

Analysis of genetic diversity in a commercially important line of oysters selected for fast growth 613

Matthew P. Gordon and Paul D. Rawson

Patterns of nucleotide variation at the GPI locus in the blue mussel, Mytilus edidis 614

Ximing Quo, Guofan Zhang. Brenda J. Landau, Louise English and Yongping Wang

Aneuploidy in the Pacific oyster, Crassostrea gigas Thunberg and its effects on growth 614

Dennis Hedgecock and Jonathan P. Davis

Improving Pacific oyster broodstock through crossbreeding 614

Sophie Hubert, Louise J. English, Brenda J. Landau, Ximing Guo and Dennis Hedgecock

Microsatellite analysis of trisomic families in the Pacific oyster, Crassostrea gigas Thunberg 615

Ellen Kenchington, Liqin Cao and Eleftherios Zouros

Nuclear control of sex ratio bias in the mussel, Mytilus edidis 615

Manfred Kittel and Kenneth K. Chew

Growth, shell morphology, reproductive physiology and molecular genetic analysis of Tasmanian Pacific oysters,

Crassostrea gigas. in Washington state 615

Chris J. Langdon, Dave P. Jacobson, Ford Evans and Mike S. Blouin

The Molluscan Broodstock Program — improving Pacific oyster broodstock through genetic selection 616

J. Moal, S. F. Samain, J. Y. Daniel, P. Boudry, S. Bougrier, D. Sellos, A. Van Wormhoudt

Evidence of ab.sorption efficiency differences in two subpopulations of Crassostrea gigas. A first approach of their

amylase gene polymorphism 616

Douglas A. Pace and Donal T. Manahan

Genotype dependent differences in feeding rates and growth in oyster larvae 616

Kimberly S. Reece, Wenda L. Ribeiro, Patrick M. Gaffney and James Pierce

Development of molecular markers for constructing a genetic linkage map of the Easter oyster Crassostrea virginica . . 617
Rejean Tremblay, Thomas Landry, Bruno Myrand and Jean-Marie Sevigny

Genetic characteristics of wild and cultured mussels, Mytilus eduUs and Mytilus trossuhis in Prince Edward Island

(Gulf of St. Lawrence) 617

Brent A. Vadopalas, Are Strom and Paul Bentzen

Microsatellite variation in geoduck clams (Panopea abrupta) in Puget Sound, Washington 617

Yongping Wang, Zhe Xu, Ximing Guo, James C. Pierce and Patrick M. Gaffney

Chromosomal location of some repetitive DNA in Crassostrea oysters as determined by FISH 618

590 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association, Seattle, Washington

INVERTEBRATE FISHERIES

Bruce E. Adkins

The British Columbia fishery for northern abalone, Haliotis kamtschatkana: management from inception to closure

and beyond 618

Alex Bradbury

Stock assessment and management of red sea urchins 618

Therese A. Cain and Jay G. Odell

Shrimp fisheries and management in Hood Canal and Paget Sound 619

Jennifer Cahalan

Application of hypothesis testing and power analysis in the Puget Sound crab fishery: closure decisions

with confidence 619

James I. Child and William W. Campbell

Management of intertidal bivalves in Puget Sound. Washington 619

Bruce Clapp

Underwater harvester's association geoduck enhancement program 620

Rick Harbo, L. Convey and J. A. Boutillier

Co-management and assessment programs in the shrimp trawl fishery of British Columbia 620

Assane Diagne, Walter R. Keithly Jr. and David Lavergne

An economic analysis of the Gulf of Mexico oyster processing sector 620

Rick Harbo and Randy Webb

Management strategies for commercial intertidal clam fisheries in British Columbia. Canada 621

Stephen Heizer

The commercial geoduck (Panopea ahnipta) fishery in British Columbia. Canada — an operational perspective of a

limited entry fishery with individual quotas 621

Peter E. Kalvass

Riding the rollercoaster: boom and decline in the California red sea urchin fishery 621

Michael D. Kaplowitz

Uncovering benefits of West Indian crown conch or 'chivita" (Melongena melongena) in Yucatan. Mexico 622

Jonathan A. Keogh and David J. Fletcher

Temporal and spatial variation in spawning oysters {Tiosirea chileiisis) distribution in Foveaux Strait, New Zealand ... 622
Carlo Medeiros y Araiijo and lara L. G. Brasileiro

Clam fishery in Brazil 622

Carol McCollough, Stephen J. Jordan and Mark L. Homer

Chesapeake Bay Oysters; Trends in relative abundance and biomass 623

Jim Morrison

Management of the Canadian Pacific Coast prawn trap fishery: recent changes, present status and future options 623

Guy Parker, Kim West and Ivan Winther

Management of the Dungeness crab fishery in British Columbia 623

R. Ian Perry, Guy Parker and Juanita Rogers

Science and management of green sea urchins in British Columbia - a rebuilding fishery? 623

Juanita Rogers and Guy Parker

Management of the red sea urchin fishery in British Columbia 624

Leonard ./. Rogers and David B. Rouse

Coupling a conipulational fluid dynamic model with a habitat suitability index model to manage Eastern oysters in

Mobile Bay. Alabama 624

Scoresby A. Shepherd

The price of sustainability is eternal vigilance 624

Doug Simons and Dan L. Ayres

Management of the Pacific razor clam and the current marine toxin threat in Washington State 62.S

Bob Sizemore

Management of geoduck clams l Panopea ahnipia) in Washington State 625

National Shellfisheries Association. Seattle. Washington Abstracts. 2000 Annual Meeting. March 19-23. 2000 591

Bradley G. Stevens

Research acti\ ities in support of Alasl^an crab fisheries 625

Derrick R. Toha

Dungeness crab ( Cancer magister) management in Puget Sound. Washington 625

Mia J. Tegiier

California abalone fisheries: what we" ve learned and where we go from here 626

Donn A. Tracy and Brad Stevens

Biology and management of Eastern Bering Sea king and tanner crab fisheries 626

Tracy Vassiliev, William Congleton, Brian Beal and Stephen Fegley

Larval recruitment of Mvn arenaria (soft-shell clams) in Eastern and Southern Maine 626

Lauran Cole Warner and Eileen P. Visser

But the crabs keep coming: trials and successes of the Grays Harbor Dungeness crab mitigation program 627

James Weinberg. Paul Rago. Charles Keith. Lisa Hendrickson. Steve Murawski. Eric Powell. Roger Mann and
Chris Weidman

Stock assessment of surfclams along the East Coast of the United States: the importance of estimating

dredge efficiency 627

Charles A. Wilson, Harry H. Roberts and John Supan

MHACS: Marine habitat acoustic characterization systems. A program for the acquisition and interpretation of digital

acoustics to characterize marine habitat 627

MANAGING SHELLFISH CULTURE IN COASTAL WATERS

William F. Dewey

Endangered Species Act and Sustainable Fisheries Act implications for molluscan shellfish culture management 628

Mark W. Luckenbach

Resource management issues facing shellfish aquaculture in the Mid- Atlantic Coast 628

Ed Rhodes

The Department of Commerce aquaculture program — implications and opportunities for shellfish culture 628

Ruth Salmon

The development of an environmental management system for the BC shellfish farming industry 629

Derrick R. Toba

Impacts of the Stevens Treaties on Western Washington Tribal shellfish culture 629

NUISANCE SPECIES

Elizabeth M. Carr and Brett R. Dumbauld

Status of the European green crab invasion in Washington coastal estuaries: can expansion be prevented? 629

Anita E. Cook and Sandra Hanson

Progress implementing a plan to monitor for presence of the European green crab iCarcinus maenas) in Puget

Sound. Washington 630

Paul A. Dinnel and Erika Yates

Biological and ecological assessments of Niitlallia obscurata in North Puget Sound 630

Andrew N. Cohen

Biological invasions in coastal waters 630

Carolynn S. Culver and Armand M. Kitris

Pro-active management of introduced marine pests: lessons from the apparently successful eradication of the sabellid

worm in California 631

Edwin Grosholz and Paul Olin

Predation by European green crabs on Manila clams in central California 63 1

Edwin Grosholz and Gregory Ruiz

The impact of European green crab in central California 631

Chris Hunt

Potential limitations of the European green crab. Carcimis maenas. in habitat suitable for the native red rock crab.

Cancer prodiictiis "3/

592 Abstracts. 2000 Annual Meeting, March 19-23. 2000 National Shellfisheries Association, Seattle, Washington

Gregory C. Jensen, P. Sean McDonald and David A. Armstrong

East meets West: competitive interactions between green crab and Heinigrcipsiis 632

P. Sean McDonald, Gregory C. Jensen and David A. Armstrong

The potential impacts of Carcinus maenas introduction on juvenile Dungeness crab. Cancer mat;ister. survival 632

Pam Meacham

The status of aquatic nuisance species prevention in Washington State 633

Kelly C. Palacios and Steven P. Ferraro

The European green crab bivalve consumption rates and prey preferences 633

Melissa J. Southworth, Juliana M. Harding and Roger Mann

Abundance of small predatory gastropods (Urosalpinx cinera. Euplciira caudate. Rapaiia veiuisa) in relation to lower

Chesapeake Bay oyster ( Crassostrea virginica ) populations 633

Tanya C. Veldhuizon

Status of the Chinese mitten crab in California 633

William C. Walton

Mitigating effects of nonindigenous marine species: evaluation of selective harvest of the European green crab.

Carcinus maenas 634

Miranda Wecker, Donald Strong and Fritzi Grevstad

Integrating biological control in the integrated pest management program for Spartina allcrniflcira in Willapa Bay 634

Sylvia Behrens Yamada, Chris Hunt and Alex Kalin

Growth of the 1997/1998 year class of the European green crab. Carcinus maenas. in Oregon estuaries 634

PHYTOPLANKTON HARMFUL TO SHELLFISH AND CONSUMERS

V. Monica Bricelj, Betty M. Twarog, Scott P. MacQuarrie, Pamela Chang and Vera L. Trainer

Does the history of toxin exposure influence bivalve population responses to PSP toxins in Mya arenarial: I)

burrowing and nerve responses 635

Rita A. Horner, Frank H. Cox and Linda D. Hanson

Harmful algal blooms and shellfish toxicity in Washington State 635

Yukihiko Matsuyama, Taktiji Uchida and Tsuneo Honjo

Impact of harmful dinoflagellate Heterocapsa circularisijuaina on shellfish aquaculture in Japan 636

James Hiingerford, Ronald M. Manger, Sue Lee, Linda Leja, Charles Kaysner, and Marleen Wekell

Methods for detecting marine toxins 636

Scott P. MacQuarrie and V. Monica Bricelj

Does the history of toxin exposure influence bivalve population responses to PSP toxins in Mya arenaria''!: II)

feeding, survival and toxin accumulation 636

John S. Ramsdell

Domoic acid toxicity: practical solutions for organizations to reduce the impact 637

Chris A. Scholin

Application of DNA probes for detection of harmful algae 637

Sandra E. Sham way

Harmful uigal blooms and shellfish aquaculture: implications for the future of the industry 637

Jeffrey Springer, .Sandra E. Shumway and JoAnn Burkholder

Behavioral variability of the toxic dinotlagellate. Pficsleria piscicida. wlien introduced to larval and adult shellfish — 637
Vera L. Trainer, Nicolaus G. Adams, John C. Wekell and Mitch Lesoing

Domoic acid production by Pseudo-nit-schia pseudodelicalissima off the central Washington coast is linked to record

levels of toxin in razor clams 638

John C. Wekell, Vera Trainer, Dan Ayres and Doug Simons

The distribution of domoic acid concentrations in razor clams as a lunclion of ele\ation between high and low tides

at Kalaloch Beach. Washington 638

J. N. C. Whyte, N. G. Ginther, L J. Keddy and R. Chiang

Variance in amnesic shellfish poisoning in geographically discrete |iopulatiniis of razor clams {Silit/ua pauila) in

British Columbia 638

Gary H. Wikfors, Jennifer H. Alix, Sandra E. Shumway, Sara Barcia, Julie Cullum, and Roxanna M. Smolowitz

Experimental exposures of bay scallops to cultures of suspected harmful microalgae 639

National Shellfisheries Association. Seattle. Washington Abstracts. 2000 Annual Meeting. March 19-23. 2000 593

SHELLFISH BIOLOGY

Lewis E. Deatoii, Bruce E. Felgenhauer and Daniel W. Duhon

The bulbus arteriosus of the clam Mercenaria niercenaria: anatomy and pharmacology 639

F. Scott Rikard, Richard K. Wallace, David Rouse and Imad Saoud

The effect of low oxygen on oyster survival during reef restoration efforts in Bon Secour Bay. Alabama 640

Ellen L. Kenchington, Kenneth R. Freeman, Scott P. Macquarrie and Shawn M. C. Robinson

Use of DNA markers to detect differential larval settlement patterns of Mytilus ediilis and M. trossulus 640

Gretta O'Sullivan, Mdire F. Mulcahy

Reproductive biology of Pacific oysters: some enigmas 640

Bradley G. Stevens

Moonlight madness and larval launch pads: tidal synchronization of mound fonnation and hatching by tanner crabs.

Chionoecetes bairdi 640

SHELLFISH HEALTH MANAGEMENT
Robert S. Anderson and Amy E. Beaven

Antimicrobial activity in cell-free hemolymph of oysters and mussels 64 1

Bruce J. Barber, Christopher V. Davis, Ryan B. Carnegie and Katherine J. Boettcher

Management of juvenile oyster disease (JOD) in Maine 641

Susan M. Bower and Gary R. Meyer

Description of an unusual parasite in prawns. Pandalus pkityceros. in British Columbia. Canada 642

L. E. Burnett and C. S. Milardo

Inside the shell of an intertidal oyster: liabilities and benefits'? 642

Eugene M. Burreson

Disease diagnosis by PCR: foolproof or foolhardy? 642

David Bushek, Jennifer Keesee, Ben Jones, Dave White, Matt Neet and Dwayne Porter

Shellfish health management: a system level perspective for Perkinsus mariniis 642

Gustavo W. Calvo, Mark W. Luckenbach and Eugene M. Burreson

High performance of Crassostrea uriakcnsis in Chesapeake Bay 643

Ryan B. Carnegie, Bruce J. Barber, Daniel L. Distel and Sarah C. Culloty

Development of a PCR assay for detection of Bonamia ostreae in fiat oysters. Ostrea edidis 643

Christopher F. Dungan, Rosalee Hamilton, David Bushek, Jennifer Cardinal and Alan Lewitus

Serological affinities between Perkinsus mariniis and some parasitic dinoflagellates 643

Rebecca Ellin and David Bushek

An examination of ecological factors governing planktonic abundance and dispersal of Perkinsus marinus 644

Ralph Elston, Arthur Gee and Russell P. Herwig

Bacterial pathogens, diseases and their control in bivalve seed culture 644

Carl A. Finley and Carolyn S. Friedman

Life history of an exotic sabellid polychaete. Terehrasaljella heteroiincinata: infiuence of temperature and

fertilization strategy 645

Susan E. Ford, Zhe Xu and Gregory Debrosse

Field transmission studies of Huplospondium nelsoni (MSX) using specific primers and PCR technology 645

Carolyn S. Friedman, Thea T. Robbins. James D. Moore, Jeffrey D. Shields, Karl B. Andree, Katherine A. Beauchamp,
Dolores B. Antonio and Ronald P. Hedrick

Candidaliis xenohaliotis califoriensis. a newly described bacterial pathogen and etiological agent of abalone

withering syndrome 645

Jerome F. La Peyre and Yanli Li

Isolation and primary culture of Eastern oyster hemocytes 646

James D. Moore, Viviane Boulo, Jane C. Burns and Carolyn S. Friedman

Retroviral vector-mediated oncogene transfer to create Crassostrea virginica cell lines 646

J. Frank Morado, Theodore R. Meyers and Robert S. Otto

Distribution and prevalence of bitter crab syndrome in snow (Chionoecetes opilio) and tanner (C. hairdi) crabs of the

Serine Sea. 1988-1996 646

594 Abstracts. 2000 Annual Meetine. March 19-23, 2000

National Shellfisheries Association, Seattle, Washington

Amy D. Nickens, Terrence R. Tiersch and Jerome F. La Peyre

Effect of a lytic peptide and protease inhibitors on Perkinstis marianus in infected hemocytes of Eastern oysters 647

Kimberly S. Reece, Eugene M. Burreson, Susan M. Bower and Christopher F. Dungan

Molecular analysis of a parasite in prawns (Paiulalits platyceros) from British Coluinbia, Canada 647

Spencer Russell, Kristen Hobble, Tom Barrage, Claudia Koerting, Sylvain De Guise, Salvatore Frasca Jr. and
Richard A. French

Identification of a protozoan parasite in the American lobster. Hoinanis americdiuis. from Long Island Sound 648

Spencer Russell, Soledad Penna and Richard A. French

Comparative evaluation of the multiplex PCR with conventional detection methods for Haplosporidiwn nelsoiii

(MSC). Haplosporidiwn costale (SSO), and Peikinsiis inariniis (Dermo) in the Eastern oyster, Crassostrea virginica .. 648
Roxanna Smolowitz, Ernest Marks, Chris Brothers, Dale Leavitt and Bruce Lancaster

Results of QPX field studies 648

Nancy A. Stokes, Lisa M. Ragone Calvo and Eugene M. Burreson

DNA-based molecular diagnostics for the hard clam parasite QPX (Quahog parasite unknown) 649

Antonio Villalba, Sandra M. Casus, Maria J. Carballal and Carmen Lopez

Effects of Perkinsosis on the clam Ruditapes decussates industry of Galica (NW Spain) 649

Gary H. Wikfors, Barry C. Smith and Loy Wilkinson

Design criteria for microalgal feeds production systems and the GRAMPS experience 649

TECHNOLOGICAL ADVANCES IN MOLLUSCAN AQUACULTURE

A. O. Alabi

The use of probiotic techniques for controlling bacterial diseases in murine invertebrate hatcheries 650

John Bayes

Recent developments in mollusk hatchery techniques 650

S. Chen, B. B. Saucier, J. S. Zhu and E. Durfey

Recirculation system design for shellfish wet storage or depuration 650

John W. Brake, Jeffrey Davidson, Jonathan Davis

Triploid production of Mylilus edidis in Prince Edward Island 651

Daniel P. Cheney and John L. Pitts

Application of a shellfish science club model in Puget Sound, Washington 651

Carolyn S. Culver, John B. Richards and Henry M. Page

Manipulation of the cementing process of the purple-hinge rock scallop. Crassadomci gigantea 651

T. Jeffrey Davidson and Richard K. Gallant

PEI mussel aquaculture: changing technologies 652

Christopher V. Davis, Dale F. Leavitt and Joseph A. Mariano

Design and evaluation of tloating upweller systems for nursery culture of juvenile clams and oysters 652

Jonathan P. Davis and Clea R. Barenberg

The use of a Schizochyirimn based dry feed for juvenile rearing and broodstock conditioning of bivalve molluscs 652

George E. Flimlin, Jr.

Applied technological development lor hard clam {Merccnariii incnciiarid) aquaculture in New Jersey 653

A. L. Mallet and C. E. Carver

Flat oyster culture In Nova Scotia: strategies to optimize the growout operation 653

Carter R. Newell

Cultivation of native oysters and native mussels on rafts, trays and on the bottom in Maine. USA 653

Francis X. O'Ueirn and Mark W. Luckenhach

A study investigating the potential iif an alternative oyster seed source for Virginia aquacullurists 653

Carmen G. Paniagua-Chavez. John T. Buchanan, Terence R. Tiersch and John E. Supan

Advances in the cryopreser\'ation of gametes and larvae of the Eastern oyster 654

Gregg Rivara, Kim TetrauU and Michael Patricio

A low cost. Hoaling avial-llovv upweller shellfish nurserv svsteni 654

Anja Robinson

Kumamalo ovsler hroodslcick 654

National Shellfisheries Association. Seattle. Washington Abstracts. 2000 Annual Meeting. March 19-23, 2000 595

Samia Sarkis, Doerete Horsfield, Greg Wells, Charles King and Karen Smith

Growth of juvenile Calico scallop, Argopecten gibbus, in Bermuda, and its implications for aquacuiture 655

John E. Siipan, Standish K. Allen, Jr. and Charles A. Wilson

Tetraploid Eastern oysters: an arduous effort 655

WATER QUALITY AND HARMFUL BACTERIA

Haejung An, Hakan Calik, Haian He, Roger Adams and Michael Morrissey

Use of high hydrostatic pressure to control pathogens in raw oysters 655

Deborah Cannon and Kim Hatfield

Can the Tillamook County performance partnership restore the ecological balance of Tillamook Bay? 656

William F. Dewey

The various relationships between shellfish and water quality 656

Stuart D. Glasoe and Duane Fagergren

Shellfish water quality trends and threats in Puget Sound 656

G. /. Scott, M. H. Fulton, B. C. Thompson, L. F. Webster, A. K. Leight, E. F. Wirth, J. Stewart, G. P. Richards,
D. Chestnut, R. F. Van Dolah, S. Paneen

The use of multiple antibiotic resistance and molecular techniques (pulsed field gel electrophoresis and ribotyping) for

identifying coliform pollution sources 657

Russel P. Herwig, Robyn M. Estes, Cindy L. Messey and Daniel P. Cheney

Distribution of Vibrio parahaemolylicus in Puget Sound oysters, water and sediments during summer 1999 657

Charles A. Kaysner and Angelo DePaola, Jr.

Outbreaks of Vibrio parahaemolyticits gastroenteritis from raw oyster consumption: assessing the risk of consumption

and genetic methods for detection of pathogenic strains 657

T. L. King

Does community involvement lead to long-term pollution solutions? 658

Ken B. Moore

ISSC's research initiatives 658

D. L. Park, L. S. Andrews and Y-P Chen

Elimination of Vibrio contamination in raw in-shell oysters through low temperature pasteurization 658

POSTERS

Richard R. Alexander and Gregory Dietl

Frequency of shell repairs in common clams from New Jersey 658

Troy D. Alphin, Martin H. Posey, David W. Freshwater and Robert A. York

Selection and growth of Cras.sostrea virginica based on water quality 659

Ingrid Ardjosoediro, Nyanti Lee, John Supan and Terrence R. Tiersch

Gamma irradiation effects on early life stages of the Eastern oyster 659

Shirley M. Baker and Jeffrey S. Levinton

Feeding selectivity of native freshwater mussels ( Unionidae) and competition with Zebra mussels 659

Wafa Birbari, Anita Wright and Gary Rodrick

Viable but non-culturable response for phase variants of Vibrio vulnificus in clams 660

Jodi Brewster, Dave Bushek and Richard Dame

Perkinsus marinus population dynamics in North Inlet. South Carolina - an ecosystem model 660

Diane J. Brousseau and Jenny A. Baglivo

Perkinsus disease progression in field oysters: a modeling study 660

Gwynne D. Brown and Kimberly S. Reece

Identification of a serine protease gene in Perkinsus marinus 660

John T. Buchanan, Carmen G. Paniagua, Terrence R. Tiersch and Richard K. Cooper

Research-scale culture of oyster larvae 661

Emily Butsic, Richard Dame and David Bushek

The effect of oyster removal on intensities of Perkinsus marinus infections in native oyster populations 66 1

Lisa M. Ragone Calvo and Eugene M. Burreson

Development and verification of a simple model for Perkinsus marinus abundance in Chesapeake Bay oysters 66 1

596 Abstracts, 2000 Annual Meeting. March 19-23, 2000

National Shelifisheries Association, Seattle, Washington

Gregory M. Coates and John E. Supan

Potential triploid production of oysters using second metaphase oocytes 662

V. G. Encomia, S. Stickler and F. L. Chu

Energy reserves in Perkinsus mariniis infected and uninfected oysters 662

Elizabeth A. Francis, Kimberly S. Reece, Standish K. Allen and Patrick M. Gaffney

Species designation among sympatric oysters Ciassoslrea ariakensis. C. i^igas and C. sikamaea 662

Dane Frank, Lisa Ewert, Sandra Shumway, and J. Evan Ward

Effect of clay suspensions on clearance rate in three species of benthic invertebrates 663

Ray Grizzle and Richard Langan

Open ocean, submerged longline culture of the blue mussel in New England: a first-year progress report 663

Walter R. Keithly Jr. and Hamady Diop

How have the warning labels and negative publicity associated with Vibrio ridnificus impacted demand for Gulf of

Mexico produced oysters? 663

Yanli Li and Jerome F. LaPeyre

Development of a defined medium for cells of the Eastern oyster Crassostrea virginica 664

Roger Mann and Juliana M. Harding

Veined Rapa whelks {Rapana venosa) in the Chesapeake Bay: current status and preliminary reports on larval growth

and development 664

Michael P. McKee, J. Evan Ward, Lisa M. Milke and Bruce A. MacDonald

Release of mucopolysaccharides by bivalved mollusks and their contribution to the production of transparent

exopolymer particles (TEP) in near shore waters 664

Amy D. Nickens, Eric Wagner and Jerome F. La Peyre

Improved procedure to count Perkinsus marinus in Eastern oyster hemolymph 665

Katsuyuki Namaguchi

Tidal fluctuation in phytopigment concentrations and sediment load at a Manila clam. Ruditapes philippinarium,

farming ground 665

Kelly Palacios, Sylvia Yamada, Laura Hauck, Alex Kalin, Chris Hunt

1999 Oregon State University studies conducted on the European green crab, Carciniis maenas. supported by Oregon

Sea Grant 665

A. J. Paul and J. M. Paul

The reproductive cycle of captive female golden king crab. Liihodes aequispiniis 665

K. T. Paynter

Oyster restoration in Chesapeake Bay: effects of oyster density on the associated benthic community 666

S. M. C. Schreiber and W. H. Watson IH

Cardioregulatory nerves are not the source of temperature-induced heart rate modulation in the American lobster

{Homarus americanus) 666

Thomas M. Sonial, Enrique V. Kortright and Sammy M. Ray

Dermowatch: a new tool for managing Perkinsus nuirintis disease in Eastern oysters, Crassostrea virginica 666

S. M. Stickler, V. G. Encomia, F.-L. Chu and S. K. Allen. Jr.

Growth, mortality and defense against Perkinsus marinus in Eastern oysters, Crassostrea virginica 666

Derrick R. Toba and Kenneth K. Chew

Western regional aquaculturc industry situation and outlook report: a shellfish perspective 667

Donn Tracy

Application of underwater time-lapsed \ ideo technology to observe King and Tanner crab behavior in and around

commercial crab pots 667

Ami E. Wilbur, William S. Arnold and Theresa M. Bert

The genetic assessment of an "enhanced" bay scallop population: do hatchery scallops produce successful recruits'? 667

Rob Zisette, Walter T. Trail and Mansour Samadpour

Tracking fecal sources in Drayton Harbor 668

National Shellfisheries Association. Seattle. Washinaton

Abstracts. 2000 Annual Meeting. March 19-23. 2000 .^97

ENVIRONMENTAL AND

ANTHROPOGENIC INFLUENCES

ON SHELLFISH

PCB ASSIMILATION IN OYSTERS {CRASSOSTREA VIR-
GIMCA): AN IMPLICATION FOR REPRODUCTIVE IM-
PAIRMENT. Fu-Lin E. Chu,* Philippe Soudant, and Robert

C. Hale, Virginia Institute of Marine Science. School of Marine
Science. College of William and Mary. Gloucester Point. VA 23062.
Polychlorinated biphenyls (PCBs) are of concern, in part due to
their high persistence and bioaccumulation potential. This may be
particularly acute for filter feeders due to association of PCBs with
natural particulate organic matter. Two experiments were con-
ducted to examine: (1) PCB assimilation in reproductively active
oysters fed daily with 0. 1 g PCB-sorbed algal paste containing 0.
0.1 or 1.0 g PCBs (mixture of Aroclor 1242. 1254. and 1260) for
15 and 30 days: and (2) PCB assimilation and its impact on re-
production in conditioned oysters fed. prior to gametogenesis,
daily 0.7 g PCB-sorbed algal paste containing 0, 0.35 or 3.5 g
PCBs. Changes in lipid and fatty composition in oyster tissues and
gametes were also analysed. Results revealed that: ( 1 1 PCB accu-
mulation was dose and time dependent and tissue (organ) specific;
(2) Higher PCB contents were found in organs (gonad and visceral
mass) rich in reserve lipids than adductor muscle and gills, which
are dominated by structural lipids; (3) PCBs were transported to
eggs, which had PCBs contents ranged from 247 to 671 ng PCBs/g
tissue DW; (4) PCB exposure reduced slightly the weight percent-
age of certain polyunsaturated fatty acids (20:4n-3. 20:5n-3 and
22:6n-3) in eggs; (5) After exposure to 3.5 g PCBs daily for 8
weeks, structural lipids (e.g.. phospholipids) in gonad, adductor
muscle and mantle decreased, while triacylglycerol (TAG) in-
creased in digestive gland; and (6) Compared to controls. PCB
exposure resulted in fewer females and spawned females in con-
ditioned oysters. Impairments of structural lipid synthesis and the
mobilization/transport of reserve lipids, particularly TAG. are be-
lieved to be the cause of the delaying and/or inhibition of oogen-
esis in PCB-exposed oysters.

ADHERENCE AND INVASION MECHANISMS OF
VIBRIO VULNIFICUS WITH OYSTER AND FISH CUL-
TURED CELLS. Gaskov Clerge, Mahendra H. Kothary.
Marianne D. Miliotis, Darcv E. Hanes. Seynabou Fall, Jeffrey
W. Bier, Dhirendra B. Shah, and B. D. Tall.* JIFSAN, US FDA.
Washington. D.C. 20204: Broderick Eribo, Howard Univ.. Wash-
ington. D.C. 20059; Jerome F. La Peyre and Mohamed Faisal,
VIMS. Gloucester Point, VA 23062.

Vibrio vulnificus (V\) causes systemic infections in many sea-
food hosts; in humans it causes gastroenteritis, wound infections.
and septicemia. To investigate the pathogenic mechanisms in-
volved, we examined several strains by electron microscopy (EM)

for presence of adherence factors. These studies revealed 3.5 nm
fibrillar structures composed of linear strands, multiple strand
bundles or wiry aggregates radiating from the bacterial surface.
Using a KSCN/(NH4)2S04 precipitation procedure, we obtained a
crude fibrillar extract (CFE) which consisted of single filaments,
filaments in bundles, which also possessed hemagglutination (HA)
activity. CFE obtained from both biotypes of Vr hemagglutinated
sheep, chicken, bovine, human O and eel RBCs. However. CFE
obtained from biotype 1 cells only hemagglutinated human A and
B RBCs. Maximal expression of the adhesin occurred when cells
were grown for 18 h on Thiaproline-NaCl-Glutamate-Agar ad-
justed to a pH 6 or 8. and incubated at 30 °C. Cells grown at a pH
of 7.4 or at a temperature of 17° or 37 °C. or under anaerobic
conditions were HA negative. These results suggest that expres-
sion of this fibrillar adhesin is controlled by environmental signals.
To determine its role in adherence and invasion. TnplioA inutagen-
esis was carried out and transconjugants were screened for lack of
HA activity. Comparison of these mutants with the parental strain
in adherence and invasion assays with primary oyster mantle,
heart, intestinal, and hemocyte cells demonstrated that adhesin
expression is needed for adherence and invasion into primary oys-
ter cells. Studies performed with Mummichog (Fundulus helero-
clitus) primary anterior kidney and liver cells showed similar re-
sults. However, different cell affinities were noted. To further
investigate the invasion mechanisms involved, uptake assays were
performed with Atlantic menhaden liver (AMD cells. Results
from these studies suggest that uptake of Vi' occurs at a limited
number of sites. However, invasion efficiency of the afibrillated
mutants was not significantly different from that of the parental
strain suggesting the exi.stence of host receptor differences among
primary and immortalized cultured cells or that more than one
ligand may be involved in the invasion of Vv into AML cells.
Inhibitors of actin. microtubulin. and receptor-mediated endocyto-
sis showed that invasion of stationary grown Vv was dependent
only on the microtubulin pathway. However, uptake of log phase
(LP) grown Vv was dependent on both actin and microtubulin
suggesting that bacterial ligand expression differs with infective
growth stage of Vv. Studies focused on the role of signal trans-
duction in invasion of LP grown cells showed that the activation of
s-protein tyrosine kinase (sPTK) and protein kinase C (PKC) are
involved in V'\' entry into AML cells. If invasion and cytotoxicity
are sequential events, then inhibitors that block invasion should
also reduce cytotoxicity. Using lactate dehydrogenase (LDH) re-
lease, a stable cytosolic enzyme as a measure of cytotoxicity in
inhibitor experiments showed that inhibition of uptake by colchi-
cine (microtubulin inhibitor) did not prevent the release of LDH
and. hence, did not reduce cytotoxicity. In contrast, inhibition of
protein kinase activity caused a significant decrease in release of
LDH suggesting that protein kinase activity i.s involved in Vv-
mediated cytolysis. and invasion and cytotoxicity are two mutually
exclusive events. In summary, these data provide evidence indi-
cating that uptake of Vr differs among cultured cells; occurs at a

598 Abslmcts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

limited number of sites on the AML cell surface; was dependent on
expression of bacterial surface ligands and on the involvement of
host cytosi>;eletal elements and protein kinase activities. These data
also indicate that Vi' uptake and bacterial-mediated cytotoxicity of
AML cells are independent events.

STRESS PROTEIN (HSP70) RESPONSE IN OYSTERS
CRASSOSTREA VIRGINICA EXPOSED TO VARIOUS
STRESS AGENTS. Luis A. Cruz-Rodn'guez,* Fu-Lin E. Chu,
and Philippe Soudant, Virginia Institute of Marine Sciences,
School of Marine Sciences. College of William and Mary,
Gloucester Point. VA 23062.

Application of stress proteins (or heat shock proteins) as cel-
lular biomarkers of exposure to environmental pollutants have
been investigated in many aquatic organisms. This study investi-
gated the stress protein (Hsp70) response to various stress agents
(contaminated sediments. PCBs. and Cd'*) in oysters iCrassostrea
virginica). Oysters were exposed to 0. 1. 1.5. or 2 g contaminated
sediments (CS) for 5. 10. 20. and 40 days. A significant increase
in Hsp70 response was noted after 40 days exposure compared to
non-exposed oysters. Oysters exposed to 2 g CS showed the largest
increase in Hsp70. but no dose dependency in the response was
noted. Reproductively active oysters fed 0.1 g PCBs-sorbed algal
paste daily containing 0, 0.1, or 1.0 ixg PCBs for 15 and 30 days
showed a decrease in Hsp70 in oysters exposed to 1 (jig PCBs for
15 days. This reflected total soluble protein trends. No significant
difference in Hsp70 levels was observed in those exposed for 30
days, although an increasing trend was noted. Reproductive inac-
tive oysters fed 0.7 g PCBs-sorbed to algal paste daily containing
0, 0.35. or 3.5 p,g PCBs for 8 weeks with or without 0.3 g artificial
sediments added, showed no significant increases in Hsp70. How-
ever, those exposed to the additional 0.3 g artificial sediments
showed increases in the Hsp70 levels compared to those without.
The absolute value in the magnitude of the response observed is
greater in those exposed to sediments and PCBs than to sediments
alone. Oysters exposed to 0. 15. or 25 ppb Cd~* showed a signifi-
cant increase in Hsp70. but no dose dependency was noted. Gen-
erally, in oysters as in other organisms, the stress protein response
is elicited in instances where the stress agent causes protein dam-
age. Thus, stress proteins can be used in oysters as an indicator of
exposure to proteotoxic stress.

TEMPERATURE AND RANGE EXTENSION BY PERKIN-

SUS MARIiWS. Susan E. Ford,'* Roxanna Smolowitz," and
Marnita M. Chintala.'"' 'Haskin Shellfish Research Laboratory.
Rutgers University. Port Norris. NJ 08349; "Marine Biological
Laboratory. Woods Hole. MA 02543; "Atlantic Ecology Di\ ision.
U.S. EPA. Narragansott. RI 02882.

Between 1990 and 1992. Dermo disease of oysters, caused by
Perkiiisiis mariiuis. experienced a 50()-km northward range exten-
sion and is now established as far north as Massachusetts. Climate
wannini; diuini; the I98()s and earlv I99()s. combined with his-

torical introductions of infected oysters, has been hypothesized as
the cause. Surprisingly, anecdotal reports of oyster growers indi-
cated that the disease was causing few deleterious effects in the
Northeast. To document and investigate possible causes for this
assertion, we monitored numerous oyster stocks between Delaware
and Cape Cod Bays to describe disease cycles, to measure Dermo
effects on oysters, and to compare results with data from more
southern regions. We also investigated whether a low-temperature-
tolerant strain of P. marinus is now present in the Northeast. Re-
sults of this two-year study showed that P. marinus behaves in its
new range very much as it does in southern areas where it has been
enzootic for decades. Seasonal cycles are similar, as is the 2-3 year
progression to a full epizootic. Mortality during the present study
was a least as great as in the south. Temperatures in most of the
growing areas examined readily became warm enough to sustain
high P. marinus proliferation and winters were not cold enough to
limit disease cycles. Data from an in vitro growth assay of P.
marinus isolates from North Carolina to Massachusetts, suggest
responses to temperature that vary along a latitudinal cline; how-
ever, there was no consistent evidence from this assay, or from in
vivo proliferation, that a low-temperature-tolerant strain has in-
vaded the Northeast.

RESPONSES OF OYSTERS AND THEIR HEMOCYTES TO
CLINICAL AND ENVIRONMENTAL ISOLATES OF

VIBRIO PARAHAEMOLYTICUS. Fred J. Genthner* and Wil-
liam S. Fisher, US EPA. Gulf Breeze. PL 32561; Aswani K.
Volety, Florida Gulf Coast University, Fort Meyers, PL 33965;
Ben D. Tall and Sherill K. Curtis, JIPSAN, US PDA. Washing-
ton. D.C. 20204; Susan A. McCarthy, US PDA. Dauphin Island.
AL 36528.

Interactions of Vibrio parahacniolyticus with oysters and oyster
hemocytes were studied using three environmental isolates ( 1094.
1163 and ATCC 17802) and three clinical isolates (2030. 2062.
21071. Clinical isolates were from patients who became ill during
the June 1998 food poisoning outbreak involving oysters from
Galveston Bay in Texas. Environmental isolates were from oys-
ters, crabs or sardines. All V. partihcwmnlylicus isolates possessed
the thermolabile direct hemolysin (tlh) gene; only the clinical iso-
lates had the thermostable direct hemolysin (tdh) gene (a putative
virulence determinant). The capacity of oyster hemocytes to kill
each V. paraliacmolxiicus isolate was examined in vilro using a
novel dye reduction assay. Differences in killing by oyster
hemocytes existed between and among en\ ironmenlal and clinical
isolates. On average, environmental isolates were more susceptible
to hemocytc killing (ban clinical isolalcs. Clinical isolate 2062 was
more susceptible to killing by oyster hemocytes than the other two
clinical isolates (2030. 21071 and displayed the most diffuse
colons morphology on nutrient agar plates. Also, unlike the other
two isolates, it lacked identifiable Alcian Blue stabilized capsular
material that appears as irregularly distributed, spike-like, elec-
tron-dense deposits often obser\ed spanning gaps between cells.

National Shellt'isheries Association. Seattle. Washinsiton

Abstracts. 2000 Annual Meetinsz. March 19-23. 2000 599

Additional experiments showed that when oysters were exposed to
mixtures of a clinical (2030) and an environmental ( 1 163) isolate,
higher numbers of the clinical isolate were found in tissue and
hemolymph. The significance of this research is that differences in
V. parahaemolyticus isolates are described that influence ways in
which these bacterial pathogens interact with oysters.

SUMMER STRESS PROTEIN RESPONSES OF CUL-
TURED PACIFIC OYSTERS: DOES CHRONIC STRESS
REDUCE TOLERANCE? Aniro M. Hamdoun," Daniel
Cheney," Ralph Elston," Brian McDonald." and Gary N.
Cherr,' 'Bodega Marine Laboratory. University of California
Davis. Bodega Bay. CA 94923 and "Pacific Shellfish Institute.
Olympia. WA 98501.

Pacific oysters (Crassostrea gigas) cultured in South Puget
Sound routinely experience mass mortalities during the summer
months. One factor thought to be associated with 'summer mor-
tality' events is the combination of acute and chronic thermal
stress often experienced during the summer. The responses of C.
gigas to acute thermal stress have been well characterized. Briefly,
these include induction of several members of the heat shock pro-
tein (HSP) 70 family and associated 'thermotolerance' to other-
wise lethal temperatures. In contrast, relatively little is known
about stress protein responses of chronically stressed Pacific oys-
ters in culture. We tested the hypothesis that this summer stress
alters the ability of Pacific oysters to mount normal heat shock
responses. Neither constitutive nor inducible members of the HSP
70 family appear to be expressed at significantly elevated levels
during the summer months. Moreover, oysters from one culture
site did not induce HSP 69 after sublethal heat shock, for the
duration of the summer. Most oysters were able to acquire ther-
motolerance after sublethal heat shock. However, some appeared
to have already acquired some degree of thermotolerance prior to
sublethal heat shock. Additionally we found that the normal stress
protein response can be similarly inhibited by exposure to moder-
ate levels of xenobiotics such as chromium. Thus, we suggest that
oysters encountering natural and/or anthropogenic stressors in the
field may acquire tolerance to chronic stress, but may lose the
ability to mount a functional, rapid stress response to elevated
temperature.

IMMUNE RESPONSES OF TWO SPECIES OF MUSSELS
(MYTILUS CALIFORNIANVS AND MYTILUS GALLOPRO-
VINCIALIS/TROSSULUS HYBRID) TO POLLUTANTS IN
SAN FRANCISCO BAY. CA. Allison C. Luengen.* ETOX De-
partment, University of California at Santa Cruz, Santa Cruz. CA
95064; Carolyn S. Friedman. Bodega Marine Lab, Bodega Bay,
CA 94923; A. R. Flegal, ETOX Department. University of Cali-
fornia at Santa Cruz. Santa Cruz. CA 95064.

Since mussels (Mytilus californiamis) are routinely deployed in
San Francisco Bay. California to monitor concentrations of con-
taminants in their tissues, this study was initiated to determine

whether the elevated concentrations of some of those contaminants
correlated with measures of variations in their immune response.
Preliminary data from the latter measurements indicate that mus-
sels from relatively contaminated sites exhibit elevated immune
responses when compared with mussels from relatively pristine
sites. This includes the following immune parameters: ( I ) number
of hemocytes, (2) percentage of cells that phagocytosed particles,
and (3) a phagocytic index, which describes how many particles
were engulfed by phagocytic cells. Additionally. M. californianus,
which does not live naturally in the Bay. appeared to show el-
evated immune responses when compared to M. galloprovincialis/
trossuhis hybrids that are endemic to the Bay. This disparity also
indicates that M. califoniianus, which has been the species his-
torically deployed as a biomonitor in the Bay. may not be the most
appropriate species. Finally, this preliminary research has lead to
the development of a new technique to evaluate phagocytosis in
the mussel cell's hemolymph because the cells were too sensitive
to the centrifugation step and the washing steps used in established
methods.

RELATIONSHIPS BETWEEN OYSTER (CRASSOSTREA
VIRGINICA) DEFENSE MEASUREMENTS AND TISSUE
CONTAMINANTS. L. M. Oliver,*' W. S. Fisher.' A. K. Vo-
lety," and Z. Malaeb,"' 'U.S. Environmental Protection Agency.
National Health and Environmental Effects Research Laboratory.
Gulf Ecology Division. I Sabine Island Drive. Gulf Breeze, IT..
32561-5299. "College of Arts and Sciences, Florida Gulf Coast
University, 10501 FGCU Blvd.. Fort Myers. PL 33965-6565.
"^United States Geological Survey. Biological Resources Division.
National Wetlands Research Center. Gulf Breeze Project Office, 1
Sabine Island Drive. Gulf Breeze, FL 32561-5239.

Bivalve mollusks such as Crassostrea virginica typically in-
habit estuaries and coastal areas that are increasingly contaminated
with anthropogenic chemicals. Oysters may bioaccumulate large
quantities of metals, polyaromatic hydrocarbons (PAHs). and
polychlorinated biphenyls (PCBs) without evident ill effects, but
various measurements of bivalve defense activity show alterations
from experimental chemical exposures and longer-term, field ex-
posure to chemical mixtures. Forty oysters were collected from
Bayou Chico and East Bay, two sites in Pensacola Bay, FL. known
to differ in the type and magnitude of chemical contaminants.
Tissue concentrations of metals, tri- and di-butylin (TBT. DBT).
PAHs and PCBs were measured along with hemocyte number,
phagocytic (PI) and bactericidal (Kl) indices, and serum lysozyme
and total protein levels. Hemocyte PI was significantly higher in
East Bay oysters, which also had low tissue levels of PAHs. PCBs,
TBT. DBT. and the metals Al. Cr. Fe. Ag. Cd. and Hg. Average
hemocyte number, Kl, serum lysozyme and protein were signifi-
cantly higher in Bayou Chico oysters which also had high tissue
concentrations of organic contaminants, butyltins, and Mn, Cu, Zn,
and Sn. Canonical correlation analysis was used to examine rela-

600 Ahsinwts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle, Washington

tionships between tissue metals and defense measurements using
linearly combined sets of variables. The highest possible correla-
tion was positive: r = .934, between canonical variables com-
posed of hemocyte number, PI, serum protein and ly.sozyme for
defense, and Cd, Fe, Al, Pb, Zn, Mn, Sb, Ni, and Cr for metals.
This suggestion of heightened defense activities in oysters from
metal-contaminated sites is consistent with previous ob.servations.
The likelihood of complex relationships between oyster immune
measurements and contaminant stress suggests that single chemi-
cal exposures and univariate analyses may be inadequate or mis-
leading.

IN VITRO KILLING OF PERKINSUS MARINUS BY
HEMOCYTES OF OYSTERS CRASSOSTREA VIRGINICA.
Aswani K. Volety,* College of Ails and Sciences, Florida Gulf
Coast University, 10501 FGCU Blvd. Fort Myers, FL 33907; Wil-
liam S. Fisher, US Environmental Protection Agency, Gulf Ecol-
ogy Division, 1 Sabine Island Drive, Gulf Breeze, FL 32561.

A colorimetric microbicidal assay was adapted, optimized and
used in experiments to characterize the capacity of eastern oyster
(Crassostrea viri^inica) hemocytes to kill cultured isolates oi Per-
kinsii.s mariinis, a protozoan parasite causing a highly destructive
di.sease of oysters throughout U.S. Atlantic and Gulf of Mexico
coastal waters. //; vilro challenges showed that hemocytes from
two geographically distinct oyster stocks (Florida and Rhode Is-
land) were able to decrease viable P. marimis cells by 45-52%.
Variability in killing was most likely due to differences in suscep-
tibility among the seven cultured isolates, which ranged in origin
from Long Island Sound (CT) to Laguna Madre (TX). Hemocytes
from oysters collected in E.scambia Bay, FL, exhibited a relatively
consistent mean killing capacity throughout a year-long period,
averaging 57% across all months monitored with a range of 21-
90%. Application of this technique demonstrated the in vilro ca-
pacity of hemocytes to kill P. inaiimis. but does not necessarily
retlect their ability under natural conditions where the disease is
widespread.

STRESS PROTEINS AS BIOMARKERS IN ESTUARINE

SHELLFISH SPECIES. Inge Werner, School of Veterinary
Medicine. Dcpl. of .\natomy. Physiology and Cell Biology, Uni-
versity of Caliloniia al Davis, Davis, CA.

The application of stress proteins (or heat shock proteins) as
cellular biomarkers of exposure to aiul/or elfecl of environmental
polUilaiils lias liccii proposed and investigated lor a niMiiber of
years. Members ot this group of proteins arc induced by a variety
of stressors which either ilamage cellular proteins directly or cause
cells to synthesize aberrant proteins. They have been detcclcd in all
organisms investigated, from bacteria and plants to humans, and
are highly conserved across phyla, l-unctions include the stabili-
zation of unlokled protein precursors before assembly, transloca-
lion of proteins into organelles, rearrangenieiil of prolein oligo-

mers, dissolution of protein aggregates, and refolding or degrada-
tion of denatured proteins.

Numerous studies showed induction of hsp70 or hsp60 by labo-
ratory exposure to chemicals which are known toxicants, e.g.
heavy metals and several pesticides. Few studies, however, have
examined the linkage of hsp induction to contaminant induced
deleterious effect in the organism, or the hsp response to multiple
stressors, chemical and physical, encountered in field situations.
As more research is being conducted, new questions arise. Our
laboratory has been investigating the suitability of hsp70 and
hsp60 protein(s) in a variety of aquatic organisms as a field bio-
marker. Results indicate that the hsp response to stressors appears
to be well suited as a biomarker of exposure and effect in some
cases and not in others. This presentation will give insight into the
pros and cons of using stress proteins as a biomarker in field
studies, and tackle the question of whether induction of hsps in-
dicates exposure to — and protection from potentially toxic com-
pounds, or if it can predict deleterious effect in the organism.

FEEDING AND NUTRITION

EVOLUTIONARY AND FUNCTIONAL TRAJECTORIES
OF THE BIVALVE GILL ABFRONTAL SURFACE: LES-
SONS FROM CONTEMPORARY CILIA AND MUCOCYTE
DISTRIBUTIONS. Peter G. Beninger,* Laboratoire de Biologic

Marine, Faculte des Sciences, Universite de Nantes. 44322 Nantes
Cedex France; Suzanne C. Dufour, Scripps Institution of Ocean-
ography, University of Califomia, San Diego, La Jolla. CA 92093-
0202.

Recent data on the distributions of cilia and mucocytes on the
bivalve gill abfrontal surface are analyzed with respect to evolu-
tionary relationships of the principal Autobranch gill types. From
the primitive function as a mucociliary cleaning surface in the
Protobranchs. two evolutionary trajectories are evident: ( I ) pro-
gressive reduction of both cilia and mucocytes with resultant loss
of surface function, .seen in the homorhabdic filibranchs studied,
and (2) reduction of cilia but retention or increase in acid muco-
polysaccharide-secreting mucocyte density in the eulamelli-
branchs. corresponding to the assumption of a new function, prob-
ably in the reduction of Irictional resistance to How in the water
canals. The hclerorhabdic gill abfrontal surfaces present a mixture
of these characteristics, corresponding to the staggered ontological
and phylogenetic devclopmcnl ol the two filament types; reduction
of cilia and mucocytes on the ordinary filaments, retention of both
on the principal filaments. The difference between heterorhabdic
filibranchs and pseudolamellibranchs in degree of inter-lamellar
fusion may be rcllected in the functions of the retained mucocytes
on the abfrontal surface of Iheir respective principal filaments:
reduction of resistance to water How in the pseudolamellibranchs,
lubricalion for retraction of the gill during xalve clapping for the

National Shelltlsheries Association. Seattle. Washington

Abstracts. 2000 Annual Meeting. March 19-23. 2000 601

heterorhabdic filibranchs. Although the original function of the
abfrontal surface has been rendered redundant by the various de-
grees of folding and fusion of the bivalve gill, the polyvalent
potential of the mucocytes has resulted in the emergence of new
functions.

A BIOCHEMICALLY-BASED MODEL OF THE GROWTH
AND DEVELOPMENT OF PACIFIC OYSTER CRASSOS-
TREA GIGAS LARVAE. Eleanor Bochenek,* N.J. Sea Grant
College Program. Sandy Hook Field Station. Fort Hancock. NJ
07732: Eric Powell, Haskin Shellfish Lab.. Rutgers Univ.. Port
Norris. NJ 08349: John Klinck and Eileen Hofmann, CCPO. Old
Dominion Univ.. Norfolk. VA 23529.

A biochemically-based model was used to simulate the growth
and metamorphosis of Crassostrea gigas larvae. This model,
which is the first of its type, includes parameterizations of the
metabolic costs associated with larval filtration, ingestion, and
respiration. The initial biochemical content of the larva is deter-
mined by the composition of the egg. Changes in the initial ratios
of protein, carbohydrate, neutral lipid and polar lipid occur as the
larva grows in response to environmental conditions. The model
obtains realistic larval life spans and success rates at metamorpho-
sis under a range of environmental conditions based on a metabolic
trigger of metamorphosis defined as a reduction in the ratio of
storage products to structural components modulated by a reduc-
tion in filtration rate. Simulations show that larger eggs produce
larvae that are more able to withstand poor food environments over
the larva's life, suggesting that egg size is one variable accounting
for the range of larval sizes at which metamorphosis is attempted
and the success rate for metamorphosis. However, eggs can be too
large. Optimal size, around 50 |jLm. yields greatest metamorphosis
success. Other simulations show that food supply and environmen-
tal conditions also control the size range and success rate for
metamorphosis by influencing the ratio of storage products (imple-
mented as neutral lipid) to structural products (chiefly protein and
polar lipid). For example, temperatures above 20 °C (and £30 °C)
and salinities a20%r (and <309tc) result in a large fraction of
larvae successfully surviving metamorphosis. High temperature
can spare low food supply up to a point because filtration rate
increases with increasing temperature: however high food supply
cannot spare low temperature.

USING CHLORELLA TO STUDY POSTINGESTIVE SE-
LECTION IN BIVALVES. Martha G. S. Brillant* and Bruce
A. MacDonald, Biology Department and Centre for Coastal Stud-
ies and Aquaculture. University of New Brunswick. Saint John.
NB. Canada E2L 4L5.

Postingestive selection is known to occur in several species of
bivalves, however the factors responsible for selection have not
been established. We have shown that size and density of particles

play a role in postingestive selection in the sea scallop (Pla-
copecten mageUunicus). Determining the role of particle chemistry
or quality is more challenging. Particles chosen to study postinges-
tive selection by chemical properties should appear physically
identical but chemically distinct to the bivalve and must be trace-
able and quantifiable after passage through the bivalve. Also, the
integrity of these particles within the bivalve stomach should be
similar. We have addressed these factors by using Clilorella
(CCMP 1227) to study postingestive selection. Clilorella has a
thick cell wall and therefore can be killed by heat and remain
intact. After heat treatment Clilorella has significantly lower car-
bon, nitrogen and photosynthetic pigments than fresh Clilorella.
Scallops will be fed heat-killed algae and live algae simultaneously
with one treatment labeled with ''^C. The feces will be collected
and analyzed on a scintillation counter. Gut retention times of the
two treatments will be compared to determine if postingestive
selection has occurred. This method should provide a means of
determining whether scallops can distinguish particles within the
stomach on the basis of chemical properties alone.

NATURAL DIET EFFECTS ON FOOD UTILIZATION BY
SEA SCALLOPS AND BLUE MUSSELS. Peter J. Cranford,*
Shelley L. Armsworthy. Michael J. White, and Timothy G.
Milligan, Fisheries and Oceans Canada. Bedford Institute of
Oceanography. P.O. Box 1006, Dartmouth. NS, B2Y 4A2.

The widespread expansion of bivalve culture operations in es-
tuarine and coastal systems is increasing the potential for bivalve
filter feeders to affect regional trophic structure. It is therefore
essential that bivalve food utilization be more fully comprehended
to determine effects on coastal ecosystems and the sustainability of
existing and expanding culture operations. Until recently, much of
the information on bivalve functional responses to diet variability
has been obtained using artificial diets (e.g. cultured algae) so that
feeding conditions could be strictly controlled. However, recent
studies on the responsiveness of feeding behaviour to natural diets
have demonstrated the importance of conducting this work under
more environmentally realistic conditions. In our paper, we will
review recent studies we conducted on the effects of natural diets
on food acquisition by Placopecten magellanicus and Mytilus edu-
lis and the consequences to growth. The focus was on seeking
generality on bioenergetic responses at sites characterized by low
seston loads (<5 mg L"'). Measured responses to ambient food
supplies were used to construct hypothesis on: the effect of the
different time-scales of variation in food supplies (hourly to inter-
annually) on food acquisifion processes: the relative importance of
exogenous and endogenous forcing: the effect of particle floccu-
lation on bivalve trophic resources and feeding behaviour: and the
food utilization strategies of different bivalves.

602

Ahslracts. 2000 Annual Meetinc. March 19-23, 2000

National Shellfisheries Association, Seattle. Washington

ENHANCED PRODUCTION OF PACIFIC DULSE (PAL-
MARIA MOLLIS) FOR CO-CULTURE WITH RED ABA-
LONE (HALIOTIS RUFESCENS) IN A LAND-BASED SYS-
TEM. C. L. Demetropoulos* and C. J. Langdon, Hattleld Ma-
rine Science Center. Oregon State University. Newport. OR
97365.

Pacific dulse (Pabnaria mollis) has shown itself to be a valu-
able algal feed for red abalone {Haliolis ntfescens). Land-based
tumble culture techniques capable of producing commercial quan-
tities of P. mollis are still in development. An understanding of
specific nutrient requirements and the importance of the relative
velocities of algal rosettes to their culture medium are essential to
increasing yields of P. mollis.

Under high photon flux densities, additions of a combination of
nitrate and ammonia, as sources of nitrogen, to dulse cultures
resulted in no significant difference in growth compared with ad-
ditions of nitrate alone. A nitrogen to phosphorus (N:P) ratio of 1 1
provided the most economical u.se of phosphorus. Addition of trace
metals, Fe, Mn, and Zn significantly increased dulse growth. Cul-
tures supplied with a combination of both CO, and NaHCO, pro-
duced higher yields compared with those supplied with either in-
organic carbon sources alone. Yields of Pabnaria mollis were
positively related to the relative velocity of rosettes to the culture
medium, up to a relative velocity of 15 cm/sec.

NUTRITIONAL STATUS OF FOUR ALGAL DIETS FOR
THE CAPTIVE CARE OF FRESHWATER MUSSELS.
Catherine M. Gatenby* and Daniel A. Kreeger. Patrick Center
for Environmental Research. Academy of Natural Sciences, Phila-
delphia. PA 19103: Vanessa A. Jones, and David M. Orcutt,
Department of Plant Pathology and Weed Science, Virginia Tech.
Blacksburg, VA 24061 ; Bruce C. Parlter. Department of Biology.
Virginia Tech, Blacksburg, VA 24061; Richard J. Neves, Vir-
ginia Cooperative Fish and Wildlife Research Unit. Department of
Fisheries and Wildlife Sciences. Virginia Tech. Blacksburg. VA
24061.

The success of conservation efforts to restore dwindling fresh-
water mussel populations through culture and propagation requires
an understanding of their nutritional requirements. We quantified
key nutritional components of several freshwater algae to identify
suitable diets for the care of unionid mussels in captivity. Total
carbohydrate, protein, and lipid contents ("/r dry w/w) were com-
pared ainong three green algae. Neochloiis oU'ouhiiudans. Biac-
Icacocciis i;rtiii(lis. Sceiwdesmus spp. and the diatom Pluwodaclv-
liim iriconniiiim. at different phases of growth: log, late log. sta-
tionary, and late stationary phase. We found no difference in
protein content among species or growth phases: ho\ve\er. carbo-
hydrate content was significantly greater in slationars phase green
algae with Sccnalcsimis containing the most carbohydrate (.^O'rl.

The greatest amount of lipid was found in log phase growth for all
algae. Since the character of lipids is known to be of nutritional
importance for bivalve molluscs, we also quantified and identified
the fatty acid and sterol composition of these algae. On average. N.
oleoabundans contained more fatty acids per mg of lipid (900
(jLg/mg lipid), and B. grandis contained more sterol per mg lipid
(47 |j.g/mg lipid). The composition of fatty acids was similar
among algae, with the exception that P. tricomutum contained
greater amounts of CI 6:8, C20:0, C20:l, C20:3 and C22:5. and
smaller amounts of CI 6:6. A greater percentage of unsaturated
fatty acids was found at log phase than at stationary phase in all
algae. The relative food value of these algae for supporting cul-
tures of freshwater mussels will be discussed by comparing these
data to measured rates of algae uptake and assimilation by the
animals.

CHANGES IN THE FLUIDITY AND FATTY ACID COM-
POSITION OF CELL MEMBRANES FROM THE SEA
SCALLOP iPLACOPECTEN MAGELLANICVS) DURING
SHORT-TERM COLD ACCLIMATION. J. M. Hall, R.J.
Thompson, and C. C. Parrish, Ocean Sciences Centre. Memorial
University of Newfoundland. St. John's. Newfoundland AlC 5S7.
Canada.

Biological membranes are highly susceptible to the increases in
membrane order and reduced membrane fluidity which result from
a decrease in temperature. Ectotherms counteract these ordering
effects of reduced temperature by adjusting the structural compo-
sition of the membrane, allowing thermal compensation of mem-
brane function over wide ranges of environmental temperature, a
process known as homeoviscous adaptation. The fatty acid com-
position of structural lipids is one of the most important factors
controlling the physical state of biological membranes. In order to
further our understanding of the role of PUFAs in cold ocean
invertebrates, we incorporated a stearic acid electron spin label
into the membranes of hemocytes and gill cells of sea scallops
(Placopeclen mai;ellaiiicus) acclimated to 15C. The temperature in
the scallop holding tanks was reduced to 5C over a 3 week period,
during which cells were sampled at intervals. Membrane order
(fluidity) was measured at 20C by electron spin resonance spec-
troscopy and the fatty acid composition of membrane phospholip-
ids determined by gas chromatography. Phospholipid vesicles of
cold acclimated scallops were more disordered (i.e. more fluid)
than tho.se of warm acclimated ones, and contained proportionately
more PUFAs. The order parameter of the spin resonance signal
was highly correlated (r = -0.71, P < 0.001 ) with the proportion
ol 20:5n-3. a PUFA which is generally believed to be important
metabolically, yet no correlation was observed with 22:6n-3, a
PUFA usually considered to ha\e more of a structural function.
The modulation of membrane phospholipid structure by 20:5n-3
(eicosapentaenoic acid. EPA) may be an important mechanism for
thermal resjulalion of tunclion in marine bi\alves.

National Shellfisheries Association. Seattle. Washinston

Abstracts. 2000 Annual Meeting. March 19-23. 2000 603

NATURAL SOURCES OF NUTRITION FOR THE MUSSEL
GEVKE^SIA DEMISSA. Daniel A. Kreeger,* Patrick Center
for Environmental Research, Academy of Natural Sciences, Phila-
delphia. PA 19103; Roger I. E. Newell and Shou-Chung Huang,

Horn Point Laboratory. University of Maryland. Cambridge. MD
21613.

Ribbed mussels [Geukensia demissa) are abundant in the in-
tertidal zone of most eastern USA salt marshes where organic
carbon inputs are apparently dominated by refractory detritus from
angiosperms. such as Spartina alteniiflora. To examine how
ribbed mussels achieve such a high biomass where labile food
resources may be limited, we integrated seasonal measurements of
the availability of different constituents of natural seston with de-
tailed physiological measurements of the mussel's ability to digest
each component. Our analysis indicates that mussels are omni-
vores since no single food type can balance either their carbon or
nitrogen demands on an annual basis. The major sources of carbon
for G. demissa appear to be a mixture of phytoplankton and mi-
croheterotrophs (bacteria and bacterivorous flagellates), followed
by smaller contributions from detrital cellulose and microphyto-
benthic diatoms and cyanobacteria. Phytoplankton are estimated to
supply the bulk of the nitrogen demands of G. demissa. followed
by a mixture of microheterotrophs and microphytobenthos. The
relative ingestion, digestion and assimilation of these different
food particles varies seasonally in proportion to their natural abun-
dances, indicating that mussels feed optimally throughout the year.
Importantly, the total bioavailable carbon from all of these foods
exceeds the annual carbon demands of mussels; whereas, the ni-
trogen demands of G. demissa are not estimated to be met at any
time of the year. This suggests that G. demissa may be nitrogen
limited, and if so. nitrogen-rich foods are of paramount importance
in the natural diet.

RIBOFLAVIN SUPPLEMENTS FOR LARVAL AND
ADULT PACIFIC OYSTERS iCRASSOSTREA GIGAS) DE-
LIVERED BY LIPID SPRAY BEADS. C. J. Langdon,* Hat

field Marine Science Center. Oregon State Uni\'ersity. Newport.
OR 97365. U.S.A.. C. Seguineau, B. Ponce. J. Moal, and J. F.
Saniain, IFREMER. Laboratoire de Physiologic des Invertebres,
BP70. 29280 Plouzane. France.

Lipid spray beads (SB) were prepared containing \i7cw/w par-
ticulate riboflavin. Beads agitated with seawater at 20-22 °C re-
tained 27% riboflavin after 24 h of suspension. Oyster larvae were
fed on riboflavin-SB and observed using an epifluorescent micro-
scope. Riboflavin was released from ingested beads, causing the
stomach contents of larvae to fluoresce green. Riboflavin concen-
trations in tissues of adult oysters fed on riboflavin-SB were sig-
nificantly greater (SNK; p < 0.05) than those of oysters fed on
seawater-filled SB. Significantly elevated (Students t-test; p <

0.05). sustained concentrations of riboflavin ( 1 1.2 (j.g/gDW) were
observed in gonad tissue samples from broodstock supplemented
with riboflavin-SB compared with those from non-supplemented
controls (4.1 p,g/gDW). There were no significant differences be-
tween riboflavin concentrations of eggs released from riboflavin-
supplemented broodstock (21.7 (xg/gDW) and those of controls
(23.2 p.g/gDW); however, egg hatching rates (73% versus 23%)
and subsequent larval survival rates (90% versus 75% on day 7,
and 82% versus 63%r on day 23) were significantly higher (Stu-
dents t-test, p < 0.05) for riboflavin-supplemented broodstock than
for control broodstock.

PALLIAL CAVITY RESIDENCE TIME IN TWO SPECIES
OF BIVALVED MOLLUSCS: MYTILUS EDULIS AND
CRASSOSTREA VIRGINICA. Lisa M. Milke* and J. Evan
Ward, Department of Marine Science. University of Connecticut.
Groton. CT 06340.

Populations of bivalved molluscs can play a large ecological
role by linking benthic and pelagic systems. Previous studies have
shown that bivalves can compensate for changing food conditions
through processes such as preferential selection and ingestion of
particulate matter. Although the gross response of bivalves to
changes in the quantity and quality of food have been examined,
the underlying mechanisms responsible for these compensations
are largely unknown. To address this issue, the fine scale feeding
processes at the level of the ctenidia and labial palps were exam-
ined under conditions of differing particle quality. Pallial cavity
residence time, or amount of time it takes a tracer particle to travel
from the inhalent aperture to the stomach, was determined for M.
edulis and C. virginica. and residence times on the labial palps
were calculated.

Bivalves were offered one of three food types; Rhodomonas
lens cells, particles prepared from ground Spartina sp. detritus, or
a 50/50 mixture of both. Once actively feeding, bivalves were
delivered 10 |jLm fluorescent polystyrene beads as a tracer. Bi-
valves were then removed at intervals from 30s up to 20 min and
placed in liquid nitrogen, ensuring the cessation of particle trans-
port. Digestive systems were isolated and examined for the pres-
ence of tracer beads, and pallial cavity residence times calculated.
For mussels, it appears that food quality has little affect on pallial
cavity residence time, as the residence time was s90s regardless
of particle type. In oysters, tracer beads were initially detected at
30s when exposed to R. lens but not until 90s when feeding on the
50/50 mix. Pallial cavity residence time in oysters, when feeding
on R. lens and the 50/50 mix, was twice as long as in mussels,
perhaps due to extensive particle processing on the ctenidia. Fur-
thermore, oysters given Spartina sp. particles were still lacking the
presence of beads in their gut even after 20 minutes of feeding.
These results suggest that food quality and degree of particle pro-
cessing on the ctenidia and labial palps may affect feeding rate.

604 Abstracts. 2000 Annual Meeting. March 19-23, 2000

National Shellfisheries Association. Seattle. Washington

PARTICLE FLUX AND CONSUMPTION BY MUSSELS AT
ROQUE ISLAND, MAINE: THE IMPORTANCE OF MA-
RINE SNOW. Carter R. Newell, Great Eastern Mussel Farms.
Tenants Harbor. ME 04860; Cynthia Pilskaln, School of Marine
Sciences. University of Maine, Orono, ME 04469; Shawn Rob-
inson, St. Andrews Biological Station, Department of Fisheries
and Oceans. St. Andrews. New Brunswick. Canada EOG2XO;
Bruce MacDonald, Department of Biology, University of New
Brunswick at St. John. St. John. New Brunswick. Canada E2L4L5.
During three days in July of 1998. the flux and consumption of
seston by mussels. Mytihis editlis, was studied at a low-current
mussel bottom lease in Englishman's Bay. Maine. The experimen-
tal approach involved feeding studies in flow-through chambers,
benthic video of mussels on the bottom, periodic water grab
samples, nightly profiles of marine snow concentration, current
measurements and sediment traps deployed over 24 hours to col-
lect the settling tlux of particles. Mussels feeding on surface water
produced no pseudofeces. in contrast to the bottom mussels where
the rates were positively correlated with exhalant siphon area of
the mussels. The volume of marine snow increased with water
depth, resulting in a pulse of material, largely inorganic, to the
mussels on bottom on the ebb tide. The food supply of mussels due
to settling and vertical mixing, with respect to particulate carbon
and nitrogen was similar, but with respect to inorganic sediments
was nearly twice as high for the settling flux than for vertical
mixing. Therefore, marine snow, at least in mid-summer during
our study, had a negative effect on inussel growth in bottom cul-
ture.

FOOD QUALITY AND FEEDING STRATEGIES IN
HATCHERY REARINCJ OF PACIFIC OYSTER CRASSOS-
TREA GIGAS LARVAE; A MODELING APPROACH. Eric
Powell,* Haskin Shellfish Lab.. Rutgers Univ.. Port Norris. NJ
08.149; John Kllntk and Eileen Hofmann, CCPO. Old Dominion
Univ., Norfolk, VA 23529; Eleanor Bothenek, N.J. Sea Grant
College Program, Sandy Hook Field Station. Fort Hancock. NJ
07732.

A biochemically-based model was used to simulate the growth
and metamorphosis of hatchery-reared Cnissostrea gigas larvae.
The model includes parameteri/.ations of the metabolic costs as-
sociated with larval filtration, ingestion, and respiration. The initial
biochemical content of the larva is determined by egg composition.
Changes in the ratios ol protein, carbohydrate, neutral lipid and
polar lipid occur as the larva grows in response to environmciilal
conditions. Model simulations show increased larval survival
when low-protein diets are provided. High-protein diets do not
pro\iile ihc lipid resources necessary to rnainlain the optimal pro-

tein to polar lipid ratio in the larva's structural components while
still providing sufficient energy for metamorphosis. Thus, food
quality is an important factor controlling the ability of C. gigas
larvae to survive and metamorphose. Additional simulations show
that small (a few hours) variations in daily food supply, such as
daily or twice-daily feeding, cause large changes in survival rate.
This may provide one explanation for widely varying survival
rates under hatchery conditions. Simulations also show that larger
eggs with greater lipid content produce larvae that are more able to
withstand poor food environments over the larva's life, suggesting
that factors associated with brood stock conditioning, as they in-
fluence egg quality, may significantly influence hatchery survival
of spawn. However, eggs can be too large. Optimal size, around 50
|jim. yields greatest metamorphosis success. Many of these obser-
vations support longstanding practices in successful oyster hatch-
eries. The model provides one way to examine optimization
schemes in hatcheries without detailed and expensive experimen-
tation.

THE NUTRITIONAL VALUE OF PALMARIA MOLLIS
CULTURED UNDER DIFFERENT LIGHT INTENSITIES
AND WATER EXCHANGE RATES FOR JUVENILE RED
ABALONE HALIOTIS RUFESCENS. Gunther Rosen, Chris
J. Langdon, and Ford Evans,* Hatfield Marine Science Center.
Oregon State University. Newport. OR 97365.

The co-culture of red abalone {Huliotis nifesceiis) and the red
macroalgae. dulse (Palnuiria iikiIHs}. has been shown to be an
effective method of abalone production. In this study we examined
the effect of dulse culture conditions on it's nutritional quality for
juvenile red abalone. Culture conditions differed by seawater vol-
ume exchange rate (I. 6. or 35 d ' ) and presence or absence of
supplemental illumination (0 or 24 h d"'). creating a variety of
dulse types that differed in morphology and biochemical compo-
sition. The results showed dulse to be of high nutritional quality for
abalone. Specific growth rates (SGR) of abalone fed on all dulse
diets were higher than those of abalone fed on kelp (Nereocystis
litclkeana). the macroalgal diet coinmonly used for abalone culture
in the United States. Protein content of dulse (10.85 to 18.227r dry
wt.) generally increased with increasing seawater volume ex-
change rale. Abalone growth rate, however, was primarily affected
by light supplementation (ANOVA; P < 0.01 ) and not water vol-
ume exchange rate (ANOVA; P > 0.05). Therefore, other variables
ap:ut Irom protein content, such as the abundance of epiphytic
diatoms and morphological differences of thalli. may have eon-
irihulcd lo higher growth rales of abalone fed on lighl-
Mipplcnicnlod diets.

National Shellfisheries Association. Seattle. Washington

Ahstmcrs. 2000 Annual Meetin". March 19-23. 2000 605

GIGAS, NUTRITION AND GAMETOGENESIS: PRESEN-
TATION AND FIRST RESULTS OF THE EUROPEAN
PROJECT GIGANUGA. J. F. Samain,* C. Quere, J. R. Le
Coz, C. Seguineau, P. Soudant, and J. Moal, Laboratoire de
physiologic des invertebres. IFREMER centre de Brest. BP 70.
29280 Plouzane. France: P. Sorgeloos, M. Caers, and C. Van
R>'ckeghem, Laboratory of Aquaculture & Artemia reference cen-
ter. University of Gent. Rozier44. 9000 Gent. Belgium: O. Garcia
and J. Espinosa, Department of Biochemistry and Molecular Bi-
ology, University of Santiago de Compostela. 15706 Santiago de
Compostela. Espafia: Y. Marty, UMR/CNRS 6521. Universite de
Bretagne Occidentale. BP 809. 29285 Brest. France: M. Mathieu
and C. Berthelin, Laboratoire de Biologic et de Biotechnologies
Marines. Universite de Caen, IBB A. 14032 Caen. France.

This European project aims at the improvement of broodstock
management in oyster hatcheries through a better understanding of
the relation between broodstock nutrition and quality of early life
stages, with the following specific objectives; 1 — Document prob-
lems in current hatchery practice by comparison of nutritional
aspects of reproduction under natural and artificial conditioning.
2 — Identify critical nutrients for broodstock nutrition and define
artificial diets to supplement these nutrients to live algae, taking
into account the initial nutrient storage of broodstock. 3 — Improve
cost-efficiency of broodstock conditioning.

Five aspects of the very first results will be reported: 1/ —
Keypoints of the cellular aspects during the reproductive cycle of
C. gigas in nature and in hatchery. 2/ — What is the biochemistry of
a normal reproductive cycle today? Biochemical aspects of the
reproductive cycle in nature and in hatchery: glycogen, essential
fatty acid, sterol or vitamin (somatic, germinal and egg compart-
ments). 3/ — Supplementation methodologies, efficiency in lipo-
soluble or hydrosoluble molecule transfer of the different artificial
particles tested. 4/ — Effect of artificial supplementations during
conditioning of C. gigas fed a standard algal mixture and a low
cost algal diet (spring experiments, histology, biochemistry and
reproduction performances) progress and questions. 5/ —
Reproduction at fall, a combination of physical and nutritional
factors perspectives for a better reproduction process in hatcheries.

LIPIDS REQUIREMENTS IN SOME ECONOMICALLY
IMPORTANT MARINE BIVALVES. Philippe Soudant* and
Fu-Lin E. Chu, Virginia Institute of Marine Science. College of
William and Mary, Gloucester Point. VA 23062, USA: Jean-
Francois Samain, DRV/A. Laboratoire de physiologic des mol-
lusques, IFREMER centre de Brest, BP 70, 29280 Plouzane,
France.

Lipids play an important role in bivalves' reproduction, devel-
opment and growth. Phytoplankton. the primary food source for
bivalves, provides essential sterols and polyunsaturated fatty acids
(PUFAs). such as 22:6n-3 (DHA). 20:5n-3 (EPA), and 20:4n-6
(AA). although the amount of these components vary with species

and seasons. Dietary DHA. EPA. AA and sterols are critical for
most bivalves' growth and reproduction. Generally, the ability to
synthesize the above PUFAs and sterols in bivalves are limited.
PUFA and sterol contents in wild oysters (e.g.. Crassostrea vir-
ginicci and C. gigas) and scallops (Pecten maximus) have been
found to be associated with their diets and reproductive cycle.
Sterol and PUFA composition of microalgae used in a hatchery-
nurseries significantly influenced the fatty acid and sterol compo-
sition of the reared larvae, spat and broodstock of C. virginica, C.
gigas. and P. maximus. Results of studies focused on the phos-
pholipid fatty acid and sterol compositions in P. ma.ximus, C.
virginica. and C. gigas revealed a selective incorporation of PU-
FAs and cholesterol in structural lipids. DHA is an essential struc-
tural component, assimilated specifically in some phospholipid
classes. Its dietary deficiency is related to gametogenic, embryo-
genie, and metamorphosis impairments in P. maximus. EPA is
considered to be an energetic rather than structural component
during embryogenesis and larval growth in P. maximus. Like most
organisms. AA in scallops and oysters preferentially resides in
phosphatidylinositol and is believed to be involved in cellular sig-
naling. However, the precise functions of specific PUFAs and their
con.servation in phospholipid classes remains to be elucidated. Al-
though the qualitative lipid requirements in most economically
important species are generally known, their quantitative require-
ments are unclear. The nutritional values of various cultured algal
species, for several economically important bivalves, will be re-
viewed and discussed according to their lipid composition.

ALGAL UPTAKE RATE OF FRESHWATER MUSSELS.
Kevin R. Stuart* and Arnold G. Eversole, Department of Aqua-
culture, Fisheries and Wildlife. Clemson University, Clemson, SC
29634: David E. Brune, Department of Agriculture and Biologi-
cal Engineering, Clemson University, Clemson, SC 29634.

Algal rich water was provided at nine flow rates in six tem-
perature ranges to Elliprio complanata. Algal uptake rates were
measured using particulate organic C/kg of wet mussel tissue/hr at
4 hr intervals from 0700 to 1900 over a 72-hr period. The water
contained 12 algal taxa dominated by Scenedesmus (82%), Mer-
ismopedia (1%). and Ankistrodesmus (3%). Mussel uptake rates
increased initially after being placed in the filtering chambers be-
fore stabilizing at a rate specific for each flow rate. These mean (±
SD) uptake rates were 35.5 ± 3.54. 42.2 ±7.16, 67.9 ± 7.34. 141.8
± 16.2. 183.0 ± 13.19, 254.6 ± 46.81, 284.6 ± 27.33. 309.4 ±
13.99. and 31 1.2 ± 8.92 mg C/kg/hr at 0.07. 0.1, 0.2, 0.6, I.O. 1.5.
2.0, 2.5, and 3.0 L/min of water at 27.4 + 0.5 °C, respectively.
Uptake rates increased as water temperatures increased from <I0°
and 10-15 °C to 15-25 °C reaching a maximum uptake rate at
25-30 °C. Uptake rates at water temperatures >30 'C were reduced
to levels lower than that observed at 15-20 °C. Uptake rates in-
creased as all concentrations (mgC/L) increased until reaching an
asymptotic level distinct for each water temperature range. E. com-

606 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washineton

platmta filtered approximately 4.8% to 24.5% of their tissue wet
weight in wet weight of algae daily. Comparison with other mus-
sels species indicate species specific uptake rates.

CHANGES IN THE FATTY ACID COMPOSITION OF THE
FLAGELLATE PAVLOVA PINGUIS (CCMP459) DURING
CULTURE. S. C. Feindel, R. J. Thompson, and C. C. Parrish,

Ocean Sciences Centre. Memorial University of Newfoundland.
St. John's. Newfoundland AlC 5S7. Canada.

Our previous work, reported at the 1999 meeting of NSA.
showed that larvae of the sea scallop Placopecten inagellanicus
grew more rapidly on a diet of Pavlova sp. (CCMP459) than on
other unialgal diets examined. In addition to possessing a balanced
distribution of n-3 fatty acids, CCMP459 was unusually rich in n-6
PUFAs, especially 20:4n-6 and 22:5n-6. The observed effective-
ness of CCMP459 in supporting growth of the larvae of a cold
water bivalve may be associated with the generally high incidence
of PUFAs in the food chain in cold oceans and their importance in
maintaining membrane tluidity at low temperatures (unpublished
observations). We have now extended this work by examining the
lipid content and fatty acid composition of CCMP459 in more
detail at various phases of culture. A cell in stationary phase con-
tained twice as much lipid as one in exponential phase, a property
common to many algal species in culture. A broader array of fatty
acids was observed in CCMP459 than is seen in most food species
used in bivalve hatcheries, and although significant levels of 22:
5n-6 have been recorded in other strains of Pavlova, the richness
of CCMP4S9 in 20:4n-6 appears to be unusual. As the culture aged
and became more nutrient and light limited, levels of n-6 PUFAs
increased at the expense of 20:.'in-3 and. to a lesser degree. 22:6n-
3. Saturated FAs were not substantially elevated during the sta-
tionary phase, and PUFA content was high during all growth
phases, which is inconsistent with many published studies on a
variety of algal cultures. Further work is required to determine the
role of 22:5n-6 in the biology and culture of the sea scallop and
other cold water bivalves.

INFLUENCE OF DIET QUALITY ON PRE-INGESTIVE
FEEDING STRATEGIES OF BIVALVES: CONNECTING
PALLIAL CAVITY FUNCTION TO ECOSYSTEM PRO-
CESSES. ,1. Evan Ward,* Dcparlnicnl of Marine Sciences. Uni-
versity of Connecticut, Grolon. CT 06340; .leffrcy S. Levinton,
Department of Ecology & Evolution. S.U.N.Y.. Stony Brook. NY
11794; Sandra E. Shunnvay, Natural Science Division.
Southampton College. Soulhanipton. NY 1 1968; Terri L. Cucci,
Bigelow Laboratory for Ocean Sciences. Boothbay Harbor. ME
04.'i7.S.

The compensatory responses of bi\al\ed molluscs lo changing
did quanlity and quality have been examined lor a number ol
species. Previous studies suggest that the ability of bivalves lo

adjust ingestion rates and reject non-nutritive particles as pseud-
ofeces. for example, is critical to their survival. The mechanisms
that underlie these observed feeding compensations, however,
have not been well studied. We hypothesize that fine-scale adjust-
ments at the level of the gill and labial palps are what ultimately
define the integrated response of the individual.

To examine some of these fine-scale adjustments, we exposed
oysters (Crassostrea virgiiiica and C. gigas) and mussels {M. edii-
lis. M. trossulus) to a mixture of ground, aged Spartina sp. (3-10
p.m) and similar sized phytoplankton (Rhodomonas sp.) at three
concentrations ( 10^. 10"*. 10"' particles mP' ). We then observed the
gills and labial palps of bivalves by means of video endoscopy,
examining aspects of particle handling and transport, and collect-
ing discrete samples from pallial organs. We also performed a
number of particle depletion and residence time experiments to
measure time course of particle handling by gills and labial palps.
Our results indicate that changes in diet quality do affect pallial
organ processes, including the route (dorsal vs. ventral tracts) and
rate at which particles are transported to the labial palps, the resi-
dence time of particles on the gills and labial palps, and particle
rejection. Gut fullness mediates pallial cavity processes, and the
magnitude and type of response elicited differs between oysters
and mussels. Our data suggest that fine-scale adjustments in pallial
cavity processes are the underlying mechanisms of previously ob-
served compensatory responses to changing diet qualities. Studies
such as these will lead to a better understanding of pallial organ
function, and allow us to better model the critical limiting factors
that mediate particle-feeding in bivalves and ultimately affect the
trophic dynamics of benthic ecosystems.

AQUACULTURAL FEEDING STANDARDS FOR MOL-
LUSCAN SHELLFISH SEED: A FIRST CUT. Gary H. Wik-

fors,* Milford Laboratory. Northeast Fisheries Science Center,
NOAA Fisheries. Milford. CT 06460.

Domestication of animals for human food requires an under-
standing of the nutritional needs of the animals. Nutritional needs
include aspects that are both qualitative (what) and quantitative
(how much and how often). Decades of research in animal agri-
culture have led to the establishment of feeding standards for com-
mon livestock (cattle, poultry, swine, sheep) that list daily energy
and biochemical inputs necessary for desired growth (or other
performance characteristics), based upon the size and maturity of
an individual ammal. In the US. these feeding standards are pub-
lished, and updated periodically, by the National Research Council
(NRC) and arc used ihroughiiut the agriculture industry in formu-
lating commercial feetls and supplements. Domestication o\ mol-
luscan shellfish can benelit from application of the feeding stan-

National Shellfisheries Association. Seattle, Washineton

Abstracts. 2000 Annual Meetin". March 19-23. 2000 607

dards concept, particularly for younger stages that are reared in
land-based systems wherein control over nutritional input is pos-
sible.

Research in the Milford Laboratory has focused upon the
"what," "how much," and "how often" of molluscan nutritional
needs from the perspective of controlled aquaculture, rather than
descriptive ecology. Experiments comparing many algal strains as
feeds for juveniles of both eastern oysters, Crassostrea virginica,
and bay scallops. Argopecten irradians, have identified several.
high-lipid strains in the algal genus Tetraselmis as being the most-
nearly complete nutritionally, on a qualitative basis. Subsequent
experiments, employing a computer-controlled feeding apparatus,
sought to optimize quantitative aspects of the delivery of Tetra-
selmis diets to both oysters and scallops. Data from these experi-
ments, and from biochemical analyses of the Tetraselmis strains,
have been used to calculate provisional feeding standards for ju-
venile oysters and bay scallops in a format similar to that of the
NRC agricultural feeding standards. These provisional feeding
standards for juvenile oysters and scallops will be presented and
compared, where possible, with those developed in agriculture,
and needs for subsequent research will be identified. Development
of practical feeding standards will benefit molluscan aquaculture
by providing a framework within which both live and prepared
dietary components may be incorporated.

FORUM: BIOLOGICAL AND

TECHNOLOGICAL TRANSFERS

IN SHELLFISH

BIOLOGICAL AND TECHNOLOGICAL TRANSFERS IN
SHELLFISH AQUACULTURE. Joth Davis, Baywater Inc.
15425 Smoland Lane. Bainbridge Island. WA 981 10, USA.

Technology transfer efforts from research to industry in aqua-
culture have followed a similar pathway as in other agricultural
industries in that the route often involves the need to protect in-
tellectual property. The result is that the dissemination of infor-
mation and the timely utilization of technological innovations may
become constrained and less readily adapted by industry. Transfers
of technology may involve a small component or process which
may help to streamline or economize an operation, a genetic tech-
nique or process which produces fundamentally different plants or
animals with enhanced value for culture, or wholesale transfers or
introductions of information including technology to parts of the
world where shellfish culture is constrained or otherwise under-
developed.

Too often, transfers from academia to industry have been con-

strained due to the actual laws or regulations, but also to ethical
considerations concerning the protections afforded intellectual
property. This forum seeks to bring together a group of practition-
ers from academia and industry, and within the legal field who
have had experience in a variety of technological transfers within
the field of shellfish biology and aquaculture. In this forum we
hope to provide information and generate discussion about the
process, the pitfalls and the opportunities for streamlining technol-
ogy transfers specific to shellfish culture for the future.

FORUM: THE APPROPRIATENESS
OF CULTURING BIVALVES FOR

FOOD, PROFIT, RESOURCE
RESTORATION, HABITAT AND
WATER QUALITY MITIGATION

FORUM ON THE APPROPRIATENESS OF CULTURING
BIVALVES FOR FOOD, PROFIT, RESOURCE RESTORA-
TION, HABITAT AND WATER QUALITY MITIGATION.
William F. Dewey,* Taylor Shellfish Farms, S.E. 130 Lynch
Road, Shelton, WA 98584; Daniel P. Cheney,* Pacific Shellfish
Institute, 120 State Avenue NE #142, Olympia. WA 98501.

This forum addresses the question: Is there a role for bivalve
culture in estuarine systems for food, profit, resource restoration,
habitat and water quality mitigation? As we enter the 21st century
the future of shellfish culture in estuaries around the world could
be described as very bleak or ultimately promising. If one is trying
to produce safe, wholesome shellfish on a farm for profit, along
urbanizing shorelines, with declining water quality, endangered
species, demands for pristine views and increased spatial demands
by competing users, the future looks bleak. On the other hand, if
one is attempting to restore oysters as a keystone species to the
Chesapeake Bay or New York Harbor to help consume excess
algae and create reef structures and fish habitat or reestablish the
Native Olympia oyster in Puget Sound the future looks intriguing.
With recent trends towards ecosystem management a greater di-
versity of individuals with varying professional backgrounds and
opinions are involved in making resource management decisions.
Support for shellfish culture varies dramatically and depends on
the region of the country and/or the backgrounds of the individuals
influencing local resource management decisions. This forum is
intended to facilitate a dialogue that explores the pros and cons of
culturing shellfish for food, profit, resource restoration, habitat and
water quality mitigation, and address the nature of the varying
positions and attitudes regarding the role of bivalve culture in
estuaries.

608 Abstracts. 2000 Annual Meeting, March 19-23, 2000

National Shellfisheries Association, Seattle, Washington

FUNCTIONAL ROLE OF BIVALVES
IN MARINE ENVIRONMENTS

OYSTER REEFS AS ESSENTIAL FISH HABITAT FOR
FINFISH AND DECAPOD CRUSTACEANS: A COMPARI-
SON FROM NATURAL AND DEVELOPING REEFS. Loren

D. Coen,* Marine Resources Research Institute, South Carolina
Department of Natural Resources, Charleston. SC 29412; Mark
W. Luckenbach, VIMS, Eastern Shore Lab, P.O. Box 350.
Wachapreague. VA 23480; Denise Breitburg. The Academy of
Natural Sciences. Estuarine Research Center. 1054.5 Mackall Rd..
St. Leonard. MD 20685.

Until recently our knowledge base on the value of oyster-
dominated habitats for the maintenance of economically- and eco-
logically-important species was extremely limited, especially rela-
tive to other biogenic habitats such as seagrasses, mangroves or
saltmarsh. In most cases the assigned value of shellfish habitats
and their conservation/protection was based exclusively on re-
source value, accessibility and public health, but of late this has
begun to change. Here we summarize the current status of oyster
reefs as "Essential Fish Habitats" (or EFH) by: (I) first making a
case for broadening our understanding of the ecological functions
of shellfish habitats; (2) summarizing the current state of our
knowledge on oyster habitat utilization patterns by both resident
and transient finfish and decapod crustaceans from the Chesapeake
Bay to the Gulf of Mexico; (3) discussing how current restoration
projects are enhancing our perspective; and finally (4) emphasiz-
ing the necessity of an integrated ecosystem (or adaptive) man-
agement approach regarding their management and restoration.
We also make recommendations for future EFH efforts.

BIVALVES OR NEKTON? IS THAT THE QUESTION?
Richard Dame,* David Bushek, Dennis Allen, Alan Lewitus,
Eric Koepfler, Leah Gregory, and Don Edwards, Baruch Ma-
rine Field Laboratory and Department of Statistics. University of
South Carolina, Georgetown, SC 29442 and Coastal Carolina Uni-
versity, Conway, SC 29528.

An ongoing ecosystem scale experiment in which oysters are
completely removed from tidal creeks is described and used as a
case study. The experimental design takes estimates of the sys-
tem's carrying capacity into account. Using the population or spe-
cies approach to monitor the oysters, the only observable change
after the experimental manipulation was a slight increase in sum-
mer somatic growth and elevated recruitment of oysters in creeks
with oyster reefs removed. These data are interpreted as an indi-
cation that the creeks with oysters present arc below or near car-
rying capacity. However, when nekton, plankton and water chem-
istry data are also examined a much more complicated picture
emerges.

During the summer growing season, nekton biomass in all creeks
is often greater than oyster biomass. Also, our calculations show
that oysters do not produce enough ammonium to satisfy phy-
toplankton productivity, but nekton, water column remineraliza-
tion and sediments can account for most of the deficit. Finally,
nanoHagellates, which are a preferred food for the oysters, domi-
nate the phytoplankton during the summer growing season and
diatoms dominate the colder months. The change in phase of phy-
toplankton dominance coincides with the seasonal arrival and de-
parture of nekton in the creeks.

We argue that dense bivalve reefs and beds are indicative of
intense positive feedback loops that make their ecosystems fragile
and susceptible to dramatic changes in structure. Such changes
have not been reported for natural systems, but are found in sys-
tems influenced by over-fishing, nutrient loading and pollution.
Thus, the management of sustainable fisheries in coastal ecosys-
tems requires an understanding of the ecosystem science and the
realization that tidal creek systems exhibit complex responses that
are not easily explained by linear dynamics.

OYSTER AQUACULTURE AND BENTHIC INVERTE-
BRATE COMMUNITIES IN WEST COAST ESTUARIES:
AN UPDATE. Brett R. Dumbauld,* Washington State Depart-
ment of Fish and Wildlife. P.O. Box 190. Ocean Park, WA 98640;
Steven P. Ferraro and Faith A. Cole, U.S. Environmental Pro-
tection Agency, 2111 S.E. Marine Drive, Newport, OR 97365.

A review of a limited number of field studies suggests that
oyster aquaculture practices play a key role in structuring the
benthic macro-invertebrate community in west coast estuaries.
Oysters are "'bioengineers" as they change the structure of the
substrate and create habitat for other organisms. Macro-
invertebrate communities are typically enhanced in intertidal
ground culture oyster habitat as compared to other estuarine habi-
tats, in particular intertidal mud and burrowing thalassinid shrimp
dominated habitats. Oysters add structure for macro-algal, mussel
and barnacle attachment which in turn provide protection and/or
food for juvenile Dungeness crab, shore crabs Hemi)>rupsiis. tube
building gammarid ainphipods such ixf, Amphitlwe and Corophhtm.
caprellid amphipods. tanaids. and some annelids such as the scale-
worm Harmothoe. Two other bioengineers, the ghost shrimp Neo-
irypcwa lalifoniicnsis and the mud shrimp Upogehia pugettensis,
dominate large portions of the intertidal in some west coast estu-
aries and compete for space with oysters. These thalassinid shrimp
create a soft, highly burrowed habitat suitable for other burrowing
organisms like the amphipods Eolumstorius and Eohrolgus, the
polychaete Mediomastiis, and some commensal organisms like the
clam Cryploniya. but support fewer filter feeders and much lower
species di\'ersity than oyster habitat. Preliminary results of a recent
study in Willapa Bay. WA comparing the macro-infaunal conmiu-
nilv in ground culture oyster habitat with that in six other estuarine
liahilals arc presented along with a review of previous studies in

National Shellfisheries Association. Seattle. Washington

Abstracts. 2000 Annual Meeting. March 19-23. 2000 609

West coast estuaries. To date, little has been done to estimate
functional effects of these changes at the larger estuarine ecosys-
tem scale, but some proposed work aims to investigate the func-
tional role of these habitats for the estuarine fish community.

JUVENILE OYSTER GROWTH AND CARRYING CAPAC-
ITY OF INTERTIDAL CREEKS IN NORTH INLET, SC.
A. J. Erskine* and David Bushek, Baruch Marine Field Labora-
tory. University of South Carolina. Georgetown. SC 29442; Rich-
ard Dame, Department of Marine Science. Coastal Carolina Uni-
versity. Conway. SC 29528; Nancy Hadley and Loren Coen,
Marine Resources Research Institute. South Carolina Department
of Natural Resources, Charleston. SC 29412.

In North Inlet Estuary. South Carolina, natural populations of
eastern oysters grow in dense assemblages that form extensive
intertidal beds and reefs. High oyster recruitment leads to intense
fouling of hard structures placed in the intertidal zone. These ob-
servations indicate a highly productive system. The density of
oysters in small ( 100-400 m long) intertidal creeks ranges from 2.3
to 27.5 g dry body wt m""" (or about 7 to 100 adult oysters m"')
with a mean of 10 g and median of 6.3 g. Based on this informa-
tion, the density of oysters in eight intertidal creeks was adjusted
to 8 g dry body wt m"' as part of an NSF-funded study (designated
'CREEK" Study) to determine the overall ecological role of oys-
ters in tidal creek ecosystems. We hypothesized that this density
was near the average carrying capacity of oysters for these tidal
creeks. To test this hypothesis, we examined the growth of juvenile
SPF-oysters deployed in cages in the eight intertidal creeks, before
and after the removal of native oysters from four of the creeks.
Prior to removal of native oysters there was little difference in
juvenile oyster growth rates deployed in the creeks. Following
removal of native oysters, deployed oysters grew faster in removal
creeks vs. those deployed in control creeks (oysters present at 8 g
dry body wt m""*). These data indicate that oysters in these small
tidal creeks may be at or near the carrying capacity for the system.

NATURAL INTERTIDAL OYSTER REEFS IN FLORIDA:
CAN THEY TEACH US ANYTHING ABOUT CON-
STRUCTED/RESTORED REEFS? Ray Grizzle. Jackson Es-
tuarine Laboratory, University of New Hampshire. Durham, NH
03824: Mike Castagna. Virginia Institute of Marine Science.
Eastern Shore Laboratory. Wachapreague, VA 23480-0350.

Distribution and abundance patterns at various spatial scales of
the natural, intertidal oyster reefs in the Canaveral National Sea-
shore, Florida may be instructive with respect to the design of
constructed/restored reefs, whether intertidal or subtidal. Areawide
reef patterns were characterized using low-altitude aerial imagery
and CIS-based mapping. Inter- and Intra-reef patterns in oyster
size and abundance were characterized using quadrat sampling on
10 reefs. Lagoon-wide there was a strong south-to-north increase

in areal coverages by the reefs correlated with increasing tidal
ranges. Tide range was also positively correlated with adult and
spat densities, but not oyster size. Although a quantitative analysis
has not been done, localized, inter-reef patterns showed a strong
relation to tidal tlows. The largest reefs and many smaller reefs
were oriented parallel to and/or along the edges of major tidal
channels, as commonly reported in other areas. In some areas,
however, there were clusters of reefs arranged in dendritic patterns
associated with multiple tidal channels. Theoretical models indi-
cate that such a pattern can cause greater mixing of the water
column and thus of food transport compared to single reefs of
similar total size. Intra-reef patterns included an "edge effect" on
some reefs with much greater spat settlement and oyster densities
within a 2 to 3 m fringe. There was no clear relationship, however,
between intra-reef spatial variations and reef size or location. Pat-
terns on all three spatial scales indicate that water movements are
of major importance to reef development and maintenance. They
also reflect the complexities involved in cause-and-effect relation-
ships and may provide insight into the design of constructed reefs.

THE ECOLOGICAL IMPLICATIONS OF HIGH DENSITY
HARD CLAM (MERCENARIA MERCENARIA) MARICUL-
TURE ON TIDAL CREEK ENVIRONMENTS. Michael L.

Judge,* Department of Biology. Manhattan College. Riverdale.
NY 10471; Loren D. Coen, Marine Resources Research Institute.
SCDNR. Charieston. SC 29412; Kamille Hammerstrom, NOS.
Beaufort Lab. Beaufort, NC 28516.

Hard clams, common along the Atlantic U.S. coast and north-
em Gulf of Mexico, have historically supported a valuable fishery.
Although wildstock landings have remained constant or decreased
over the last decade, mariculture production has increased steadily,
with several large clam aquaculture operations established in VA,
NC, SC and FL. The deployment of hundreds to thousands of clam
culture pens, each with tens of thousands of clams has the potential
to affect: ( 1 ) local hydrodynamics, (2) sediment characteristics. (3)
associated benthos. (4) food quality and quantity and. (5) ulti-
mately the carrying capacity of the local habitat. The consequences
of the above can have both direct and indirect impacts for both the
mariculture industry and the environment. In SC, hard clam culture
is performed on low intertidal mudflats within tidal creeks that are
typically surrounded by dense oyster reefs (Crassostrea virginica).
In 1997, we initiated a study, in conjunction with a large clam
aquaculture facility to address in part the above concerns within an
intertidal soft-bottom system typical of southeastern U.S. The
structural presence of cages imparted profound changes in the
hydrodynamic regimes within and around clam pens, thereby al-
tering numerous sediment attributes (such as. grain size, chloro-
phyll a concentrations, and C/N ratios). Moreover, the localized
de-coupling of the benthic boundary layer owing to cage-induced
mixing dramatically affected the temporal variation of re-
suspended algal food supplies. Over longer time scales, the pens

610 Abstracts. 2000 Annual Meeting. March 19-23, 2000

National Shellfisheries Association. Seattle, Washington

themselves provide additional hard substrate habitat for non-target
species (oysters, sponges, tunicates). The maintenance and ulti-
mate removal of caging materials present additional future con-
cerns.

TEMPORAL PATTERNS OF FISH AND DECAPOD UTILI-
ZATION OF OYSTER REEFS: COMPARISONS ACROSS
AN ESTUARINE GRADIENT. Mark Luckenbach* and Fran-
cis O'Beirn. Virginia Institute of Marine Science. Eastern Shore
Lab, College of William and Mary, Wachapreague VA 23480;
Juliana Harding, Roger Mann, and Janet Nestlerode, Virginia
Institute of Marine Science. College of William and Mary. Glouces-
ter Point. VA 23062.

Biogenic reefs created by the eastern oyster Crassostrea vir-
ginica are increasingly being recognized for their associated biodi-
versity. Yet, few details of the use of oyster reefs as habitat for
mobile species offish and decapods are available. Specifically, the
temporal patterns of species use of reefs and the nature of that use
(e.g., refugia or foraging) are largely unknown. At two restored
reef sites in the Chesapeake Bay, one in a polyhaline and one in a
mesohaline environment, we have conducted multi-year studies of
the development of reef communities and their utilization by mo-
bile fauna. Here we report on variation in use of these reef habitats
by finfish and crabs on several temporal scales: sea.sonal. fort-
nightly, diel and tidal. Using fixed and towed nets and underwater
video, we compare the abundances of fish and crabs on the reef
with that of adjacent unstructured habitats. Direct observations of
feeding activity and gut analysis are used to clarify trophic link-
ages. Our findings indicate that oysters and the resident assem-
blages which they support may play a significant role in supporting
several commercially and recreationally important species.

GRAZING OF NATURAL PARTICULATES BY BLUE
MUSSELS ON RAFTS: SIMULATIONS USING FLOW-3D.
Carter R. Newell,* Great Eastern Mussel Farms. Tenants Harbor.
ME 04860; John E. Richardson, Earth Tech. Concord. NH
03301.

Since the early 198()"s blue mussels have been cultivated on the
bottom in Maine, and more recently mussel culture on rafts has
become popular on both the west and east coasts of the U.S. The
supply and demand of particulate food to mussels on the bottom
has been previously quantified in a model MUSMOD. which has
recently been improved to include the settling tlux of particles as
well as those supplied lo the bottom by advection and vertical
mixing. On rafts, the effects of the mussel lines and predator nets
on current speed result in a complex pattern of flow around and
through the rafts, which wc have successfully simulated using
FLOW-3D. Food availability within and around the rafts was mea-
sured by water grab samples and using a CTD u itii a lluoromcter.
Depletion of food particles by as much as MVi v\ere measured in

the field, and simulated in a model relating particle consumption
by the mussels to their biomass on the ropes. Initial model runs
were then used to optimize mussel growth rates and yield by
changing raft orientation to flow, rope spacing and seed density.
Sedimentation rates of mass, carbon and nitrogen were also mea-
sured and compared with control stations.

Adequate currents are required to provide food to the middle of
mussel rafts, and also to provide oxygen to benthic bioturbators
feeding on the organic matter accumulation below the rafts.

ROLE OF SUSPENSION FEEDING BIVALVES IN MEDI-
ATING ESTUARINE NUTRIENT CYCLING. Roger I. E.
Newell* and Jeff C. Cornwell, Horn Point Laboratory. UMCES,
PC Box 775, Cambridge, MD 21631, USA.

Although it is apparent that changes in the abundance of
benthic suspension feeding bivalve molluscs will proportionally
alter the degree of benthic pelagic-coupling the ensuing ecosystem
changes are less predictable. That is. will an increase in bivalves
just result in faster inorganic nutrient recycling, thereby stimulat-
ing phytoplankton production, or will the rate of nutrient recycling
be different than if the same amount of organic material was de-
graded in the water column? In laboratory mesocosms under oxic
and anoxic conditions in the dark we measured changes in sedi-
ment geochemistry, nutrient tluxes. and denitrification in response
to loading by different amounts of algal paste, an experimental
analog of oyster biodeposits. Increased organic loading to the sedi-
ment under oxidized conditions resulted in higher rates of coupled
nitrification/denitrification. In contrast, coupled nitrification/
denitrification was suppressed under anoxic conditions. Similar
incubations in the light which permitted the growth of benthic
microalgae showed negligible ammonium fluxes from sediments,
with the algal/microbial community efficiently retaining ammo-
nium and fixing nitrogen. Because no DIN was recycled to the
water column under oxic conditions we conclude that increasing
the stocks of suspension feeders stocks v\ill have the beneficial
effect of removing phytoplankton frotn the water column without
stimulating further phytoplankton production. Furthermore, net
rates of nitrogen loss via denitrification will be enhanced in areas
with higher levels of benthic-pelagic coupling.

INTERRELATIONSHIPS BETWEEN SEAGRASSES AND
BENTHIC SUSPENSION FEEDERS. Bradley J. Peterson,*

Department of Biological Sciences. Florida International Univer-
sity. Miami. FL 33 199; Kenneth L. Heck, Jr., Dauphin Island Sea
Lab, Dauphin Island, AL .^6528.

Two simultaneously conducted field experiments using live
mussel density manipulations and a 3 x 3 factorial incomplete
randomized design utilizing mussel mimics and nutrient enrich-
ment of the sediments were conducted to examine the effect of
mussels on meadows ot the seasjrass TliciUissia ti'stitdiiniin.

National Shellfisheries Association. Seattle. Washinaton

Abslracrs. 2000 Annual Meeting, March 19-23. 2000 61 1

The live mussel density manipulations resulted in significantly
increased nutrient concentration of sediment porewaters. signifi-
cantly reduced leaf tissue C:N. N:P and C:P ratios and reduced
epiphytic loads, and increased seagrass production when mussels
were present. The 3 x 3 factorial design tested the separate factors
of increased habitat structure and increased nutrient enrichment
resulting from the presence of the mussels. Structure had a sig-
nificantly negative effect on epiphytic biomass. Nutrient had a
significantly positive effect on sediment porewater nutrient con-
centrations and a significantly negative effect on leaf tissue N:P
and C:P ratios. The strength of the positive response to mussel
presence in seagrass productivity appeared to progress along a
continuum from early reliance on nutrient enrichment to the in-
creasing role of habitat complexity as the growing season ad-
vanced.

Finally, a field experiment evaluating the effects of seagrass on
the survivorship of the associated mussel. Modiolus americwnis
was conducted. Mean survival was significantly greater in veg-
etated habitats than in unvegetated sediments. Thus, this study
demonstrates the reciprocal positive interactions of these organ-
isms when associated and suggests that seagrass meadows may
exist as a mosaic of patches of differing productivity when sus-
pension feeding organisms are present.

USE OF OYSTER REEFS BY MOBILE FAUNA: CONSE-
QUENCES FOR ADJACENT SANDFLAT HABITATS. Mar-
tin H. Posey and Troy D. Alphin, Depart. Biological Sciences.
UNC-Wilmington. Wilmington. NC 28403: Christopher M.

Powell. Center for Marine Science Research. UNC-Wilmington.
Wilmington. NC 28403; John M. Rhoads, Barry A. Vittor &
Assoc. 271 Zena Rd.. Kingston. NY 12401.

There has been increasing recognition of the importance of
oyster reefs as habitat for benthic fauna and nekton. However, the
importance of landscape parameters in the function of oyster reefs
is less well understood. Among these landscape considerations are
the potential interactions between organisms inhabiting oyster reef
communities and those in adjacent habitats. Oyster reefs provide
refuge for a variety of resident predators, such as rock crabs,
gobies and certain shrimp and transient predators such as blue
crabs and pinfish. Research on coral reefs, hardbottom outcrops,
and artificial reefs indicates that reef-associated predators often
increase predation intensity on adjacent sandtlat habitats, suggest-
ing the possibility for similar linkages between oyster reefs and
sandflat areas adjacent to the reefs. We have conducted a variety
of field observations, field manipulations, and laboratory experi-
ments to determine what predators may be utilizing oyster reefs
and how their presence may affect infauna in adjacent habitats.
Sampling over a variety of reefs emphasizes their importance as
habitat for predatory fish and decapods. There is a trend towards
lower abundance of certain infaunal groups near oyster reefs with

evidence for stronger effects of predator exclusion immediately
adjacent to a reef compared to several meters distant. Laboratory
experiments confirm the potential for off-reef foraging by reef-
associated predators. These results emphasize the need to consider
habitats as interconnected units in management efforts.

NITROGEN EXCRETION BY THE PACIFIC OYSTER,
CRASSOSTREA GIGAS; A CONTRIBUTOR TO ESTUA-
RINE NUTRIENT CYCLING IN TOMALES BAY, CA.
Linda Righetti. Romberg Tiburon Center, San Francisco State
University. PC Box 8.')5. Tiburon CA 94925.

Because of its importance as an aquaculture commodity, the
filtering capacity and dietary requirements of the Pacific Oyster.
Crassostrea gigas. have been studied in some detail. Most inqui-
ries have focused on studies of food ingestion by the animal, and
the portion of their intake that is converted to meat production.
Very little attention has been given to the fate of excreted nutrients.
Excreted matter may be returned to nutrient cycling systems, in
such forms as ammonium (NHj), which is readily taken up by
phytoplankton and bacteria. This investigation found that C. gigas
sampled from Tomales Bay (TB), California, provided with an
excess of algal food excretes ammonium at a rate of 3.07 |Jig/g/h.
Based on recorded biomass values for cultured oysters, C. gigas
may be contributing ammonium to TB at a rate upward of 0.007
|j.M/h. The ambient phytoplankton population of 1 |jig/l chloro-
phyll in TB is capable of using this NHj. thus C. gigas excretion
could account for up to 58% of N taken up by phytoplankton.
Cleariy the activity of filter feeders is important in determining the
composition of available nutrients in the water column, impacting
phytoplankton-based food webs in Tomales Bay. Future research
will examine the extent to which nutrients may be removed from
these systems, e.g. in the ingestion of diatoms resulting in the loss
of unregenerated silica.

MUSSELS: SPACE MONOPOLISERS OR ECOSYSTEM-
ENGINEERS? Ray Seed, School of Ocean Sciences, University
of Wales, Menai Bridge, Anglesey, UK. LL59 5EY.

Mussels are extremely successful and widely distributed bi-
valve molluscs occurring in freshwater and estuarine habitats
throughout the world and ranging from the high intertidal zone in
coastal seas to mid ocean depths at sites of hydrothermal vent
activity. This paper will explore some of the many reasons that
underpin the evolutionary and ecological success of mussels and
will briefly consider, by way of selected examples, the significance
of their success to humans. As competitively dominant species
mussels can potentially monopolise certain epibenthic communi-
ties with a consequent reduction in diversity of the primary space-
occupying species. However, dense patches of mussels can dras-

612 Abstracts. 2000 Annual Meeting, March 19-23. 2000

National Shellfisheries Association, Seattle, Washington

tically modify the local environment, especially through biodepo-
sition and the provision of additional habitat, features which serve
to encourage species enrichment. The relative importance of mus-
sels as space monopolisers and/or ecosystem engineers will pro-
vide a particular focus of this paper.

TWO STORIES OF PHYTOPLANKTON CONTROL BY BI-
VALVES IN SAN FRANCISCO BAY: THE IMPORTANCE
OF SPATIAL AND TEMPORAL DISTRIBUTION OF BI-
VALVES. Janet K. Thompson, U.S. Geological Survey. Menlo
Park. CA 94025.

The introduction of the Asian clam, Potamocorbiiki ainureusis.
into San Francisco Bay has resulted in changes to the food web
within the northern bay (NB) but not within the southern bay (SB).
P. aiintrensis invaded the bay in 1986, became the dominant mem-
ber of the benthic community within one year in NB and within
three years in SB. Large declines in phytoplanklon biomass in NB
appear to be due to "over-grazing" by P. ainurensis populations
which are estimated to filter the shallow reaches of NB in excess
of twice a day. Because high turbidity restricts net positive primary
production to the shallow reaches of NB and limits the net primary
production in the deep areas of SB, shallow water grazing controls
system-wide phytoplankton biomass throughout the system. SB
phytoplankton biomass has not changed with the invasion of P.
amiirensi. despite similar density and biomass levels of P. atiui-
rensis in the deep water throughout the system. There are. how-
ever, large differences in the temporal and spatial distribution of
shallow water P. ainurensis in the NB and SB. Shallow water P.
ainurensis live 1 '/2-2 years in the NB but only 8-9 months in the
SB, and the annual phytoplankton bloom in SB occurs during the
three month period when P. ainurensis are absent from the shallow
water.

GENETICS AND BREEDING

RESEARCH AND DEVELOPMENT ON SUMINOEGAKI,
CRASSOSTREA ARIAhENSIS. FOR AQUACULTURE IN
VIRGINIA, AND OTHER ACTIVITIES WITH NON-
NATIVES. Standish K. Allen, ,Ir.,* Aquaculture Genetics and
Breeding Technology Center, Virginia Institute of Marine Science,
College of William & Mary, Gloucester Point. VA.

For several years, VIMS has been examining the biology and
potential of non-native species for aquacullure development in the
Chesapeake Bay. Earlier work has shown potential for C. f;ii;as in
higher salinity sites only and C. arial<ensis generally throughout
the Bay. With the goal of developing "put-and-takc" aquacullure
using sterile Iriploids, the Aquaculture Genetics and Breeding
Technology Center has begun more extensive R&D on a luinibcr

of fronts for C. ariakensis. Using stocks brought to the East Coast
about 8 years ago, in 1999 we produced triploids for field trials,
specifically to examine reversion and aspects of their marketabil-
ity. From previous experiments, it is clear that reversion is a fea-
ture in triploid C. ariakensis as well. We have also attempted to
produce tetraploid C. ariakensis with some difficulty. After dozens
of attempts, some dozen or so putative tetraploids are in hand. We
are also examining population genetic structure in collections
throughout Southeast Asia. Early evidence points to discreet popu-
lation structure among locales. Native Suminoegaki from southern
and northern China were imported in 1999 and we produced F,
diploids and triploids from the southern population. Larval culture
for this species is still problematic in our hands and so we will be
trying to optimize this fundamental step by working closely with
collaborators in China. For C ariakensis and also for more general
work with non-natives, we have upgraded .several key aspects of
our physical plant, including development of a dedicated, land-
based holding facility for long-term non-native research.

MICROSATELLITE MARKERS AS A TOOL TO STUDY
REPRODUCTIVE SUCCESS IN THE PACIFIC OYSTER,
CRASSOSTREA GIGAS (THUNBERG). CROSSED UNDER
CONTROLLED HATCHERY CONDITION. Pierre Boudry
and Bertrand Collet, Laboratoire IFREMER de Genetique et Pa-
thologic. BPI.^.3. 17390 La Tremblade. France; Florence Cor-
nette, Veronique Hervouet and Francois Bonhomme, Labora-
toire Genome. Populations, Interactions. 1 quai de la Daurade,
34200 Sete. France.

Oysters, like many marine species have a very high fecundity.
Previous studies have shown that populations, from both hatcher-
ies and the natural environment, have very low Ne/N ratios. These
observations reveal high variation in reproductive success. In order
to study individual reproductive success under controlled condi-
tions, we used microsatellite markers to quantify parental contri-
butions in in vitro crosses (5 males and 5 females) of Crassostrea
gii-as. the Pacific oyster. High polymorphism of the microsatellites
(more that 50 alleles per locus) eased the parentage identifications.
The results of a cross allowing gametic competition were com-
pared with the results from a second cross where the gametes of
the same parents were kept separate for each parental combination
until after fertilization. The progeny were then sampled at different
stages of development and the parental contributions determined to
follow their evolution through time. Despite the fact that equal
numbers of gametes were mixed for each male and each female,
the contributions of these parents to the resulting progeny was
highly unbalanced at both lar\al and juvenile stages in both
crosses. We demonstrated that variation in individual reproductive
success is due lo both spermatic competition and selective phe-
nomena at early stages.

National Shellt'isheries Association. Seattle. Washington

Ahstracts. 2000 Annual Meeting. March l9-2,\ 2000 613

BEAD-BASED GENOMICS TECHNOLOGIES AT LYNX:
APPLICATIONS FOR PACIFIC OYSTER BREEDING. Ben
Bowen, Lynx Therapeutics, Inc.. Hayward, CA 94543.

Lynx has developed a method (Megacione"^') for cloning am-
plified DNA fragments on the suiface of 5-rnicron plastic beads
rather than in E. coli. Beads harboring differentially expressed
genes can be identified rapidly using a fiuorescence activated cell
sorter in a process called Megasort™'. An automated procedure for
determining 16-20 bases of signature sequence from upto one
million beads simultaneously (Massively Parallel Signature Se-
quencing or MPSST''') has also been devised. These signature se-
quences assign an identity to each bead-based clone, and, in many
cases, allow matching of each clone in a bead array with genes in
a sequence database. Finally, a new application of Lynx's bead-
sorting technology (Megatype'") is being developed to identify
polymorphic genomic DNA fragments that correspond to alleles
present at different frequencies in two phenotypically distinct
populations. I will discuss applications of these technologies for
the genetics and breeding of Pacific oysters, especially understand-
ing the phenomenon of heterosis or hybrid vigor.

TRANSFECTION OF EASTERN OYSTER EMBRYOS.
John T. Buchanan,^ Department of Oceanography and Coastal
Sciences, Louisiana State University, Baton Rouge, LA 70803;
Amy D. Nickens and Terrence R. Tiersch, Aquaculture Research
Station, Louisiana State University Agricultural Center, Baton
Rouge, LA 70820; Richard K. Cooper. Department of Veterinary
Science, Louisiana State University, Baton Rouge, LA 70803.

There is a need for research in disease resistance and microbial
elimination in eastern oysters, Crassostrea virginica. Gene transfer
research may lead to advances in this area, and a means of select-
ing transfected larvae would be useful. We transfected 3-h-old
embryos with the bacterial gene aminoglycoside phosphotrans-
ferase II ineo' ). which confers resistance to neomycin and related
antibiotics such as G4I8. The antibiotic G418 was examined as a
potential selective agent. A neutral red assay was used to deter-
mine survival after 48-h exposure to various concentrations of
G418 (0 to 4 mg/ml). We examined the effects of electroporation
and chemically mediated transfection (SuperFect""'; Qiagen) of
3-h-old embryos on survival to D-stage larvae. DNA alone was
found to have no effect on survival (.P > 0.05). For electroporation,
we found that increasing voltage and pulse duration decreased
survival (P < 0.05). Chemically mediated transfection did not sig-
nificantly affect survival [P = 0.5172). Transgenic larvae were
produced by electroporation or chemically mediated transfection
of 3-h-old oyster embryos with neo'. These embryos were reared
for 24 h and exposed to G4I8 at 0.3 mg/ml for 48 h. Significant
differences in survival between transfected and nontransfected lar-
vae were detected for electroporation (P = 0.0147) and chemi-
cally mediated transfection (P = 0.037). This study documents the

successful insertion and expression of foreign DNA in eastern
oyster larvae.

ESTIMATION OF NARROW-SENSE HERITABILITY FOR
LARVAL AND JUVENILE GROWTH TRAITS IN SE-
LECTED AND UNSELECTED SUB-LINES OF EASTERN
OYSTERS, CRASSOSTREA VIRGINICA. Christopher V.

Davis,* Darling Marine Center. University of Maine. 193 Clarks
Cove Road. Walpole, ME 04573, USA.

When embarking on a selective breeding program, prior knowl-
edge of the heritability for the selected trait is useful in developing
an optimal breeding design. Narrow-.sense estimates of heritability
describe the proportion of phenotypic variation due to additive
gene effects. These estimates are useful in predicting potential
gains that may result from exploitation of additive genetic variance
in selection programs. The goal of this study was to determine the
narrow-sense heritability for juvenile growth traits in hatchery
propagated lines of eastern oysters, Crassostrea virginicd (Gmelin
1 79 1 ) selected for rapid growth.

Heritabilities for growth traits were determined using half-sib
analysis of twenty-five families produced from 5x5 diallele
crosses. Estimates were made from a population having undergone
three generations of selection for rapid growth and from an unse-
lected control sub-line. Both sub-lines were originally derived
from a common source population. Sire-based estimates of heri-
tability for larval shell length were 0.44 (±0.14) and 0.14 (±0.07)
in the selected and unselected control sub-lines, respectively. Cor-
responding estimates for live weight at 7 months were 0.51 (±0.15)
and 0.10 (±0.05). respectively. These results suggest that selective
breeding efforts may significantly increase growth rates in oysters,
although the magnitude of improvement may vary considerably,
depending on the degree of exploitable additive genetic variance
within the population.

ANALYSIS OF GENETIC DIVERSITY IN A COMMER-
CIALLY IMPORTANT LINE OF OYSTERS SELECTED
FOR FAST GROWTH. Daniel DenDanto,* University of

Maine, Orono. ME 04469; Bonnie L. Brown, Virginia Common-
wealth University, Richmond. VA 23284; Chris Davis, Pemequid
Oyster. Waldaboro, ME 04572; Irving Kornfield, University of
Maine, Orono, ME 04469.

Levels of heterozygosity and allelic diversity at discrete mic-
rosatellite loci are compared among two hatchery derived lines and
a wild population of Eastern Oyster, {Crassostrea virginica) to
assess the effects of "selection" for fast growth among the hatchery
lines. Wild samples and ""selected" experimental cohorts of ""Mil-
ford"" and ""Flowers" oyster lines are evaluated for generational
loss of genetic variation within and between the groups. A com-
mercially important line of oyster, improved for growth on the
Maine coast, is investigated for changes in its genetic background

614 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

over four generations of selection and over larvel and juvenile
phases of an Fj cohort. Comparison of these hatchery lines to wild
populations of oysters in the Gulf of Maine allows for a much
needed evaluation of genetic consequences incurred during the
selection experiments for fast growth. Information on the genetic
change across life history stages within the "Flowers" Fj cohort
addresses unique deviations from population genetic expectations
as observed by other investigators for this genus.

PATTERNS OF NUCLEOTIDE VARIATION AT THE GPl
LOCUS IN THE BLUE MUSSEL MYTILUS EDULIS. Mat-
thew P. Gordon* and Paul D. Rawson, School of Marine Sci-
ences, Murray Hall. University of Maine. Orono. ME 04469.

Glucose-6-phosphate isomerase (GPI) is an enzyme that func-
tions as a branch point between the glycolytic pathway and the
pentose shunt pathway. In many coastal marine taxa along the
Atlantic Coast of North America there is a high degree of allelic
polymorphism as well as concordant patterns of allelic distribution
for this enzyme. These concordant patterns suggest that adaptation
is important in the maintenance of variation at the Gpi locus. For
Mytihis echtlis. Hall (1985) demonstrated temperature related ki-
netic differences between two common Gpi alleles, GPI' "", which
is most common in the Mid-Atlantic, and GPI" '''', which increases
in frequency with latitude. This evidence is consistent but not
conclusive with regard to the hypothesis that Gpi is thermally
adapted in M. edulis. The objective of this study was to examine
DNA sequence variation for Gpi in M. edulis to further investigate
the role of selection in the maintenance of variation at this locus.
From preliminary sequence information obtained by using RT-
PCR and 5' IV Race methodologies we have designed primers that
amplify complete coding segments of M. edulis Gpi. Individual
mussels from Merrick. NY and Walpole, ME have been allotyped
at GPI by electrophoresis. From individuals homozygous for either
gpi' "" or GPI"'"' we have isolated complete coding sequences.
We will present an analysis of patterns of nucleic and amino acid
variation among the sequences using a Gpi sequence from the
congener M. trossulus as an outgroup.

ANEUPLOIDY IN THE PACIFIC OYSTER, CRASSOSTREA
GIGAS THUNBERG AND ITS EFFECTS ON GROWTH.

\iming (Juo,'* (luoi'an Zhang." Brcnda .1. Landau.' Louise
Enghsh,' and ^'ongpini; Wang,"' 'llaskin Shellfish Research
Laboratory, Rutgers University, 6959 Miller Avenue. Port Norris,
NJ 08.349. USA; "Key Laboratory of Aquacultural licology.
Dalian Fisheries University, Dalian, Liaoning I 16025. PRC; 'Hx-
perimental Marine Biology Laboratory, Institute of Oceanology
CAS. Qingdao, Shandong 266071. PRC.

We previously described the incitlental induction of aneuploids
in the Pacific oyster. Cnissasireu f;incis Thunberg, from tctraploiti
anti triploid prcKliiclion. Here we report the intentional production

of aneuploids and isolation of trisomic families, with observations
on their growth performance. The first generation of aneuploids
was produced from triploid x diploid crosses. Two types of crosses
were made: diploid $ x triploid 6 (DTA) and the reciprocal
triploid ? X diploid 6 (TDA) crosses. DTA crosses were highly
effective in producing aneuploids. and 80-95% of the DTA prog-
eny were aneuploids as determined by chromosome counts at one
year of age. Aneuploid conditions included 2n -i- 1 , 2n -t- 2, 2n -i- 3,
3n - 2, and 3n - I. TDA crosses produced fewer aneuploids
(16-20%) and more triploids (20-53%) than DTA crosses. Aneu-
ploids as a group are significantly smaller in size than normal
diploids. DTA progeny with an approximate diploid DNA content
were separated using flow cytometry and considered putative tri-
somies. The putative trisomies were crossed with normal diploids
in single-pair matings. Sixty putative trisomic families were pro-
duced, and 20 of them were confirmed as trisomic families using
chromosome counts of embryos at the 2-cell stage. In most fami-
lies, the frequency of trisomies sharply declined, from about 50%
at the 2-cell stage to 5-25% at one year of age, possibly due to
mortality or chromosome loss. In some families, the trisomies
remained at 40-61%. Trisomic oysters are smaller on average than
normal diploids in most families, but not different from normal
diploids in others. Results of this study show that the Pacific oyster
can tolerate aneuploidy up to 15% of its genome. Aneuploids as a
group have growth retardation, but certain aneuploid conditions
grow as well as normal diploids.

IMPROVING PACIFIC OYSTER BROODSTOCK
THROUGH CROSSBREEDING. Dennis Hedgecock,* Univer-
sity of California. Davis. Bodega Marine Laboratory. Bodega Bay,
CA 94923-0247; Jonathan P. Davis, Taylor Resources, Inc., 701
Broad Spit Rd., Quilcene, WA 98376.

Controlled crosses among Inbred lines of the Pacific oyster
Cnissostrea ^ifids reveal much hybrid vigor or heterosis for larval
and adult growth rate. Evidence for a large genetic load in this
oyster suggest that hybrids are superior because they inherit dom-
inant alleles that mask deleterious recessive mutations in many
functional genes. Physiologically, hybrids appear to have greater
efficiencies than inbreds in energy and protein metabolism and
feeding. How growth or yield of hybrid oysters compares to that of
farmed Pacific oysters has been the focus of a project funded by
the USDA's Western Regional Aqiiacullure Center since 1993.
Initial large-scale comparisons of inbred and hybrid with farmed
oysters suggest that crossbreeding can improve commercial brood-
slocks. Inbred offspring, which were made b\ mating siblings from
the first selected generation of oysters in the Molluscan Brood-
stock Program, Hatfield Marine Science Center, Newport. OR,
show inbreeding depression, as expected. Growth trials comparing
WRAC livhrids and MBP select families are in progress. Hybrid
larvae produced in l'^).S at the Taylor Shellfish Hatchery,
Quilcene. WA. grew faster and set 4-5 days earlier than larvae

National Shellfisheries Association. Seattle, Washington

Ahsrracts. 2000 Annual Meeting. March 19-23, 2000 615

from commercial control spawns. Body-size data for these hybrids,
which are growing on long-lines in Samish Bay. WA, will be
obtained in December 1999. Despite the promi.se indicated by
e\'idence for heterosis and WRAC results to date, commercial
implementation of crossbreeding will require testing crosses
among hundreds if not thousands of inbred lines. As it is not
possible to rear such a large number of groups in commercial
culture facilities, very early physiological or molecular indicators
of hybrid performance are needed to improve the efficiency of
testing. Differences in respiration between inbred and hybrid oys-
ters, for example, are evident at the early trochophore stage; com-
parisons of respiration and protein turnover among different hy-
brids at the trochophore stage are therefore planned. Patterns of
early gene expression are also being explored for their potential
use in identifying elite inbred lines for hybrid oyster production.

MICROSATELLITE ANALYSIS OF TRISOMIC FAMILIES
IN THE PACIFIC OYSTER, CRASSOSTREA GIGAS THUN-
BERG. Sophie Hubert,'* Louise J. English,' Brenda J. Lan-
dau," Ximing Guo," and Dennis Hedgecock,' 'Bodega Marine
Laboratory. University of California at Davis. P.O. Box 247.
Bodega Bay. CA 94923; and "Haskin Shellfish Research Labora-
tory. Institute of Marine and Coastal Science. Rutgers University,
6959 Miller Avenue. Port Norris. NJ 08349.

Trisomy {2n -I- 1 ) is an aneuploid condition where one chro-
mosome is represented by three copies instead of the normal two
copies. Change in copy number may affect the expression of genes
located on the trisoniic chromosome and therefore, analysis of
trisomies may be useful for the chromosomal assignment of mark-
ers and quantitative trait loci. We produced trisomic families in the
Pacific oyster, Crassostrea gigas Thunberg, and tested microsat-
ellite jiiarkers for trisomic identification and analysis. Trisomic
families were produced in two steps. First, diploid x triploid
crosses were made, producing a mixture of normal diploids, trip-
loids. trisomies and other aneuploids. Individuals with an approxi-
mate diploid DNA content were separated with flow cytometry
and considered as putative trisomies. Then putative trisomies were
crossed with each other or with normal diploids in single-pair
matings. Sixty putative trisomic families were produced, and 20 of
them were confirmed as trisomic families using chromosome
counts of embryos at 2-cell stage. Parents from 16 trisomic fami-
lies were screened with 14 microsatellite markers. Tri-allelism (3
alieles/locus/individual) was observed at three loci in six trisomic
families. The tri-allelism was found only in the putative trisomic
parent, not in normal diploids. One locus was tri-allelic in three of
the 16 families, suggesting that the chromosome carrying this lo-
cus may be over-represented among the trisomic families. Progeny
from the trisomic families are being analyzed for confirmation of
trisomic inheritance. Results so far indicate that trisomic families
can be readily produced, and microsatellite markers are useful in
trisomic identification because of their high polymorphism.

NUCLEAR CONTROL OF SEX RATIO BIAS IN THE MUS-
SEL MYTILVS EDULIS. Ellen Kenchington* and Liqin Cao,

Department of Biology. Dalhousie University, Halifax. Nova
Scotia B3H 4JI. Canada; Eleftherios Zouros, Institute of Marine
Biology of Crete. Greece.

Previous studies have shown that in pair-matings of Mytilus
edidis, M. trossiilus and M. galloprovincialis there can be a large
sex-ratio bias in favor of either males or females. The degree of
bias is a characteristic property of the female parent, as matings of
the same female with different males produce the same sex ratio,
but matings of the same male with different females produce dif-
ferent sex ratios. All three species possess the unusual feature of
doubly uniparental inheritance of mitochondrial DNA (mtDNA),
i.e.. they contain two distinct types of mtDNA. one that is trans-
mitted palrilinearly (the M type), and one that is transmitted matri-
linearly (the F type). This coupling of sex and mtDNA inheritance
raises the possibility that a female's sex-ratio is under the control
of the female's mtDNA. Here we present data from controlled pair
matings that are incompatible with this hypothesis, but are consis-
tent with a nuclear control of sex ratio.

GROWTH, SHELL MORPHOLOGY, REPRODUCTIVE
PHYSIOLOGY, AND MOLECULAR GENETIC ANALYSIS
OF TASMANIAN PACIFIC OYSTERS, CRASSOSTREA GI-
GAS, IN WASHINGTON STATE. Manfred Kittel* and Ken-
neth K. Chew, School of Fisheries, University of Washington,
Seattle, WA 98195,

Controlled introductions of shellfish populations may be eco-
nomically beneficial to the aquaculture industry by providing de-
sirable traits and improved productivity through hybrid vigor. A
small number of deep-cupped Pacific oysters {Crassostrea gigas)
was transferred from a Tasmanian shellfish hatchery to the state of
Washington in 1994. Oysters were spawned artificially in two
separate mass spawns with maximum effective population sizes
(NJ of 18 and 6.4, respectively. The resulting F, oysters were
compared to control C. gigas of local origin with respect to overall
survival, growth rate, shell morphology, gonadal maturation, and
glycogen storage. A molecular genetic analysis was performed to
determine the species status of the introduced oysters, investigate
the possible loss of genetic variability due to the founder effect,
and to develop a DNA-based molecular population marker.

Results from this study indicate that the Tasmanian F, oysters
experienced a 40% reduction in cumulative mortalities and at-
tained significantly greater length, weight, and volume than con-
trols. There was no decrease in heterozygosity or polymorphism
but allelic variation was reduced by 28% due to the loss of several

616 Ahslracts. 2000 Annual Meeting. March 19-23, 2000

National Shellfisheries Association, Seattle, Washington

rare alleles. Restriction of a 2.100 bp mitochondrial (ml) ribosomal
DNA segment (including ITS-1 and ITS-2) with 45 restriction
endonucleases did not produce population-specific haplotypes.
However, 50% of a small sample (N = 14) of Tasmanian F,
oysters were characterized by a C — > T transition at one specific
nucleotide position within a 524 bp PCR-amplified DNA fragment
of the mt cytochrome h locus.

THE MOLLUSCAN BROODSTOCK PROGRAM-
IMPROVING PACIFIC OYSTER BROODSTOCK
THROUGH GENETIC SELECTION. Chris J. Langdon.*
Dave P. Jacobson, and Ford Evans, Hatfield Marine Science
Center. Oregon State University. Newport, OR 97365; Mike S.
Blouin, Zoology Department, Oregon State University, Corvallis,
OR 97331.

The Molluscan Broodstock Program (MBP) was established in
1995 to improve yields of Pacific oysters on the West Coast, U.S.,
by genetic .selection. Currently, about 400 full-sib families have
been produced and planted at commercial sites in West Coast
states.

The performance of top-performing families (expressed in
terms of live weight per bag) is up to five times greater than that
of poorer performing families. There is a strong environment-
genetic interaction effect on relative family performance, although
"generalist" families are evident that perform well across a range
of culture environments.

Yields (live weight per bag) of progeny from crosses among
top-performing founder MBP families are significantly greater
(Fisher's PLSD test; p < 0.05) than those of progeny from non-
.selected "wild" oysters but not significantly different from those of
industry stocks. The heritability value for yield was estimated to be
0.54, indicating genetic selection should result in significant, long-
term improvements in comtiiercial oyster production.

EVIDENCE OF ABSORPTION EFFICIENCY DIFFER-
ENCES IN TWO SUBPOPULATIONS OF CRASSOSTREA
GIGAS. A FIRST APPROACH OF THEIR AMYLASE GENE
POLYMORPHISM. .|. Moal, ,1. F. Samain,* and J. Y. Daniel,

Laboratoire dc Physiologic des hncrlcbrcS. Ilremcr Centre dc
Brest BP 70, 29280 Plouzane. France; P. Boudry, Laboratoire de
gcnetique Ifremer Centre de I. a Trombladc; S. Bougrier, CRIiM.A
Ifrcmer-CNRS LHoumeau; 1). Sellos and A. Van Wormhoudt,

MNHN, laboratoire de biologic marine. BP 225. 29182, Concar-
neaii.

A Cil population from three 5 x 5 crosses of Cniwusirfd ,i,'/^'((.v
oysters from three origins was reared at low ticnsity and was feti in
the same controlled coiKlilions lor two years to studs origin of

growth variability in the frame of the European genetic programme
GENEPHYS (Genetic and Physiology). Relationships between di-
gestive enzyme activities, ingestion, absorption efficiency and
polymorphism of amylase genes, were studied on individuals of
this C. gigas generation. Two sub populations among the 60 in-
dividuals were evidenced, based on two different relationships
observed between absorption efficiency, ingested food, and diges-
tive amyla.se activities. A first approach showed differences in the
relationships between ingestion, enzyme activities, and in Michae-
lis constants (Km). A study on amylase genes, as a model, was
undertaken to study a possible genetic origin of these physiological
and catalytic traits. Gene structure was determined using different
sets of primers deduced from the amylase cDNA sequence,
previously determined. Two different amylase genes (A and B)
were characterized through their differences in nucleotide se-
quences. A first approach of individual polymorphism of amylase
genes was performed on the two physiologically characterized
subpopulations. using PCR and RFLP on the genomic DNA of
individuals, and specific primers of both genes. Two different
variants were observed for gene A and three for gene B corre-
sponding to one or two EcoRl restriction sites. Differences in
frequencies of the different observed variants were evidenced be-
tween the two oyster subpopulations. These first results and inter-
est of such an approach to identify functional genetic markers for
selection are discussed.

GENOTYPE DEPENDENT DIFFERENCES IN FEEDING
RATES AND GROWTH IN OYSTER LARVAE. Douglas A.
Pace and Dnnal T. Manahan, Department of Biological Sciences.
University of Southern California. Los Angeles. CA 90089.

A major biological question concerning heterosis is the physi-
ological e\planalion(s) for the observed differences in growth.
Differences in growth rates and feeding rates on algae were mea-
sured for larvae of the Pacific oyster. Crassostrea gigas. that had
relatively high (hybrids) and low (inbreds) levels of heterozygos-
ity. In 4 independent experiments, involving the crossing of 2 or
more inbred lines, heterosis for growth was observed. Levels of
heterozygosity also had a significant effect on size-specific feeding
rates of larvae. The slopes of the relationships between the increase
in algal clearance rales with lar\al growth (shell length) were the
same between hybrid and inbred lar\ac ( ANOVA. p > 0.05). How-
ever, there was a significant difference in the y-iiucrcept \alues
between the two groups (ANOVA, p < 0.001 I, such thai at any
given shell length hybrid larvae were feeding faster than their
inbred counteiparls. For instance, at a shell length of 280 |j.m.
hybrid larvae had a mean clearance rale (based on 4 different
experiments) that was 95'* higher than inbred lar\ae. Additional

National Shellfisheries Associatii)ii. Seattle. Washinsiton

Abstracts. 2()()() Annual Meetins;. March 19-23. 2000 617

measurements showed that rates of oxygen consumption and ci-
trate synthase (index of mitochondrial activity) were the same for
both inbred and hybrid larvae. We conclude that faster growing
hybrid larvae have higher size-specific feeding rates, but similar
metabolic rates, and so have a higher scope for growth than inbred
larvae.

DEVELOPMENT OF MOLECULAR MARKERS FOR
CONSTRUCTING A GENETIC LINKAGE MAP OF THE
EASTERN OYSTER CRASSOSTREA VIRGINICA. Kimberly
S. Reece* and Wenda L. Ribeiro, Virginia Institute of Marine
Science, The College of William and Mary. Gloucester Point, VA
23062; Patrick M. Gaffney, College of Marine Studies, Univer-
sity of Delaware, Lewes, DE 19958; James Pierce. University of
the Sciences in Philadelphia, Philadelphia, PA 19104.

The oyster diseases Dermo and MSX continue to plague the
eastern oyster Crassostrea virginica. dramatically reducing wild
harvests and discouraging the establishment of aquaculture opera-
tions in affected waters. A potential solution to this problem is
development of genetically improved disease-resistant strains of C
virginica that can grow to market size despite disease challenge.
One means of accelerating the selective breeding process is to
identify genetic markers associated with traits such as disease re-
sistance or growth rate. The goal of this project is to develop
genetic markers for constructing a linkage map and to eventually
identify markers associated with specific traits. To date six allo-
zyme and nine nuclear DNA polymorphisms have been scored in
parents and Fl progeny of a panel of ten reference families of
known pedigree. We are currently developing additional DNA
markers (single-copy, micro- and minisatellite). 768 C. virginica
clones from a small insert genomic DNA library have been par-
tially sequenced, providing =700.000 bp for marker development.
Both unknown sequences and putative coding regions are being
screened for polymorphisms by denaturing gradient gel electro-
phoresis analysis of PCR amplified fragments. PCR primers have
also been designed to anneal to regions flanking identified repeat
sequences. Amplification reactions have been optimized for analy-
sis of size variation on the automated DNA sequencer at fourteen
repetitive sequence loci, which includes one tetra-, three tri- and
four di-nucleotide niicrosatellite sequences. In addition, we are
developing a 384-well microtiter plate PCR-based assay to facili-
tate identification of specific short tandem repeats (STRs). Pre-
liminary screening of the genomic library has identified a number
of trinucleotide STR candidates. Genotypes for the allozyme and
nuclear DNA loci are being generated for the parents and 35-40 Fl
individuals of the ten reference families in order to construct a
preliminary genetic linkage map.

GENETIC CHARACTERISTICS OF WILD AND CUL-
TURED MUSSELS, MYTILUS EDULIS AND MYTILUS
TROSSULUS IN PRINCE EDWARD ISLAND (GULF OF ST.
LAWRENCE). Rejean Tremblay,* Centre Aquicole Marin.
MAPAQ — Universite du Quebec a Rimouski, Grande-Riviere,
Que., GOC IVO; Thomas Landry, Gulf Fisheries Center, DFO,
Moncton, N.B., EIC 986; Bruno Myrand, Station Technologique
Maricole des Iles-de-la-Madeleine, MAPAQ, Cap-aux-Meules,
Que., GOB I BO; Jean-Marie Sevigny, Institut Maurice-
Lamontagne. MPO. Mont-Joli, Que., G5H 3Z4.

For the first time, Mytilus trossulus was observed in popula-
tions generally considered as totally Mytilus edulis in Prince Ed-
ward Island but at low level (only 9.7% in 1997 and 2% in 1999).
Furthermore, we observed in both years (1997 and 1999), genetic
differentiations in Mytilus edulis populations from different sites in
Prince Edward Island, particularly between wild and cultivated
mussels. The genetic differences, observed by electrophoretic data
on multiple loci, were not related to allelic frequencies, but to the
genotype structure, particularly to the proportion of heterozygous
individuals. We observed that off-bottom cultivated mussels ex-
hibited an important heterozygote deficiency, comparatively to
wild mussels, in sites where no cultures were practised. As gene
flow is most likely important between sites, we suggest that the
genetic differentiation was in relation with culture practice. These
results would be discussed in relation with our studies in Magdalen
Island, where we have demonstrated the energetic advantages of
more heterozygous individuals and the impact of suspension-
cultured methods on mean heterozygosity of mussel populations.
Finally, gene flow of Mytilus edulis is discussed between the 250
km separating the Prince Edward Island and the Magdalen Island.

MICROSATELLITE VARIATION IN GEODUCK CLAMS
{PANOPEA ABRUPTA) IN PUGET SOUND, WASHING-
TON. Brent A. Vadopalas,* Are Strom, and Paul Bentzen,

School of Fisheries, University of Washington. Seattle. WA
98105.

Population differentiation is often assumed to be non-existent
among marine invertebrate species with high dispersal potential
due to a protracted pelagic larval phase. Using high resolution
DNA microsatellites and other molecular markers, however, ge-
netic differences between populations of some marine inverte-
brates have been demonstrated on both macro- and microgeo-
graphic scales. Similar analyses of geoduck clam population
genetics are problematic, in that these extremely long-lived clams
occur in contagious distributions in Puget Sound, Washington,
with each patch comprised of many overlapping generations. The
effects of temporal variation and sweepstakes recruitment must be
considered in the interpretation of spatial genetic variation in pre-

618 Abstracts. 2000 Annual Meeting, March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

sumed neutral microsatellite loci. Both age and genetic data are
necessary to examine this hypothesis.

Microsatellite allele frequency data were collected from 100
individuals each from semi-isolated populations in Hood Canal.
South Puget Sound, and the Straight of Juan de Fuca. in addition
to an outgroup from Southeast Alaska. The populations were
screened for allelic variation using seven tetranucleotide and three
dinucleotide microsatellite loci developed via magnetic bead hy-
bridization selection methods. Puget Sound samples were aged by
counting hinge plate annuli on thin-sections from the right valve.
A significant deficiency of heterozygotes was detected at many
loci necessitating the use of alternate, less powerful tests of genetic
differentiation independent of assumptions of Hardy-Weinburg
equilibrium. Analysis of age and microsatellite data may provide
valuable insight into the genetic population structure of this spe-
cies.

CHROMOSOMAL LOCATION OF SOME REPETITIVE
DNA IN CRASSOSTREA OYSTERS AS DETERMINED BY
FISH. Yongping Wang,'* Zhe Xu," Ximing Guo." James C.
Pierce,-' and Patrick M. Gaffney,'' 'Experimental Marine Biology
Laboratory. Institute of Oceanology CAS. Qingdao. Shandong
266071. PRC; "Haskin Shellfish Research Laboratory. Rutgers
University. 6959 Miller Avenue, Port Norris. NJ 08349; 'Depart-
ment of Biological Sciences. University of Science in Philadel-
phia, Philadelphia. PA 19104; "'College of Marine Studies. Uni-
versity of Delaware. Lewes. DE 19958.

Characterization and identification of chromosomes are needed
for several types of genomic analyses and mapping. Although
oysters have a low haploid number of 10. oyster chromosomes are
difficult to characterize because of their similarities in size and
shape. Traditional banding techniques in oy.sters have been diffi-
cult and unreliable. Fluorescence in situ hybridization (FISH), on
the other hand, may provide a powerful tool for the identification
and physical mapping of oyster chromosomes. We tested FISH on
oyster chromosomes with several repetitive DNA sequences using
chromosomes from early embryos. All probes were made by PCR
amplification and incorporation of DIG-11-dUTP. Meiaphase
chromosomes prepared from early embryos were adequate for use
in FISH analysis. In C. virginica. an anonymous repetitive DNA
fragment produced strong signals on several chromosomes, al-
though some locations were not as stable as others. Two short
repetitive sequences (156 and 283 bp) hybridized to all regions of
all chromosomes, suggesting that these two elements are dispersed
throughout the genome. In C. aiaas. a short repetitive sequence
was mapped to centromeric regions of 5-7 chromosomes. FISH
signals were small or weak for all repetitive sequences studied so
far, possibly suggesting that Crassosirea oyster genomes ha\e
relatively little repetitive DNA. Nevertheless, this study shows that
FISH with repctili\'C DNA is useful for chromosome identification.

INVERTEBRATE FISHERIES

THE BRITISH COLUMBIA FISHERY FOR NORTHERN
ABALONE, HALIOTIS KAMTSCHATKANA: MANAGE-
MENT FROM INCEPTION TO CLOSURE AND BEYOND.
Bruce E. Adkins, Fisheries and Oceans Canada, Pacific Biologi-
cal Station, Nanaimo. B.C. V9R 5K6 Canada.

Northern abalone (Haliotis kamtschatkana) have been har-
vested by First Nations in British Columbia for food, social and
ceremonial purposes since pre-recorded time. While recreational
and commercial abalone fisheries have occurred in British Colum-
bia since as early as 1900. these were small and largely unregu-
lated up to 1972 when a directed commercial dive fishery began.
The commercial abalone fishery, which developed during the
1970"s. typifies an emerging fishery. Annual landings increased
rapidly to 433 tonnes in 1978 but then declined quickly to less than
100 tonnes by 198! and then to 47 tonnes in 1985 as increasingly
restrictive management measures were applied in an attempt to
establish sustainable harvests. Despite the restrictive management
measures, however, abalone stocks measured at key index sites
continued to decline during the course of this fishery. As a result,
in 1990. conservation concerns led Fisheries and Ocean Canada to
close the abalone fishery to harvesting by all user groups.

Aboriginal, commercial and recreational fisheries have re-
mained closed as assessment surveys since 1990 have shown a
further decline in abalone abundance at key index sites. While this
is likely biologically related, illegal fishing is considered to be a
contributing factor.

Concerns with respect to the continued declines in abalone
stocks since the fishery closure has resulted in the northern abalone
being designated as "threatened" in British Columbia by the Com-
mittee on Status of Endangered Wildlife in Canada (COSEWIC).
A stock rehabilitation initiative currently being developed for
northern abalone in British Columbia is discussed.

The abalone fishery, the management measures and the post
closure acti\ ities are described.

STOCK ASSESSMENT AND MANAGEMENT OF RED SEA
URCHINS iSTRONGYLOCENTROTUS FRANCISCA.\US) IN
WASHIN(;T0N. Alex Bradbury.* Washington Department of
Fish and Wildlilc. Poiiil Whitney Shellfish Laboratory. Brinnon
WA 98320.

Washington's commercial red sea urchin fishery began in 1 97 1 .
The first regulations in 1977 included a restricted winter season,
area rotation, minimum and maximum size limits, and mandatory
logbooks. Exploralory surveys began in 1978. and annual surveys
at index stations on the commercial beds began in 1984. Annual
landiniis peaked at 3.658 I in 1988. followed by restricted seasons

National Shell fisheries Association. Seattle. Washington

Abstracts. 2000 Annual Meeting. March 19-23. 2000 619

and limited entry which reduced fleet size by 61%. Until 1993,
managers made aii hoc adjustments to season length based on the
obsersed trends in urchin density and size at index stations. Be-
ginning in 1993. a size-structured model based on survey data was
used to recommend harvest rates in five management regions.
Biomass has been estimated using one of four methods; 1 ) Under-
water video scans at systematically-spaced sites; 2) Adaptive Clus-
ter Sampling (ACS) dive surveys, modified to permit a fixed
sample size: 3) Change-In-Ratio (CIR) estimates based on known
catch and the observed change in urchin density from surveys; and
4) Leslie estimates based on declining CPUE. The CIR and Leslie
methods are problematic because only high harvest rates provide
suitable data for making biomass estimates. Similarly, long-term
stability in CPUE has prevented the use of surplus production
models. Direct sampling methods, such as video or ACS, are cur-
rently considered the only reliable ways to estimate biomass.
Funding problems ended surveys in most regions in 1995, and the
last direct biomass estimate occurred in 1997. In 1998, TACs were
reduced \5% from the 1997 levels as an arbitrary precaution in the
absence of survey data.

where fishing previously occurred. Quotas were based on histori-
cal harvests with adjustments based on recent fishery performance.
A description of other methods used to manage these fisheries is
also presented.

SHRIMP FISHERIES AND MANAGEMENT IN HOOD CA-
NAL AND PUGET SOUND. Therese A. Cain* and Jay G.
Odell, Washington Department of Fish and Wildlife, Point Whit-
ney Shellfish Laboratory, Point Whitney Road. Brinnon. WA
98320.

Shrimp stocks in the Hood Canal and Puget Sound have sup-
ported important shrimp fisheries for much of the past century.
This paper describes historical trends in relative stock abundance
of spot shrimp in Hood Canal as well as the present status of those
stocks. These trends are illustrated using data from preseason test
fisheries conducted to provide annual estimates of total allowable
catch. A 1994 federal court order requires co-management to pro-
vide for equitable state/tribal sharing of Hood Canal shrimp be-
tween the state recreational fishery and tribal commercial fishery.
Methods the Washington Department of Fish and Wildlife uses to
provide recreational harvest opportunity while meeting allocation
and conservation requirements are described.

Puget Sound (excluding Hood Canal) shrimp fisheries are man-
aged with a more passive management scheme. This presentation
will discuss trends in landings and the current status of the state
commercial and recreational fisheries, and the tribal commercial
fishery which has been developing since 1995. Because of rapidly
increasing participation in the state commercial fishery, the
Emerging Fisheries Act was implemented in 1994 to reduce the
state commercial shrimp pot fleet from 73 to 18 boats and the
shrimp trawl fleet from 15 to 10 boats. In 1996, WDFW began
comanaging the shrimp resource with treaty tribes, and quotas
were established for pandalid shrimp in areas of Puget Sound

APPLICATION OF HYPOTHESIS TESTING AND POWER
ANALYSIS IN THE PUGET SOUND CRAB FISHERY:
CLOSURE DECISIONS WITH CONFIDENCE. Jennifer Ca-
halan,"^ Washington Department of Fish and Wildlife, Point Whit-
ney Shellfish Laboratory, Brinnon, WA 98320.

Traditional fisheries" methods often rely on point estimators to
determine fishery decisions. This includes closure of fishing areas
due to the presence of an undesirable characteristic, such as a toxin
accumulation, fish condition, presence of disease, or low indexes
of population abundance. In many cases, a hypothesis test, which
considers the probabilities of making correct and incorrect deci-
sions, is better suited to the problem than the use of a single point
estimate of the characteristic in question. The Dungeness crab
fishery in Puget Sound is managed through a combination of size
limits, daily recreational harvest limits, and area closures during
periods when the crabs are in a soft-shell condition. In order to
determine the appropriate soft-shell closure periods sampling is
conducted in areas of t"ishing activity. When the point estimate of
the proportion of crabs in soft-shell condition exceeds a critical
point, the fishery is closed. A sample design for shell condition
testing was developed to test the null hypothesis that the sampled
crabs came from a population of soft-crabs. Sample size was de-
termined beforehand to control both the probability of opening the
fishery when the crabs are soft (type I error), and the probability of
closing the fishery when the crabs are hard (type II error). The
hypothesis test allows us to control sample size so that the prob-
ability of making a wrong decision is within an acceptable range.
Point estimates do not provide this additional information and
probability of wrong decisions cannot be readily assessed. Given
that sample data has uncertainty, this hypothesis test can improve
fishery management decisions.

MANAGEMENT OF INTERTIDAL BIVALVES IN PUGET
SOUND, WASHINGTON. James I. Child,* Squaxin Island
Tribe. Natural Resources Department, 2752 Old Olympic High-
way, Shelton, WA 98584; William W. Campbell, Washington
State Department of Fish and Wildlife, Point Whitney Shellfish
Laboratory, 1000 Point Whitney Road, Brinnon, WA 98320.

With over 35,000 acres of public beach in Washington State,
intertidal clams and oysters provide a high source of economic and
social benefit for recreational and commercial harvesters. Western

620 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheiies Association. Seattle. Washington

Washington Treaty Tribes reserved the right to harvest shellfish
under the Stevens Treaties in the 1850"s. A 1994 federal district
court ruling recognized the Tribes' right to harvest up to 50% of
the harvestable biomass of shellfish found within their usual and
accustomed fishing grounds. Intertidal clams and oysters are co-
operatively managed in the State of Washington between the af-
fected Treaty Tribes in a given region and the State of Washington.
There are a total of 8 intertidal management regions encompassed
within the waters of Puget Sound.

With Treaty rights to shellfish established and an increasing
public demand for shellfish, there is a need for a more intensive
beach management strategy. This paper will give a brief overview
of the management activities directed at beach-by-beach manage-
ment and identify some alterations from past State wide manage-
ment practices. Processes include a State wide bivalve agreement,
regional annual management plans, population estimation, calcu-
lation of "harvestable biomass", fishery monitoring and catch re-
porting.

Using a regional approach to intertidal management has al-
lowed for identification of the differing biological requirements
that exist from region to region. In various regions, the need to
utilize adaptive management practices to meet the biological re-
quirements of the species is being considered.

UNDERWATER HARVESTER'S ASSOCIATION GEO-
DUCK ENHANCEMENT PROGRAM. Bruce Clapp. Bruce
Clapp Biological Consulting. Ladysmith. B. C. VOR 2E0.

The Underwater Harvester's Association (UHA) is comprised
of all the licensed geoduck and horseclam fishermen in British
Columbia (BC). The geoduck fishery started in BC in 1976. the
UHA was formed in 1981 and in 1989 the fishery changed to an
Individual Vessel Quota (IVQ) system, which was initiated by the
UHA. From 1989 to 1998 the UHA saw a continual reduction in
their annual quota. The reduction in quota was a result of changes
in the knowledge of bed areas and geoduck density estimates. In
1994 the UHA funded and initiated an enhancement program to
plant hatchery raised juvenile geoducks in existing beds to enhance
the local populations. The initial objectives for the program were
to explore the feasibility of geoduck enhancement, to increase
stock for brood and eventually to offset harvest (partially). This
was the first shellfish fishery to try enhancement in BC. There
were no hatchery facilities in BC and no planting technology avail-
able for the UHA to copy. In 1999. there was a successful geoduck
hatchery and the UHA has a machine that can plant up to .SO.OOO
juvenile geoducks per day. The delails ot how this enhancement
program would be incorporated into the wild geoduck manage-
ment plans base not been explored.

CO-MANAGEMENT AND ASSESSMENT PROGRAMS IN
THE SHRIMP TRAWL FISHERY OF BRITISH COLUM-
BIA. Rick Harbo and L. Convey, Fisheries and Oceans Canada,
Operations Branch, Fisheries Management. Pacific Region. 3225
Stephenson Point Road. Nanaimo. B.C. V9T 1K3; J. A.
Boutillier, Fisheries and Oceans Canada, Science Branch, Pacific
Region, Pacific Biological Station. 3190 Hammond Bay Rd..
Nanaimo, B.C. V9R 5K6.

The diverse and complex Pacific shrimp trawl fishery takes
place along the British Columbia coastline, in a number of small
inshore areas and large offshore grounds. The fleet of 248 licences
is a mix of beam and otter trawls. There are seven Pandalid species
harvested commercially and fisheries vary in complexity from
single to multiple species fisheries with a variety of markets, in-
cluding machine-peeled, hand-peeled, frozen-at-sea. fresh and live
shrimp.

Landings peaked at over 7.300 tonnes, with annual landed val-
ues reaching $Cdn 13.6 million. Landings have declined since
1996. to annual levels ranging from 2.000 to 3.000 t @ SCdn 5 to
7 million, due to low stock levels in offshore areas and more
re.strictive, precautionary management practices.

In response to a dramatic increase in effort on this fishery,
management has developed rapidly from passive management at
relatively low levels of efforts to a complex suite of management
programs starting in 1997. The offshore pink shrimp fishery on the
west coast of Vancouver Island is managed by a seasonal opening.
For the inshore fisheries and the remaining offshore areas, fixed
arbitrary, historically based or forecast catch ceilings (TACs) are
initially assigned to more than 30 individual Shrimp Management
Areas. These initial catch ceilings are adjusted in-.season when
information from the fishery or a biomass survey indicate the area
can sustain fishing pressure either less than or greater than the
initial levels.

Development towards a biologically based management strat-
egy is ongoing. A long-term collaborative management and as-
sessment program has been initiated with stakeholders that in-
cludes catch monitoring, catch sampling, biological sampling, and
fishery independent surveys. An index system of assessment is
being combined with an experimental management approach to
develop the critical data necessary to model and manage this di-
verse and complex set of shrimp fisheries.

AN ECONOMIC ANALYSIS OF THE GULF OF MEXICO
OYSTER PROCESSIN(; SECTOR. Assane Diagne,* Louisi
ana Department of Wildlife and Fisheries (LDWF) — Socio-
Economic Section. 2()()() Quail Dr. P.O. Box 98()()(). Baton Rouge,
LA 70898-9000; Walter R. Kelthly. Jr., Center for Coastal, En-
ergy, and Environmental Resources, Louisiana State University.
Baton Rouge. LA 70803: David Lavergne (LDWFl.

Annual o\ster landings In ihc Gulf of Mexico account lor ap-
proxImalL'lv 60 percent of annual landings In the United States.
0\er the past fi\e \ears. annual oNsler landings in the Gulf aver-

National Shcllt'isheries Association. Seattle. Wasliini;lon

Ahslracts. 2000 Annual Meeting, March 19-23. 2000 621

aged 26 million pounds, valued at $40 million. Along with other
seafood species harvested, this sizeable supply of fresh oysters
helps support the dynamic seafood industry that has developed in
the Gulf and throughout the southeastern United States. In 1997.
the oyster processing sector generated in excess of $60 million.
This study evaluates the market structure of the oyster processing
industry in the Gulf of Mexico. Structural parameters used to
analyze the oyster processing industry include the number of deal-
ers operating in the industry, their size distribution, degree of
diversification, and the concentration in the industry as measured
by several concentration indices. Calculated structural parameters
are used to draw economic inferences on market conduct and to
make comparisons between the different states.

MANAGEMENT STRATEGIES FOR COMMERCIAL IN-
TERTIDAL CLAM FISHERIES IN BRITISH COLUMBIA.
CANADA. Rick Harbo' and Randy Webb, Fisheries and Oceans
Canada. 3225 Stephenson Point Road. Nanaimo, B.C. V9T 1K3
Canada. '

There are a number of intertidal clam fisheries in B.C. includ-
ing First Nations fisheries for food, social and ceremonial pur-
poses, commercial fisheries and recreational fisheries managed by
the federal Department of Fisheries and Oceans. The four com-
mercially harvested species (landings in 1998) are Manila clams,
Venerupis philippinanim (1,115 tonnes), native littleneck clams
Protothaca staminea (50 1), mixed (1 18 t), butter clams Saxidoirms
gigantea (40 t) and razor clams Siliqua panda (40 t).

The commercial fishery has historically been managed by mini-
mum size limits and time and area closures. Fisheries are moni-
tored in-season against historical landings and effort, and once
these ceilings are reached in any given fishing area, the fishery
may be closed. Extensive consultation takes place and an effort to
develop Community Management Boards has been initiated in two
areas. The fisheries are designed to allow openings throughout the
year in order to deal with market demands. Area management
(1989) divided the coast into 7 areas. Licence limitation (1998)
reduced the number of harvesters from approximately 2000 to a
fixed number of 1 160. Approximately 50% of these licence hold-
ers are First Nations participants. First Nations also participate in
the co-management of beaches fronting or immediately adjacent to
Reserves and pilot projects in the north coast.

Harvests for depuration are managed experimentally by quotas.
setting a variety of exploitation rates (0 to 50%). A collaborative
agreement was developed with industry that supports surveys and
stock assessment programs and a fishery manager. Five plants are
licensed to depurate in B.C.: harvesting >400 t in 1998 from vacant
crown foreshore. Additional harvests from aquaculture leases in
open and contaminated areas are managed by the province.

THE COMMERCIAL GEODUCK (PANOPEA ABRUPTA)
FISHERY IN BRITISH COLUMBIA, CANADA— AN OP-
ERATIONAL PERSPECTIVE OF A LIMITED ENTRY
FISHERY WITH INDIVIDUAL QUOTAS. Stephen Heizer,

Fisheries and Oceans Canada, 3225 Stephenson Point Road,
Nanaimo, B.C., Cda„ V9T 1K3.

The geoduck fishery in British Columbia began in 1976 as an
open access fishery without catch limits. In 1979, entry to the
fishery was limited to 55 licences and total allowable catches were
introduced. In 1989, equal individual vessel quotas were intro-
duced, at industry request, in response to excessive effort and
concerns over product supply and handling, safety and conserva-
tion. Subsequently, the fishery became BC's most valuable inver-
tebrate fishery, worth approximately CDN$40 million annual
landed value and supplying a high quality live product year round.

The IQ fishery has resulted in improved fishery management
and assessment. An industry-funded dockside monitoring program
improved the timeliness and quality of reporting of catch and effort
data. Quota overages have been limited to less than 0.1% annually
compared to overages of 55% in pre-IQ periods. Improved track-
ing of product has aided enforcement. The geoduck fisher's asso-
ciation provides over CDNSl million annually towards the man-
agement and assessment of the fishery. Funds cover water quality
certifications, biotoxin monitoring, funding for a fishery manager,
fisheries research and stock assessment activities.

More biological research is being done with the support of
industry vessels and divers, and managers have greater operational
flexibility to deal with real-time biological, enforcement and lo-
gistic issues. Health and safety in the industry has improved
greatly. Diver deaths and injuries and vessel accidents have de-
creased. Fishing plans are developed for two-year terms, and co-
management agreements for periods up to 5 years. Industry is
seeking longer and guaranteed terms of access.

RIDING THE ROLLERCOASTER: BOOM AND DECLINE
IN THE CALIFORNIA RED SEA URCHIN FISHERY. Peter

E. Kalvass,* California Department of Fish and Game. 19160 S.
Harbor Dr., Fort Bragg. CA 95437.

Initiated as an experimental fishery in southern California in
1971, the red sea urchin catch reached over 10 million pounds in
1981 prior to a three year El Nino related decline. Rapid growth of
the fishery into northern California between 1985 and 1987 fueled
concern that specific management measures were needed as permit
numbers and catch more than doubled, to 915 permits and nearly
50 million pounds. As a result, the legislature enacted additional
landing taxes to fund gathering of fishery data, research on popu-
lation parameters and resource enhancement, and the creation of an

622

Abstracts. 2000 Annual Meetin", March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

industry advisory committee to assist the Department in develop-
ing further management measures. The state Fish and Game Com-
mission adopted a formal limited entry system with a target num-
ber of fishery participants and the first minimum size requirements
in 1989. Separate harvesting closure periods in northern and south-
ern California were established in 1990. While it was the policy of
the Commission to give consideration to maximum sustained yield
in its management of marine resources, there were no scientific
management mandates prior to the adoption of the Marine Life
Management Act in 1998. Consequently, management followed a
reactive 'points of concern" approach designed primarily to reduce
harvesting pressure in the face of evidence that recruitment over-
fishing has occun'ed in northern California, and that the harvest-
able stock has been serially depleted in southern California. By
1998, following two El Ninos in the 1990s and a weakened Japa-
nese export market for uni. the statewide catch had steadily de-
clined to 10.5 million pounds valued at $8 million, from a high in
1988 of 52 million pounds, worth $35 million.

TEMPORAL AND SPATIAL VARIATION IN SPAWNING
OYSTER (TIOSTREA CHILENSIS) DISTRIBUTION IN
FOVEAUX STRAIT, NEW ZEALAND. Jonathan A. Keogh,*

Portobello Marine Laboratory. University of Otago. P O Box 8,
Portobello, New Zealand; David J. Fletcher, Centre for the Ap-
plication of Statistics and Mathematics. University of Otago. P O
Box 56, Dunedin. New Zealand.

A stratified random stock assessment survey using a standard
double-sided oyster dredge was conducted over a three week pe-
riod in October of 1999 throughout the Foveaux Strait oyster fish-
ery in southern New Zealand. As this timing was co-incident with
the austral spawning season and since the oysters surveyed brood
their larvae up to the late pedi-veliger stage, legal sized oysters
(>58 mm shell length) from the 210 stations sampled were re-
tained, landed and opened so as to assess spawning oyster occur-
rence. For each station estimates were made of the number of
non-brooding oysters and the number of oysters bearing eyed or
non-eyed larvae, the brood size and oyster condition. The relation-
ship between the number of spawning oysters at each station and
oyster density and size distribution is reported on and the impli-
cations of this for larval fishing as a source of larvae for stock
enhancement is discussed.

UNCOVERING BENEFITS OF WEST INDIAN CROWN
CONCH OR CHIVITA' (MELONGENA MELONGENA) IN
YUCATAN, MEXICO. Michael D. Kaplowitz,* Department of
Resource Development, Michigan State University, East Lansing,
MI 48824.

Focus groups and individual interviews were conducted with
local resource beneficiaries as part of the design phase for an
economic valuation study of mangrove ecosystems of Yucatan.
Mexico. The research examined how local resource beneficiaries
use. perceive of and understand the ecological services associated
with their shared mangrove ecosystem. The data revealed that
collection of West Indian Conch (Melongeiui meloii^eiui) locally
called chivita has become an increasing part of the communities"
economic activity. Surprisingly, chivita collection appears to have
replaced other resource-based subsistence strategies in these com-
munities. These findings place Mel()n)>eiui mcloni>cna in a new
light since heretofore both marine re.source beneficiaries and shell-
fish researchers have viewed this species as little more than a
predator species with little or no benefits. The reported research
also uses data collected from local beneficiaries to estimate eco-
nomic benefits of chivita collection to these communities. The
analysis demonstrates that the current extractive use benefits lo
these communities of Melongcini iiH'loiim'iici for subsistence and
commercial sale are significant. I-'urlhcniiore. the research reveals
conflicting managemcnl agendas lor ihc shared mangrove ecosys-
tem including some that threaten the contimicd viahlliiy of chiviia
collection.

CLAM FISHERY IN BRAZIL. Carla Medeiros y Araiijo,*
Universidade de Brasi'lia-IB-GEM; lara L. G. Brasileiro, Univer-
sidade de Brasilia — Centro de Desenvolvimento Sustentavel SAS
Qd.5. Bl.H. 2° andar. Brasilia-DF-Brasil.

A lot of bivalve species are easy to collect and are largely
consumed by coastal populations. Since 1920 some researchers
(Edmondson. 1920: Quayle, 1943, Eldridge et al. 1979) have been
worrying about this situation and nowadays sustainable develop-
ment politics is linking conservation and exploitation. Brown mus-
sel Perna perna cultures and clam Annmalocardia hrasiliana man-
agement fishery in South of Brazil are examples of new political
strategies. Dosinia concentiica. Liicimi pectiitata. Trachycurdiwn
mitricatum. Iphigenia hnisiliuna and Pnnhothacci pcctorina are
yet consumed without any fishery management control in north-
east and south regions of Brazil. The aim of Sustainable Devel-
opment Centre is lo obtain more data about fishermen communi-
ties in social, health and economical aspects. A number of speci-
mens were collected in Florianopolis (27°38'SE. 48°33'05"W)
(Santa Catarina) and Maragojipe (I2°33'S. 38°00'W) (Bahia) in
order to make preliminary studies. Both places show no controlled
fishery and few biological Information. But in Bahia. clam con-
sumption Is more Intensively allied to precarious commercial
structure and health control. Improved biological intbrmatlon
about the Brazilian clam fishery is needed. This will lead us to
better comprehension of the social situation of fishermen In our
commiinltv.

National Shellfisheries Association. Seattle. Washinpton

Abstracts. 2000 Annual Meetins. March 19-23. 2000 623

CHESAPEAKE BAY OYSTERS: TRENDS IN RELATIVE
ABUNDANCE AND BIOMASS. Carol McCollough. Stephen
J. Jordan,* and Mark L. Homer. Maryland Department of Natu-
ral Resources, Sarbanes Cooperative Oxford Laboratory. 904 S.
Morris St.. Oxford MD 21654.

Oyster populations are distributed patchily over more than
400.000 acres in Chesapeake Bay. so it is not feasible to assess
their absolute numbers or biomass. Traditionally, landings data,
with their inherent inaccuracies and biases, have been the only
consistent means of estimating trends. A long term monitoring
program in Maryland has recorded relative numbers and size dis-
tributions of oysters, along with other population and disease data
annually; 43 fixed sites have been monitored consistently since
1990. with many records from these sites available from earlier
years. In 1999. we obtained shell height measurements and dry
tissue weights from samples of 10 oysters from each site (selected
to represent the range of sizes present). By applying the resulting
length:weight equation to size-frequency data from earlier surveys,
we computed an index of relative biomass that varied from year to
year according to the relative abundance and size distribution of
the oyster populations. The index is useful for portraying trends
and tracking the performance of restoration efforts. It reflects in-
terannual \ariations in recruitment and growth, as well as mortal-
ity caused by the oyster parasites Haplosporidiwn nelsoni and
Perkinsiis maiinus.

MANAGEMENT OF THE CANADIAN PACIFIC COAST
PRAWN TRAP FISHERY: RECENT CHANGES, PRESENT
STATUS AND FUTURE OPTIONS. Jim Morrison, Fisheries
and Oceans Canada, 3225 Stephenson Point Road. Nanaimo. B.C.,
Cda.. V9T 1K3.

The spot prawn Pandahis pknyceros is the largest of seven
commercial pandalid shrimp species occurring in B.C. waters. In
1997 this was the sixth most valuable fishery on Canada's Pacific
coast, valued in excess of $30 M. (Cdn|. It is a competitive fishery
limited to 253 licence holders, with vessel length limits, gear
specifications and size limits. Japan is the largest market for frozen
at sea product.

Recent significant changes in this fishery began with the adop-
tion of trap limits in 1995. followed by increases in effort due to
external factors as well as changes in fishing behaviour. This re-
sulted in recent record landings and record values while the fishing
season declined in the last 6 years from 230 days to 79 days.
Simultaneously, stock protection targets have been achieved due to
in-season biological sampling provided by 1 1 industry funded
charter patrol vessels and the fishery is being managed in a more
conservative manner than historically. However, fishermen have
expressed concerns for the fishery.

Future management options are described based on a discus-
sion paper written by elected industry representatives of the advi-
sory committee. A pilot program will be implemented in 2000 to

test and compare two management regimes, one with single gear
pulls per day intended to reduce catches of undersized prawns, and
one which would continue the present intense multiple haul fishing
pattern.

Planning for the 2001 fishery remains uncertain due to chang-
ing DFO policies regarding industry funding for fishery manage-
ment. Present funding arrangements will sunset in March 2001.
Options for "partnering" with industry for co-management and
co-fundina will be described.

MANAGEMENT OF THE DUNGENESE CRAB FISHERY
IN BRITISH COLUMBIA. Guy Parker, Fisheries and Oceans
Canada. 3225 Stephenson Point Road. Nanaimo, B.C, Canada,
V9T 1K3; Kim West, Fisheries and Oceans Canada, 610 Derwent
Way, New Westminster, B.C.. Canada, V3L 5B3: Ivan Winther,
Fisheries and Oceans Canada, 417-2'"' Ave West, Prince Rupert,
B.C., Canada, V8J 1G8,

Dungeness crabs are harvested extensively throughout British
Columbia by First Nations, recreational, and commercial fishers.
The commercial fishery began back in the 1880's, and has ex-
panded to become the seventh most valuable wild fishery in British
Columbia during 1997. worth an estimated $28.7 million. Land-
ings in 1997 reached approximately 3,000 tonnes, slightly lower
than the average annual landings of 4,000 tonnes that have been
commercially harvested during the 1990's.

Until the late 1980's much of the management and conserva-
tion measures within the crab fishery were passive, relying on a
minimum size limit for harvest, non-retention of females, and a
few small time and area closures. Increases in the number of
participants in the fishery and increases in over-all fishing effort
have led to the need for more active management, including li-
cence limitation, area licencing, harvest logbook requirements,
vessel trap limits, and an increasing number of time and area
closures.

SCIENCE AND MANAGEMENT OF GREEN SEA UR-
CHINS IN BRITISH COLUMBIA— A REBUILDING FISH-
ERY? R. Ian Perry,* Fisheries & Oceans Canada. Pacific Bio-
logical Station. Nanaimo. B.C. V9R 5K6. Canada; Guy Parker,
Fisheries & Oceans Canada. 3225 Stephenson Point Rd. Nanaimo.
B.C. V9T 1K3, Canada; Juanita Rogers, Fisheries and Oceans
Canada, 417 2"" Ave. W, Prince Rupert. B.C. V8J 108. Canada.
The green sea urchin {Strongylocentrotits droebachiensis) fish-
ery in British Columbia is a small but important component of the
province's dive fisheries. It has had a typical boom and bust profile
of a developing fishery, reaching peak landings of 978 tonnes
(value Cdn$4.5 million) in 1992, then declining to about 150
tonnes in 1995. These declines were driven in part by increasingly
restrictive management regulations. Since 1995. stock assessment
activities have been conducted annually, including surplus produc-

624 Ahstnicts. 2000 Annual Meetina. March 19-23. 2000

National Shellfisheries Association. Seattle. Washincton

tion estimates of sustainable yields and fishery-independent sur-
veys (conducted jointly with industry) of abundance, size compo-
sition, and recruitment. Resulting management actions included
restricting the fishery to two core fishing areas, minimum size
limits, and area and individual quotas. Since 1995. landings have
stabilized and the catch per unit of effort has been increasing. A
program of re-opening areas to fishing has been established which
requires surveys to "prove the resource'", which the industry has
keenly adopted and is actively pursuing. The green urchin fishery
in B.C. appears to be rebuilding, although not at the same rate in
both core areas.

MANAGEMENT OF THE RED SEA URCHIN FISHERY IN
BRITISH COLUMBIA. Juanita Rogers and Guy Parker, Fish-
eries and Oceans Canada, Pacific Region.

The Red Sea Urchin fishery began in 1971 and expanded
quickly until 1992 when landings reached nearly 13,000 tonnes.
Today, there are 110 personal licences eligible to fish Red Sea
Urchins annually, with a total commercial allowable catch of ap-
proximately 5.600 tonnes. The value of the fishery is susceptible to
the Japanese markets and economy, with the average annual price
fluctuating between $1,300 and $2,000 per tonne since the imple-
mentation of an individual quota (IQ) program in 1994.

Management in the red urchin fishery has become increasingly
more active. Current management measures include a minimum
size limit of 100 mm, licence limitation and area licensing, area
quotas calculated using a fixed exploitation rate of area biomass
estimates, individual licence quotas, catch validation and monitor-
ing, and fishery-independent biomass surveys.

The Pacific Urchin Harvesters Association (PUHA) represents
all licence holders, is responsible for developing the catch valida-
tion program, and is a major contributor to research surveys, both
through direct funding and in-kind support.

COUPLING A COMPUTATIONAL FLUID DYNAMIC
MODEL WITH A HABITAT SUITABILITY INDEX
MODEL TO MANAGE EASTERN OYSTERS IN MOBILE
BAY, ALABAMA. Leonard J. Rodgers and David B. Rouse,

Department of Fisheries and Allied Aquaculturcs. Auburn L'niver-
sity. Auburn, AL 36849.

The need to quantify and qualify habitat is an important com-
ponent of resource management. Habitat suitability index (HSI)
models are used to spatially delineate essential habitat. When con-
sidering the implementation of an HSI for Eastern oysters (Cra.v-
sostrea viri^inica) in an estuarine environment, an investigator es-
timates highly dynamic spatial variables such as temperature, sa-
linity, and hydraulic tlux.

In general, extremely simplified methods of estimating spatial
variables are incorporated into HSI models of estuarine systems.
One example would be llic use of mean monthly ualer parameters

from a few fixed sites to characterize an estuary. The current study
uses a two dimensional computational fluid dynamic (CFD) model
to calculate a measure of central tendency and a variance for major
hydrodynamic variables of oyster habitat on a tessellated grid.
Spatial data from several sources were combined with the CFD to
determine suitability indices. Using the U.S. Fish and Wildlife
Service geometric mean method an HSI was calculated. Methods,
applications, implications and limitations of this modeling method
will be discussed.

THE PRICE OF SUSTAINABILITY IS ETERNAL VIGI-
LANCE. Scoresby A. Shepherd, South Australian Research and
Development Institute. PC Box 120 Henley Beach 5022. South
Australia.

The South Australian abalone fishery has been going for about
34 years. There have been .some declines, but the majority of
stocks of blacklip. Haliotis rubra, and greenlip H. laevigata, have
been sustained under fishing. The input and output measures used
to control fishing are described. However, much is to be learned
from the study of stocks which have declined. Three stocks of
greenlip abalone were monitored by survey up till the point of
collapse. They have provided remarkable insight into the effect of
fishing on population processes. Three consequences are set out
below.

1. Because divers fish for aggregations they are able to reduce
fertile gamete production much more that is indicated just by the
reduction in density. This is the Allee effect.

2. Abalone metapopulations are spatially structured and often
occupy habitats that are differentially vulnerable to fishing. Stock-
recruitment curves for vulnerable and resilient habitats within a
metapopulation indicate that higher densities must be retained in
the former than in the latter habitats to avoid spatial contraction
(and ultimately collapse) of the stock.

3. At a larger spatial scale, small metapopulations are more
vulnerable to overfishing that larger ones. The first implication
from this is that serial depletion is certain unless management
measiues are tailored to ensure the persistence of the most vulner-
able populations. Second, if even mild o\erfishing of large popu-
lations occurs, they will slide downhill as producti\ ity declines and
become more vulnerable to overfishing.

These consequences indicate that spatial measures such as roll-
ing closures and refugia are valuable for management of multiple
sedentary stocks with limited larval dispersal in addition to other
traditional measures such as quotas, and size limits.

Given the usual fuzzy understanding of the status of individual
abalone stocks the use of an ensemble of fishery indicators in a
"trouble spot thermostat" approach (inspired by Caddy 1999) is
proposed. As the temperature rises in a stock and indicators are
sequentially triggered increasingly severe management responses
arc generated, until at the extreme the area is closed. A system
dexised lor the South .Xustralian abalone fisherv is outlined.

National Shellfisheries Association. Seattle. Washinslon

Abstracts. 2000 Annual Meetina. March 19-23. 2000 625

MANAGEMENT OF THE PACIFIC RAZOR CLAM AND
THE CURRENT MARINE TOXIN THREAT IN WASHING-
TON STATE. Doug Simons and Dan L. Ayres, Washington
State Department of Fish and Wildlife. 48 Devonshire Rd.. Mon-
tesano. WA 9S563.

The Pacitlc razor clam Siliqiia patiila. (Dixon) has been closely
managed and monitored in Washington State since the early
1900's. What began as a large and important commercial fishery,
evohed into a major recreational shellfish fishery. Upwards of 15
million clams were harvested in the recreational fishery during the
peak years in the late 70"s by almost one million digger trips. This
magnitude of usage prompted Washington State to manage the
resource in a continually increasing conservative manner. Seasons
were shortened and limits lowered repeatedly when population and
catch levels continued to decline. Since the mid 70" s. the resource
has gone through more major turmoil than all the years leading up
to then. In the late 70's massive wastage of small clams occurred
due to a disproportionate recruitment in the population. In the 80' s,
a specie specific disease caused the loss of over 90% of all razor
clams in Washington State. In 1991. a new marine toxin was
discovered that totally changed the way razor clams are now man-
aged and in 1993. coastal Indian Tribes began exercising their
Federally recognized treaty rights to harvest razor clams off res-
ervation. This paper briefly summarizes the major transitions in
management with the focus on the current influence of marine
toxins and tribal co-management.

Sound. A tagging study, at one location, has been completed to
identify methods to empirically verify the yield model mortality
parameter. Poaching and high-grading have led to under-reporting,
overharvest. and wastage of the geoduck clam resource.

MANAGEMENT OF GEODUCK CLAMS (PANOPEA
ABRUPTA) IN WASHINGTON STATE. Bob Sizemore,*

Washington Department of Fish and Wildlife. Point Whitney
Shellfish Laboratory. Brinnon. WA 98320.

Washington's commercial geoduck clam fishery began in 1970.
Annual subtidal SCUBA dive surveys began in 1967. Population
size is based on surveyed harvest tracts located between the -5.5
meter (MLLW) and -21.3 meter water depth contours. Annual
landings peaked at 3901 t in 1977, followed by a restricted total
allowable catch (TAG) of 2268 t from 1979 to 1988, 1497 t from
1989 to 1995, and a range of 1678 t to 1950 t from 1996 through
1999. The average annual ex-vessel value of the commercial geo-
duck fishery in Washington state is US$14 million for years 1990
to 1998. Prior to 1997, geoduck yield for the commercial subtidal
fishery was based on a Ricker yield per recruit model. In 1997,
managers adopted a deterministic, age-structured equilibrium yield
model and recommended a TAC of 2.7% of the commercially
available biomass in six management regions. The rate of recovery
on harvested tracts is empirically evaluated by a series of post-
harvest SCUBA surveys on 15 tracts scattered throughout Puget

RESEARCH ACTIVITIES IN SUPPORT OF ALASKAN
CRAB FISHERIES. Bradley G. Stevens, National marine Fish-
eries Service, Kodiak Fisheries Research Center, 301 Research Ct.
Kodiak, Alaska, 99615.

Both the National Marine Fisheries Service (NMFS) and the
Alaska Dept. of Fish and Game (ADF&G) conduct research in
support of Alaskan crab fisheries. Research activities fall into four
general categories: ( 1 ) Studies to improve survey methodology
include estimating catch efficiency of survey trawls, effects of
variable tow length, adaptive sampling, and alternative techniques
such as video and laser scanning devices. (2) Maintenance of
healthy stocks and reproductive capacity requires research on mat-
ing behavior, size at maturity, fecundity, terminal molt, mortality
factors, shell aging, and genetic stock discrimination. (3) Under-
standing and preventing harmful effects of fishing requires studies
of pot loss, ghost fishing, discard mortality, escapement behavior
and devices to allow escapement, pot design and functionality, and
improvement of species recognition. (4) Additional research is
targeted towards understanding basic biology of crabs and long
term recruitment trends, and includes studies on reproductive de-
velopment, hatching behavior, culture techniques and larval sur-
vival, settlement and habitat use, endocrinology, studies of aggre-
gation and burial behavior, tagging, and environmental effects on
reproduction and recruitment.

DUNGENESS CRAB (CANCER MAGISTER) MANAGE-
MENT IN PUGET SOUND, WASHINGTON. Derrick R.
Toba,* The Tulalip Tribes. Tulalip Shellfish Program, 7615 To-
tem Beach Road. Marysville, WA 98271.

Dungeness crab ( Cancer magister) is one of the most important
commercial and recreational shellfish species in Washington State.
In 1994. the federal district court ruled that the Western Washing-
ton Treaty Tribes reserved the right to harvest shellfish under the
Treaties. Following prior rulings by Judge Boldt in 1974, the
Tribes had a right to harvest up to 50% of the sustainable harvest
biomass of shellfish in their usual and accustomed fishing areas.
As a result of this decision, the crab resource is cooperatively
managed by the State of Washington and affected Treaty Tribes in
a given region. The management of Dungeness crab for the Puget
Sound and Strait of Georgia area is divided into six management
areas.

626 Abstracts. 2000 Annual Meeting, March 19-23, 2000

National Shellfisheries Association. Seattle. Washington

The management of Dungeness crab is similar throughout the
West Coast of North America. One of the current management
goals is geared toward protecting soft-crab and is based on his-
torical studies that were conducted by Washington Department of
Fisheries. The State of Washington has traditionally used June 1
through July 15 as a time period to close its fishery to protect
soft-shell crab. Since the data was collected primarily from the
northern portion of Puget Sound, this closure did not fit all man-
agement areas.

Additional biological information is being collected in various
regions to determine deviations from the traditional closure period.
This includes crab hardness tests for legal sized male crab, con-
dition of females and sub-legal male crab. In addition, other indi-
cators may be used to determine the timing of the molt and closure
of the fishery. The studies indicate that there are variations within
regions and that crab management needs to conform with the bio-
logical requirements of the region.

CALIFORNIA ABALONE FISHERIES: WHAT WE'VE
LEARNED AND WHERE WE GO FROM HERE. Mia J.

Tegner, Scripps Institution of Oceanography. University of Cali-
fornia, San Diego, La Jolla, CA 92093-0201.

California once supported fisheries for five species of abalones
(Haliotis spp.). Found in predictable and accessible locations near
stands of their algal food, these animals exhibit irregular recruit-
ment, grow slowly, and, as adults, move very little. They are
preyed on by sea otters and a host of other predators; fishable stock
sizes resulted from the extirpation of otters. Human fishing, which
dates to 9,775 years bp, has an extensive history. Modern Southern
California fisheries began in the 1940s, remained high until the late
1960s, and then began a precipitous decline; all commercial and
sport fishing south of San Francisco was halted in 1997. Limited
entry to the commercial fishery and experimental enhancement
programs instituted in the 1970s failed to stem the decline. Man-
agement was based largely on size limits that should have allowed
adequate egg production; stock assessments were rare and envi-
ronmental variability was not taken into account. The importance
of maintaining patches of spawners at densities adequate to sup-
port fertilization was learned only after many local populations
collapsed to densities so low that remaining animals are function-
ally sterile. High economic value driving intense search efforts, the
sea urchin fishery, gear improvements. El Nino events affecting
feeding and reproduction, and abalonc disease all contributed to
the decline. Today, white (A/, sorenseni) and black (//. crachero-
(lii) abalones arc under evaluation for the Endangered Species List.
In contrast with Southern California, the coast north of San Fran-
cisco has been reserved for sport fishing since 1945 and gear
restrictions prohibit the use of compressed air. This fishery has
been sustained at high levels despite extensive poaching pressure;
the lie facto depth reserve has apparently maintained brood stocks
and recruilment. Recoverv efforts in Southern California arc aimed

at aggregating rare species into enforced closed areas to protect
brood stocks and allow for natural reproduction. The conundrum
of sea otters, now foraging below Point Conception, remains to be
resolved.

BIOLOGY AND MANAGEMENT OF EASTERN BERING
SEA KING AND TANNER CRAB FISHERIES. Donn A.
Tracy, Alaska Department of Fish and Game, Commercial Fish-
eries Division. Kodiak, AK 99615; Brad Stevens, National Ma-
rine Fisheries Service, Kodiak Fisheries Research Center. Kodiak,
AK 99615.

Commercial king and Tanner crab fisheries of the Eastern Ber-
ing Sea are regulated under the joint jurisdiction of the National
Marine Fisheries Service and the Alaska Department of Fish and
Game. Fishery management goals are accomplished by incorpo-
rating the 3-S principle (size-sex-season) into harvest strategies
based upon estimates of mature biomass, exploitation rates applied
to mature males and minimum abundance thresholds. Stock as-
sessment methods include trawl surveys from which area-swept
estimates of abundance are derived (in some fisheries length-
based-assessment models are applied to annual area swept esti-
mates to minimize survey measurement error). Adoption of harvest
strategies into fishery regulations create a foundation for imple-
mentation of shorter term management measures (e.g., pot limits,
reporting requirements) necessary to attain overall conservation
objectives. Application of state and federal management policy
(including remedial plans for jeopardized stocks) are illustrated by
examining two high profile fisheries: Bristol Bay red king crab and
EBS Tanner crab.

LARVAL RECRUITMENT OF MYA ARENARIA (SOFT-
SHELL CLAMS) IN EASTERN AND SOUTHERN MAINE.
Tracy Vassiliev* and William Congleton, Department of Bio-
systems Science and Engineering. University of Maine. Orono.
ME 04469; Brian Beat. University of Maine Machias, Machias,
ME 04654; Stephen Fegley, Maine Maritime Academy, Castine,
ME 04420.

Clam landings have dramatically decreased in Eastern Maine
since 1982. Densities of 0-year M aremiria recruits were sampled
in two Maine counties. Washington County (Downeast) and Cum-
berland County (Southwest), by taking intertidal core samples
(0.0133 m-) over three years (1996-1999). Each year Wa.shington
County had significantly fewer M. aremiria recruits than Cumber-
land County (P < 0.001). To determine whether this recruitment
difference was due to lack of M. areiiaria larvae in near-shore
waters or post-settlement mortality, spat bags filled \\ ith monolila-
ment were placed in Mason Bay (Eastern Maine! and the Scarbor-
ough Riser (Southern Maine), during the summer of 1998 (May-
October). The spat bags were replaced monthly and contents
sicscti through a 750 p. screen. The Scaihorough River had sig-

National Shellfisheiies Association. Seattle. Wasliinston

Ahstracrs. 2000 Annual Meeting, March 19-23. 2000 627

nificantly more M. arenaria per spat bag during the mid summer
months (P < 0.001) suggesting Eastern Maine's lack of M.
cireiiiiriti recruits is due to reduced densities of lar\ae in near-shore
waters rather than post-settlement mortality or location rejection.
These results indicate increased juvenile recruitment could en-
hance the soft-shell clam fishery in Eastern Maine.

BUT THE CRABS KEEP COMING: TRIALS AND SUC-
CESSES OF THE GRAYS HARBOR DUNGENESS CRAB
MITIGATION PROGRAM. Lauran Cole Warner.* Seattle
District Corps of Engineers. PC Box 3755, Seattle, WA 98124;
Eileen P. Visser, 14 Spring Street, Potsdam, NY 13676.

The Corps of Engineers built and maintains a navigation chan-
nel from the mouth of the Grays Harbor estuary up to ports near
Aberdeen, Washington. That navigation channel was widened and
deepened beginning in 1990 so that bigger ships could call these
ports. Environmental studies leading up to this project found that
Dungeness crabs. Cancer magister, are entrained and killed during
dredging. Mitigation for this loss consists of placing aged oyster
shell on the intertidal mud flats of Grays Harbor, providing habitat
for young-of-the-year C magister that otherwise would not sur-
vive. Since the first test plots were built in 1990, over 60 hectares
of oyster shell have been placed in the Grays Harbor intertidal
area, producing over 12 million juvenile crab. But there have been
many obstacles along the way: oyster shell disappeared under the
mud much more quickly than optimistic planners had hoped for;
green shore crab (Heinigrapsus oregonensis) overtook plots after
the first year, staying for the winter and apparently outcompeting
settling Dungeness the following summer. In addition, crab im-
pacts continued to pile up as the channel was maintained each year,
and the mitigation program fell deeply into debt. Changes in both
the mitigation and dredging programs have since been made to
both reduce impacts and increase the efficiency of mitigation.

STOCK ASSESSMENT OF SURFCLAMS ALONG THE
EAST COAST OF THE UNITED STATES: THE IMPOR-
TANCE OF ESTIMATING DREDGE EFFICIENCY. James
Weinberg, Paul Rago, Charles Keith, Lisa Hendrickson, and
Steve Murawski, NMFS. Woods Hole, MA 02543; Eric Powell.
Haskin Shellfish Lab., Rutgers University, Port Norris, NJ 08349;
Roger Mann. College of William and Mary, VIMS, Gloucester
Pnt., VA 23062; Chris Weidman, WHOl, Woods Hole, MA
02543.

The National Marine Fisheries Service (NMFS) has conducted
surveys of Atlantic surfclam (Spisula solidissima) populations
along the USA Atlantic coast since 1980 with a standardized hy-
draulic clam dredge. To estimate the absolute abundance of clams
in an area for stock assessment, the efficiency, E. (i.e.. probability

of clam capture given encounter with the gear) of the survey
dredge must be known. To compute E for the dredge on the NMFS
research vessel (R/V Delaware II), we performed five field experi-
ments in 1997 with the RN Delaware II. and three commercial
clam vessels. We compared a swept area catch estimate from the
RA^ Delaware II with density estimates from depletion experi-
ments conducted by commercial vessels at the same sites. The data
were analyzed using an extension of the traditional theory for
analyzing depletion experiments. For each tow in the depletion
experiment the expected catch was modeled as a chain binomial
process whose parameters are a function of catches in previous
tows at that site. The model also takes into account the degree of
spatial overlap among tows. Estimates of E for the NMFS survey
dredge ranged from 0.23-0.46. Results from these experiments
were used in recent surfclam stock assessments. Additional ex-
periments are being planned because E is likely to vary with depth,
sediment type and sea state.

MHACS: MARINE HABITAT ACOUSTIC CHARACTER-
IZATION SYSTEMS. A PROGRAM FOR THE ACQUISI-
TION AND INTERPRETATION OF DIGITAL ACOUSTICS
TO CHARACTERIZE MARINE HABITAT. Charles A. Wil-
son and Harry H. Roberts. Coastal Fisheries and Coastal Studies
Institutes. Department of Oceanography and Coastal Sciences,
CCEER; John Supan. Office of Sea Grant Development, Louisi-
ana State University Baton Rouge, LA 70803.

Coastal Louisiana, like many deltaic land-masses, faces con-
tinued landscape alteration from natural processes and anthropo-
genic impacts that affect fisheries production. Many steps are be-
ing taken at both State and Federal levels to slow/mitigate these
changes. Most promising of these strategies is river diversions,
which introduce freshwater and sediment to river-flanking envi-
ronments (lakes, bays, and associated marshlands). Two such di-
version projects, planned by Louisiana Department of Wildlife and
Fisheries and U.S Army Corps of Engineers (Caenarvon and Davis
Pond), are designed to nourish marshes with water and sediment as
well as to help establish ideal isohalines over historic oyster
grounds. Critical to the success of these programs is a rapid and
accurate means to qualify and quantify changes in marine habitat
in the Barataria Basin. Digital high resolution acousdc instrumen-
tation linked to state-of-the-art data acquisition and processing
software is available for building a baseline of information that can
be used for evaluating future changes in shallow and shelf water
bottoms with special emphasis on fisheries habitat.

Application of dual beam hydroacoustics (120 khZ). digital
side-scan sonar (100 and 500 kHz), a broad-spectrum sub-bottom
profiler (4-24 kHz) for rapidly acquiring water column, surflcial
and shallow subsurface data has now been accomplished. These
data sets, "calibrated" with trawling, surface sampling, coring, and

628 Abstracis. 2000 Annual Meeting, March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

other "ground-truthing" techniques, have enormous potential for
understanding (a) distributions of bottom sediment types (includ-
ing man-made objects), (b) locations of oyster reefs and distribu-
tions of scattered oyster clumps and shells, (c) fisheries habitats,
(d) areas of active sedimentation and erosion, and (e) shallow
subsurface configurations that influence surface conditions. In our
most recent study, geo-referenced side-scan sonar mosaics of sur-
veyed oyster lease areas were incorporated into a GIS database.
Using image-processing techniques to analyze mosaic reflectance
patterns, we estimated the percent and total acreage of several
bottom types. Results were calibrated with field collected ground
truth measurements.

MANAGING SHELLFISH CULTURE
IN COASTAL WATERS

ENDANGERED SPECIES ACT AND SUSTAINABLE FISH-
ERIES ACT IMPLICATIONS FOR MOLLUSCAN SHELL-
FISH CULTURE MANAGEMENT. William F. Dewey, Taylor
Shellfish Company. Inc., 130 SE Lynch Road. Shelton. WA
98584.

The Endangered Species Act and the Sustainable Fisheries Act
are two federal laws with broad reaching powers. These Acts with
their sweeping ability to protect .species and critical or essential
habitat required by the species at all costs, have the potential to
drastically alter estuarine management strategies with little regard
for the economic impact on the shellfish culture industry. Trig-
gered by the federal nexus of Army Corp permits or by receiving
federal research dollars, growers find themselves facing a whole
new layer of intimidating federal bureaucracy never before expe-
rienced. Several West Coast estuaries now have wild runs of
salmon listed as threatened or endangered under the Endangered
Species Act. Eleven species of Puget Sound bottom fish are cur-
rently being considered by the National Marine Fisheries Service
for listing. Section 9 of the ESA prohibits "take" of threatened or
endangered species. A "take" not only constitutes killing the spe-
cies but includes any activity that hurts or harms any aspect of the
species' lifecycle. including damaging critical liat>itat. Individuals
found guilty of a "take" under ESA are subject to fines and/or
pri.son.

The Sustainable Fisheries Act of 1996 reauthorized the Mag-
nusen-Stevens Fisheries Conservation and Management Act. The
Act requires regional management councils to identify essential
fish habitat (EFH) for the species in the exclusive economic /one
(EEZ) and to develop conservation and enhancement measures lo
protect that EFH.

Shellfish growers conduct a variety of activities in the day lo
day operations of their farms, with the potential to adversely im-
pact salmon or bottom fish criiicdl or cssciulal lidlnrm. Proacli\ cly

the Pacific Coast Shellfish Growers Association is developing an
environmental code of practice, pursuing research to better under-
stand the interactions with protected fish species and discussing
with federal and state resource management agencies the best ap-
proach to achieve some level of regulatory stability under these
federal acts.

RESOURCE MANAGEMENT ISSUES FACING SHELL-
FISH AQUACULTURE ON THE MID-ATLANTIC COAST.
Mark W. Luckenbach, Virginia Institute of Marine Science. Col-
lege of William and Mary, Wachapreague, VA 23480.

Bivalve aquaculture. particulariy that of hard clams Mercenaria
mercenaria, has expanded rapidly along the mid- and south-
Atlandc coasts of the United States over the past several decades.
Though generally perceived as an "environmentally-friendly" and
sustainable use of near-shore coastal waters, neither the ecological
interactions nor the resource management implications of this use
of near shore habitats have received much consideration in this
region. In this presentation. 1(1) briefiy describe the techniques
used in shellfish aquaculture in this region and (2) provide an
overview of some of the research and management issues relevant
to its further development and expansion throughout the region.
Emphasis is placed on the need for research to understand eco-
logical interactions with aquaculture to promote its wise and sus-
tainable development. Understanding issues such as the role of
shellfish culture operations on nutrient cycling and food web dy-
namics, its interactions with submerged aquatic vegetation and its
impacts on fish habitat are fundamental to developing sound poli-
cies to guide its development. The inevitable conflict between
increasing populations in the coastal zone and a growing aquacul-
ture industry underscore the need for proactive strategies to pro-
mote the development of environmentally-sound, culturally ac-
ceptable aquaculture practices.

THE DEPARTMENT OF COMMERCE AQUACULTURE
PROGRAM— IMPLICATIONS AND OPPORTUNITIES
FOR SHELLFISH CULTURE. Ed Rhodes. NOAA Fisheries.
I31.'S East-West Highway. Silver Spring. MD 20910.

During the last year the Department of Commerce (DOC) has
made progress in advancing its aquaculture program through a new
Department aquaculture policy, the development of draft legisla-
tion for aquaculture in Federal waters, and by holding a stake-
holder workshop. The shellfish aquaculture industry is a key com-
ponent of the domestic production of marine species, and some
pieces of the Commerce program have relevancy to this industry.
The Commerce Aquaculture Policy became effective in August
and outlines implementation steps for the Department that will, in
cooperation \\ ith other go\cninicntal and non-go\ernmcnlal part-

National Shellfisheries Association. Seattle. Washington

Abstracts. 2000 Annual Meetina. March 19-23. 2000 629

ners. create a business climate favorable for environmentally
sound aquaculture development. The policy recognizes that it is
the role of goNcrnment to foster economic opportunities in aqua-
culture by providing a fair regulatory framework within which the
industry can operate, and to efficiently provide other government
services, including technology development, information and fi-
nancial, marketing and trade assistance. The Department has de-
\ eloped draft legislation that, if enacted, would authorize the Sec-
retary of Commerce to issue long term leases for aquaculture fa-
cilities in U.S. Federal waters, generally between three miles and
two hundred miles from seaward State boundaries. A significant
part of this legislation is the development in a timely fashion of
environmental standards for aquaculture operations thai would ap-
ply to lease holders in Federal waters. DOC held a stakeholder
workshop in late summer to obtain guidance for its aquaculture
program. Some of the results are especially relevant to the shellfish
aquaculture industry. The workshop also spawned some regional
focus groups that may lead to enhanced cooperation across species
groups.

THE DEVELOPMENT OF AN ENVIRONMENTAL MAN-
AGEMENT SYSTEM FOR THE BC SHELLFISH FARM-
ING INDUSTRY. Ruth Salmon. Executive Director. BC Shell-
fish Growers Association.

The BC Shellfish Growers Association (BCSGA) is proceeding
with the development and implementation of an Environmental
Management System (EMS 1 for the BC shellfish farming Industry.
The EMS will comprise of an Environmental Policy (EP) and
Codes of Practice (COP). The EP will set out the philosophy or
approach that the industry will take to address a range of environ-
mental issues and the COP is the tool through which that philoso-
phy will be implemented. The development of an EMS is a key
initiative for the development of EC's shellfish farming industry.
The goal of this project is to provide BC shellfish farmers and
processors with guidance for maintaining and protecting environ-
mental quality while impro\ina production and processing effi-
ciencies. The process will allow existing farmers to objectively
assess their internal operations for possible solutions towards the
pursuit of an ecologically sustainable farming operation. Shellfish
aquaculture is a marine-based industry that is affected by other
land users such as tourism, recreation, forestry, agriculture and
urban development. The effects of these industries as well as the
shellfish aquaculture industry's impact on them need to be exam-
ined in a comprehensive manner to create an effective EMS. In
developing an EMS. consultation with the community and other
users of the marine resource will be critical to enable legitimate
concerns and issues to be raised and solutions proposed. Shellfish
industries in other regions of Canada and other countries are also
taking a proactive role in developing policies and actions to ad-
dress environmental issues.

IMPACTS OF THE STEVENS TREATIES ON WESTERN
WASHINGTON TRIBAL SHELLFISH CULTURE. Derrick
R. Toba,* The Tulalip Tribes, Tulalip Shellfish Program. 7615
Totem Beach Road. Marysville. WA 98271.

Shellfish and fish have been an important resource to Western
Washington Indian tribes for thousands of years. This was re-
flected in the reservation of fishing rights in the Stevens Treaties
signed in 1854 & 1855. In 1994. the federal district court ruled that
the Western Washington Treaty Tribes reserved the right to har-
vest up to 50'7f of the sustainable harvest biomass of .shellfish in
their usual and accustomed fishing areas. However, the treaty also
contained a proviso, which excluded lands that were "staked and
cultivated". Following the canons of treaty interpretation. Judge
Rafeedie ruled that "staked and cultivated" followed the defini-
tions used by the shellfish industry at the time of the Treaty and by
what the Indians signing the Treaty would have understood. Judge
Rafeedie ruled that the shellfish industry could not "stake and
cultivate" naturally occurring shellfish beds. In addition, the State
of Washington sold tidelands to private individuals, which may or
may not be included in the proviso.

Certiarari was denied by the U.S. Supreme Court regarding the
shellfish case. However, the 9'^ Circuit Court of Appeals re-
manded several issues back to the lower court for clarification,
several of which impact shellfish aquaculture. The current status of
the case will be discussed.

In addition, the State of Washington, Treaty Tribes, and the
Federal Government signed a shellfish sanitation consent decree in
1994. which allowed for increasing tribal responsibilities in be-
coming a Shellfish Control Authority. Until Tribes have that full
capability, the tribes will work in conjunction with the State of
Washington, which applies federal regulations regarding shellfish
sanitation.

NUISANCE SPECIES

STATUS OF THE EUROPEAN GREEN CRAB INVASION
IN WASHINGTON COASTAL ESTUARIES: CAN EXPAN-
SION BE PREVENTED? Elizabeth M. Carr* and Brett R.
Dumbauld, Washington State Department of Fish and Wildlife,
P.O. Box 190, Ocean Park, WA 98640.

The European green crab Carciniis muenas was first found in
the San Francisco estuary on the West coast of North America in
1989 and has since spread as far north as British Columbia. What
is believed to be the result of a strong coast-wide recruitment event
in 1997 resulted in the discovery of this invader in Washington
coastal estuaries in 1998. Washington state has since responded by
establishing a statewide monitoring and control program for both
the invader and native crabs. Results from the monitoring program
in the coastal estuaries of Willapa Bay and Grays Harbor suggest

630 Abstracts. 2000 Annual Meeting. March 19-23, 2000

National Shellfisheries Association, Seattle, Washington

that green crab have either declined in abundance or spread out,
since average catch per unit effort (CPUE) from a location near the
mouth of Willapa Bay declined from 0.008-1 .04 crab/trap/hr in the
summer of 1998 to 0.002-0.006 crab/trap/hr in 1999. Crabs have
grown in size and ovigerous females were noted during the winter
and spring months. The presence of a new year class was also
noted in late summer 1999. but abundance of this year class also
appeared to be much lower than that of the previous year. In
general the crab prefers low salt marsh habitat during the summer
months, where it is often most abundant in cover provided by
Spartina alterniflora (another invader in Willapa Bay) or the na-
tive Triglochin maritimum, but does not come to traps and moves
to low intertidal and subtidal habitats during the winter. Control is
expected to be difficult due to a number of factors including lo-
gistics like the vast area to be covered, but also potentially declin-
ing interest from affected parties like the shellfish growers due to
the lack of observed effects on their product to date with crabs at
such low density. Nonetheless, it seems imperative that an effort
be made to at least reduce the population and therefore further
reduce the chance and success of what have already been shown to
be intermittent recruitment events. With the help of volunteers we
expect to initiate a broader scale control effort with traps in 2000
and are considering several ways to make this effort more effec-
tive.

PROGRESS IMPLEMENTING A PLAN TO MONITOR
FOR PRESENCE OF THE EUROPEAN GREEN CRAB
(CARCINUS MAENAS) IN PUGET SOUND, WASHINGTON.
Anita E. Cook* and Sandra Hanson, Washington State Depart-
ment of Fish and Wildlife (WDFW). Point Whitney Shellfish Lab.
Brinnon. WA 98320.

A significant population of the European green crab, whose
first persistent presence on the U.S. west coast was recorded in
1989 in San Francisco, was first noted in Washington State in 1998
in coastal Willapa Bay and Grays Harbor. The green crab likely
arrived in Washington via larval drift on ocean currents. To date no
European green crab have been confirmed in Puget Sound.

A large-scale Puget Sound green crab monitoring program was
established in 1999. with WDFW as the coordinating agency. The
primary aiin of this initial phase was thorough geographical sam-
pling coverage of Puget Sound (including the Strait of Juan de
Fuca and the San Juan Islands) to maximi/e the potential of de-
tecting any green crab that might have spread to Puget Sound by
larval transport or other means. This was accomplished by enlist-
ing and training various volunteers to set crayfish traps at moni-
toring sites spread throughout the Puget Sound. Over l.S groups
sampled more than 80 monitoring stations in 1999. Participants
included non-profit volunteer organizations, shellfish growers,
tribes, marine science centers, government agencies, scht)ols. and
the general public. In addition to providing information about the
potential presence of green crab in Puget Sound, ihc trapping

supplied some general baseline data about populations of small
native crab in the sampling areas. In the year 2000 WDFW will
focus on increasing the number of sample sites (for higher poten-
tial of discovering green crab presence), identifying sites with the
highest likelihood for introductions, and examining other green
crab detection techniques.

BIOLOGICAL AND ECOLOGICAL ASSESSMENTS OF
NUTTALLIA OBSCURATA IN NORTH PUGET SOUND.
Paul A. Dinnel, Shannon Point Marine Center. 1900 Shannon
Point Road, Anacortes, WA, 98221; Erika Yates, University of
North Carolina at Pembroke. NC.

The purple varnish, or mahogany clam, Nuttallia ohscwata. is
a recent arrival to northern regions of Puget Sound. This clam is
native to Japan. Korea, and China, and was probably introduced to
the Pacific Northwest in the late 1980"s via ballast water dis-
charged in the Vancouver. BC region. This species has spread
rapidly and may now be found as far south as Port Townsend. WA.
Lack of data for this species drove this study to assess its biologi-
cal and ecological characteristics in the North Puget Sound region
of Washington State. During this study, we measured length/
weight/width characteristics, size-frequency distribution, depth in
sediment, timing of post-larval recruitment, survival and growth of
the 1999 year class, and Nuttallia' s relationship to interstitial sa-
linity. We also assessed edibility and shelf-life, as this clam may
possibly be a future sport or fishery resource. During an initial
survey of ten beaches in the Padilla Bay region of North Puget
Sound, we found Nuttallia at only three locations; one each in
Padilla Bay, Fidalgo and Samish Bays at tidal elevations between
-1-0.6 to -1-1.5 m (MLLW). The habitat preference of this species
was clean sand or mixed sand/gravel. Most post-larval recruitment
appeared to take place in late winter or early spring, with growth
from 4 mm shell width in mid-June 1999 to about II mm by
mid-October. Adult sizes ranged up to about 70 mm shell width.
Densities of first-year clams in Samish Bay were about 250 clams/
m~ in mid-June and 1 10/m" in mid-October. Densities of adult
clams have been found to be as high as about 800 clams/m" in a
localized area in Fidalgo Bay. Edibility was judged to be excellent
for clams less than about 35 inm shell width. Edibility of larger
clams was compromised by a very "creamy texture," which was
probably due to gonad development. Initial tests to assess shelf life
indicated that Nuttallia could survi\e at least 30 days in a refrig-
erator at about 4 ' C.

BIOLOGICAL INVASIONS IN COASTAL WATERS. An-
drew N. Cohen, San Francisco Estuary Institute, 1325 South 46th
Street. Richmond. CA 94804.

C)\cr the past 20 years, a rapidly accumulating btidy of knowl-
edge has demonstrated that invasions by exotic organisms threaten
the aquatic flora and fauna in the wcirld's coastal regions and the

National Sliellfisheries Association. Seattle. Washington

Abslracls. 2000 Annual Mectinsi. March 19-23. 2000 631

human activities and economies that depend on them. Various
invasions have disrupted food webs, altered the physical structure
of ecosystems, decimated fisheries, damaged water supply sys-
tems, and driven aquaculture operations into bankruptcy. The ex-
tent of these invasions has been studied most intensively in the San
Francisco Bay/Delta Estuary, which hosts over 230 exotic species
including protists. plants and animals. Exotic species dominate
several habitats in this estuary, accounting for 40% to lOO'/r of the
common species and over 90% of the biomass in some habitats.
Furthermore, the rate of invasion has been increasing, from an
average rate of about one new species a year before 1 960. to nearly
four new species a year since 1960. Pathways for the introduction
of exotic aquatic species include ships" ballast water and hull
fouling, aquaculture activities, the aquarium and ornamental plant
trades, and the live bait and seafood trades. With the continuing
expansion of international trade there will be an ever-increasing
risk of introduction of exotic organisms — including parasites and
diseases of fish and shellfish and human parasites and diseases that
may be transferred through the consumption offish and shellfish —
unless stronger measures are adopted to manage these invasion
pathways.

PRO-ACTIVE MANAGEMENT OF INTRODUCED MA-
RINE PESTS: LESSONS FROM THE APPARENTLY SUC-
CESSFUL ERADICATION OF THE SABELLID WORM IN
CALIFORNIA. Carolynn S. Culver* and Armand M. Kuris,

Marine Science Institute and Ecology, Evolution and Marine Bi-
ology, University of California. Santa Barbara. CA 93106.

Although much effort has recently been devoted to prevention
of additional introductions of non-indigenous species, little, if any-
thing, has been done to eradicate or control those pests that are
already here. This lack of a pro-active stance towards established
invaders is, in part, due to the perception that once an invader has
become established, nothing can be done to reduce its associated
impacts. In addition, others take a "wait & see" attitude, where
substantial negative impacts must be shown before even consid-
ering development or implementation of eradication/control mea-
sures. Some recent experiences suggest that these defeatist atti-
tudes may be unwarranted and result in costly delays that allow
pest populations to increase and spread. Subsequently, the chance
for successful eradication/control is decreased, while the likeli-
hood for damage to the ecosystem and the costs associated with
management of the pest are increased. To minimize such negative
outcomes, a more pro-active management stance should be con-
sidered. The apparent eradication of an introduced population of
the South African sabellid worm pest in California offers insight
towards development and implementation of a successful, cost-
effective management program. We will discuss criteria for suc-
cessful management of invasive species and review the need and
potential for eradication/control of other introduced marine pests.

PREDATION BY EUROPEAN GREEN CRABS ON MA-
NILA CLAMS IN CENTRAL CALIFORNIA. Edwin Grosh-
olz and Paul Olin, Department of Environmental Science and
Policy. University of California. Davis. CA 95616 and University
of California Sea Grant Extension Program. 2604 Ventura Avenue.
Room 100, Santa Rosa, CA 95403.

One of the key concerns regarding the recent invasion of the
European green crab. Carciniis mcienas, is the potential impact of
this species upon invertebrate fisheries such as clams, oysters, and
mussels in western North America. To investigate the potential
impacts of green crabs upon the Manila clam ( Venenipis philip-
pinanim) fishery, we conducted a field experiment to determine
size-specific rates of predation by green crabs on Manila clams.
Using commercial growout bags provided by Hog Island Oyster
Company of Marshall, CA, we placed one green crab from one of
three sizes classes (30-40 mm. 50-65 mm, or >70 mm) into a
growout bag with fifteen Manila clams chosen from one of three
size classes (<23 mm, 25-33 mm, or >36 mm), the largest size
class being market size. Five replicate bags of each of the nine
treatments (three crab sizes by three clam sizes) were placed near
MLLW on July 13. 1999 and lightly covered with sediment. We
used the five replicates of the small green crab/large Manila clam
treatment as conservative controls. After two weeks, we collected
all bags and assessed the mortality of clams and crabs. We found
very low survival of Manila clams in treatments with large green
crabs. For the smallest Manila clams in treatments with large
crabs, nearly all clams were eaten with survivors remaining in only
one bag ( 15% overall). Small clams had moderately better survival
in treatments with medium (52%) and small green crabs (71%).
The medium size class of clams had poor survival in bags with
either large (36%) or medium sized green crabs (46%). The large
market size clams had 65% survival with both medium and large
green crabs over the two week period. In summary, our results
show that even newly recruiting juvenile green crabs can quickly
reduce the numbers of juvenile Manila clams, and even market size
clams are at risk from even intermediate size green crabs. There-
fore we conclude that green crab predation may represent a sig-
nificant threat to the commercial production of Manila clams.

THE IMPACT OF EUROPEAN GREEN CRABS IN CEN-
TRAL CALIFORNIA. Edwin Groshoiz, Department of Envi-
ronmental Science and Policy. University of California. Davis. CA
95616: Gregory Ruiz, Smithsonian Environmental Research Cen-
ter. P.O. Box 28. Edgewater. MD 21037.

The European green crab, Carcinus maenas. is one of the most
potentially serious recent introductions into the coastal waters of
western North America. In this study, we measured the impacts of
green crabs on a coastal marine food web in central California. We
found that this predator exerted strong "top-down" control and
significantly reduced the abundances of several of the 20 inverte-
brate species monitored over a nine-year period. Densities of na-

632 Absinicts. 2000 Annual Meetina. March 19-23, 2000

National Shellfisheries Association, Seattle, Washington

tive clams, Nutricola tantilla and Nutricola confusa. and native
shore crabs, Hemigrapsus oregonensis, declined fivefold to tenfold
within three years of the green crab invasion. Field and laboratory
experiments indicated predation by green crabs caused these de-
clines. In addition, we tested for indirect responses of invertebrates
and vertebrates to green crab predation. We found significant in-
creases in the abundances of two polychaete taxa, Lumbrineris sp.
and Exogene sp.. and tube-building tunaid crustaceans, Lepto-
clielia diihia. most likely due to the removal of co-occurring green
crab prey. However, we observed no significant changes in shore-
bird abundances (13 species) over a nine-year period suggesting
green crabs have had no "bottom-up" effect on shorebird popula-
tions, which subsist on benthic invertebrate prey. We predict that
such "bottom-up" control may occur as both the local effects and
the geographic range of green crabs increase.

POTENTIAL LIMITATIONS OF THE EUROPEAN GREEN
CRAB, CARCINVS MAENAS, IN HABITAT SUITABLE
FOR THE NATIVE RED ROCK CRAB, CANCER PRODUC-
TUS. Chris Hunt,* Environmental Science Department, Oregon
State University, Corvallis, OR 97331-2914.

Carcinus maenas. thought to have arrived in the Western Pa-
cific in San Francisco Bay in 1989, was discovered in Coos Bay,
Oregon in 1997. By the summer of 1998 C. maenas was discov-
ered in at least eight Oregon estuaries. Trapping observations dur-
ing the summer of 1998 suggested that low C. maenas abundance
occurred in areas that were either physiologically intolerant for C.
maenas, or areas occupied by large numbers of adult Cancer pro-
ductiis. the native Red Rock crab. An intensified trapping effort in
Yaquina Bay, Oregon, during the summer of 1999 was used to
document the distribution of the estuary's crab community. These
observations indicated that although C. maenas coexisted with
adult Dungeness crab. Cancer magisler. it was much more rare in
areas where the physiologically more sensitive adult C. proditctus
were abundant. These results are further supported by lab preda-
tion studies pairing adult and juvenile crabs of both C. maenas and
C. prodiictus. These two observational studies support the theory
that in habitat suitable for adult C. productits. the invasive C.
maenas may be severely restricted. This research was supported by
Oregon Sea Grant.

EAST MEETS WEST: COMPETITIVE INTERACTIONS
BETWEEN CREEN CRAB AND HEMICRAI'SUS SPP.
Gregory C. .Icnscn,* P. Sean McDonald, and David A. Ami-
strong, School of Fisheries 3.'i.'S()2(), University of Washington,
Seattle, WA 9819.";.

Juvenile green crab iCaninits maenas) rely on inlcnidal struc-
ture (i.e., rocks, shell) for shelter, and the recent introduction of
this species to the west coast ol North .America places lliciii in

potential competition for this resource with the abundant native
grapsid, Hemigrapsus oregonensis. Similarly, the recent arrival of
a Japanese species. H. sanguineus, on the east coast of North
America also suggests the possibility for competitive interactions.
The morphological and behavioral similarities of these two
grapsids and their likely interaction with juvenile Carcinus pro-
vides an interesting contrast, with Carcinus in the role of invader
on the west coast and as "resident" on the east coast, having been
established there for 150-1- years. We conducted fine-scale sam-
pling on both coasts, examining species distributions both under
rocks and in adjacent sediments. Only 20% of the juvenile Carci-
nus sampled were found under rocks in areas occupied by either
Hemigrapsus species, while north of the present distribution of H.
sanguineus >97% of the Carcinus were under rocks. In laboratory
trials examining competition for food or space between Carciims
and Hemigrapsus of equal carapace width, H. sanguineus was
overwhelmingly dominant, and H. oregonensis also dominated in
competition for space. These findings may have important impli-
cations both for the ultimate distribution and impact of Carcinus.
and also for possible use of grapsids for biocontrol in culture
systems.

THE POTENTIAL IMPACTS OF CARCINUS MAENAS IN-
TRODUCTION ON JUVENILE DUNGENESS CRAB, CAN-
CER MAGISTER. SURVIVAL. P. Sean McDonald,* Gregory
C. Jensen, and David A. Armstrong, School of Fisheries, Uni-
versity of Washington, Seattle, WA 98195.

The spread of the European green crab, Carcinus maenas. in
the northeast Pacific represents one of many invasive introductions
that are potentially devastating to aquaculture and shellfisheries.
Discovery of the species in Washington State coastal estuaries in
1998 precipitated debate as to the impact of the invasion on local
commercial and recreational Dungeness crab. Cancer magister.
harvests. These estuaries provide appropriate habitat for C mae-
nas. yet they are important nursery grounds for C. nuigisler. The
results of laboratory experiments and infrared video observations
show that juvenile C. maenas displace C. magister of equal size
from single shells in one-on-one competition. C. maenas also con-
sistently wins nocturnal foraging trials in which the species com-
pete for freshly killed clams. Laboratory and field enclosure ex-
periments indicate that juvenile C. /»«i,'/.v;<t emigrate froin refuge
habitat as a result of competition and predation by adult C. mae-
nas. Interactions with the dominant invasive species could have a
negative influence on juvenile C. magister survival and subsequent
recruitment to the fishery. However, the real impact of the C
maenas introduction will depend on the extent to which the two
species actually overlap, a condition which may be more limited
than previous!) Ihoiighl.

National Shellt'isheries Association. Seattle. Washinaton

Abstmcrs. 2000 Annual Meetina. Maah 19-23. 2000 6.33

THE STATUS OF AQUATIC NUISANCE SPECIES PRE-
VENTION IN WASHINGTON STATE. Pani Meacham.

Washington Department of Fish and Wildlife, 600 Capitol Way N..
Olympia.WA 98501.

Aquatic nuisance species pose a threat to the ecological integ-
rity of Washington's marine and freshwater resources, and have a
significant impact on economic, social, and public health condi-
tions in Washington State. We have learned from our experience
with spartina that these species can spread rapidly, and we must do
everything in our power to prevent new introductions and mini-
mize the impact of those already present.

The state hired a full time ANS Coordinator, estabhshed a
Zebra Mussel and Green Crab Task Force, and provided funding
for ANS programs. A State ANS Management Plan has been com-
pleted, and monitoring and control plans for green crab and zebra
mussels have been put in place. Presently there is a multi-agency
focus on educating the public on the role they can play in prevent-
ing the spread of ANS plants and animals. Two pieces of legisla-
tion have been drafted for the 2000 legislature. One bill, supported
by the shipping industry, is designed to protect Washington waters
from the introduction of non-native organisms and pathogens car-
ried in ballast water and other ship vectors. The other bill creates
an Aquatic Nuisance Coordinating Committee with the intent of
minimizing the environmental and economic risks of ANS by en-
hancing cooperation and coordination among the various state and
federal agencies responsible for controlling ANS. Through the
cooperative efforts of federal, state, and local government, indus-
try, and the public. Washington State is making a significant con-
tribution toward solving a global problem.

THE EUROPEAN GREEN CRAB BIVALVE CONSUMP-
TION RATES AND PREY PREFERENCES. Kelly C. Pala-

cios,* College of Oceanic and Atmospheric Sciences. 104 Ocean
Admin. Bldg.. Corvallis. OR 97331-5503; Steven P. Ferraro,
Coastal Ecology Branch. US EPA, Newport, OR 97365.

The European green crab, Carcinus maenas. a voracious bi-
valve predator, is a recent invader to Pacific Northwest estuaries.
The objectives of this study were to determine green crab con-
sumption rates and prey preferences using four bivalve species:
Yaquina oyster {Oslrea hirida). Manila clam (Tapes phiUinanim).
bent-nosed clam (Macoma nasuta), and Cryptomya clam (Cryp-
tomya califomica). Various bivalve size classes, ranging from the
smallest (10-14 mm) to the largest (33-37 mm), were tested. In
both the consumption and preference experiments (t| = 3 - 8), one
previously starved (48 hours) green crab (CW: 60-75 mm) was
placed in a 38 1 aquaria with 13 cm of .sediment and allowed to
feed ad libitum on bivalve prey for 16 hours. For each bivalve
species being tested. 60 individuals were offered at the beginning
of the experiment and not replaced. Differences in the mean con-
sumption rates were tested by ANOVA. The null hypothesis of no
prey preference was tested by a goodnes.s-of-fit (G-test) to an equal

proportion of prey consumed. Among the prey species tested, there
was no difference in the mean consumption rate for a given prey
size class, but green crabs exhibited strong prey preferences when
offered more than one prey species choice. The results suggest that
Yaquina oysters are at greater risk of green crab predation than
bent-nosed clams and Manila clams and Cryptomya clams are at
areater risk than bent-nosed clams.

ABUNDANCE OF SMALL PREDATORY GASTROPODS
WROSALPINX CINERA, EU PLEURA CAUDATA, RAP AN A
VENOSA) IN RELATION TO LOWER CHESAPEAKE BAY
OYSTER (CRASSOSTREA VIRGINICA) POPULATIONS.
Melissa J. Southworth,* Juliana M. Harding, and Roger Mann,
Department of Fisheries Science, Virginia Institute of Marine Sci-
ence. Gloucester Point, VA 23062.

Oysters in the Virginia portion of the Chesapeake Bay have
enjoyed a relative hiatus from oyster drill (Urosalpinx cinera.
Eupleura ccntdata) predation since Hurricane Agnes dramatically
reduced oyster drill abundance in 1972. In recent years, anecdotal
reports have indicated that oyster drill abundance has been increas-
ing in Virginia waters. Increased oyster drill abundance combined
with the recent discovery of a third predatory gastropod. Veined
Rapa whelk (Rapana venosa), in the lower Chesapeake Bay, has
potentially significant consequences for the commercial oyster
fishery as well as Virginia's ongoing oyster restoration efforts.
Quantitative estimates of the abundance and distribution of small
predatory gastropods in relation to existing oyster resources were
made during Fall 1999 at >150 sites in 8 tributaries. Oyster drills
were present in <50% of sites sampled, juvenile Rapana venosa
were not observed. Both species of oyster drill were more abun-
dant in downriver habitats with salinities ranging from 15 to 25
ppt. Drill abundance ranged from 1 to 4 animals m^".

STATUS OF THE CHINESE MITTEN CRAB IN CALIFOR-
NIA. Tanya C. Veldhuizon,* California Department of Water
Resources. Environmental Services Office. Sacramento. CA
95816.

The catadromous Chinese mitten crab (Eriocheir sinensis) is
native to China and Korea and is also established in Europe and
California. First collected in south San Francisco Bay in 1992, E.
sinensis rapidly expanded in distribution and abundance. The cur-
rent distribution in California is the San Francisco Estuary and the
lower elevational reaches of the watershed. Based on the adverse
impacts of the crab in Germany, E. sinensis poses ecological,
economic, and health concerns in California. However, an assess-
ment of the degree of impact in California is required. In 1999. the
California Fish and Game Commission denied requests to com-
mercially exploit the crab. Reasons for denial ranged from poten-
tial acceleration of dispersal to increased management costs to
encouragement of future illegal introductions. Research and man-

634 Abstracts. 2000 Annual Meetina. March 19-23, 2000

National Shellfisheries Association, Seattle, Washington

agement of E. sinensis in California are facilitated through the
Interagency Ecological Program's (lEP) Chinese mitten crab Proj-
ect Work Team. For additional information, visit the lEP website
at <htlp://www.icp.ca.gov>.

MITIGATING EFFECTS OF NONINDIGENOUS MARINE
SPECIES: EVALUATION OF SELECTIVE HARVEST OF
THE EUROPEAN GREEN CRAB, CARCINVS MAENAS.
William C. Walton,* Smithsonian Environmental Research Cen-
ter, PC Box 28. Edgewater, MD 21037.

With the increasing need for management of nonindigenous
species in marine habitats, managers are considering available
mitigation methods to reduce the negative effects of established
exotic species. I briefly outline a menu of possible mitigation
methods, illustrated by management experience with the European
green crab, Carcinus niaenas: chemical control, biological control,
genetic manipulations, local physical barriers, altered maritime/
fishery practices, and selective harvest (trapping). Selective har-
vest, relative to the alternatives is generally perceived as incurring
the least negative side effects on resident species. Does selective
harvest, however, sufficiently reduce invader abundance to signifi-
cantly reduce their negative effects? As a case study, I explore the
efficacy of municipal selective harvest programs currently in use
on Martha's Vineyard, MA (USA). Current harvest programs, de-
spite considerable effort, do not appear to reduce the within- or
among-year abundance of C. maenas (verified by independent
censuses). Experimental tests of intensive, short-term trapping in
the shallow subtidal zone (every 48 hrs for 2 weeks) similarly led
to no decline. Habitat-specific trapping surveys and mark-
recapture .studies, however, suggest that populations within em-
bayments are relatively closed and therefore theoretically vulner-
able to within-year reductions in abundance given appropriate har-
vest levels. I tested this with an intensive one day trap down (6
hauls, \.5 hr immersion time) in a relatively small embayment
(-0.01 km") and observed significant declines in 1) green crab
abundance and 2) relative predation intensity on quahaug. Merce-
naria mercenaria. seed (13-17 mm shell length). The reduction in
relative predation intensity persisted for up to a month. Selective
harvest as currently practiced, therefore, does not appear to be
effective, but deserves further consideration as a possible mitiga-
tion method.

INTEGRATING BIOLOGICAL CONTROL IN THE INTE-
GRATED PEST MANAGEMENT PROGRAM FOR SPAR-
TINA ALTERNIFLORA IN WILLAPA BAY. Miranda
Wccker, Marine Program. Olympic Natural Resources Center.
University of Washington; Donald Strong, Center for Population
Biology; Fritzi Grevstad, Olympic Natural Resources Center.
University of Washington.

In 1995, the Washington Legislature unanimously declared the
spread of invasive exotic Spartina "an environmental disaster."
(RCW 17.26.()().'i). Extensive research supported the conclusion

that Spartina species are causing profound structural and, if not
controlled, in-eversible alterations to estuarine areas. Since 1993,
over $ 1 ,000,000 has been spent each biennium by state and federal
agencies on an "integrated pest management" (IPM) approach to
Spartina control. Still the pace of spread outstrips the rate of con-
trol. Ecologically .sound, effective, and affordable new control
techniques are needed to achieve the program's goals. Biological
control is considered the most promising new tool for Spartina
control in Willapa Bay, the site of the largest infestation. Green-
house studies carried out in the early 1990s demonstrated that
Willapa Spartina clones were severely stressed or killed by mod-
erate populations of Prokelisia marginata, a leafhopper common to
Spartina's home range (Daehler & Strong 1997).

Research was undertaken during the past two years to evaluate
the risks of releasing P. marginata in Washington state. Host speci-
ficity studies included choice, no-choice and preference trials dur-
ing which the most likely non-target hosts were exposed to P.
marginata. Tests were also conducted to determine whether P.
marginata serves as a vector for pathogens responsible for ob-
served mortality of Willapa Spartina. Preparations are underway
for release of the insects. Pre-release monitoring has begun and a
release strategy is being formulated. Project participants are also
testing new approaches to the transfer of scientific information
generated through research activities. The team will prepare so-
phisticated models that will allow participants to project and track
the spread and impacts of the insects. Using these planning tools,
state agency officials will be able to comprehensively target the
use of other control techniques and generate a more efficient over-
all plan. Extensive outreach activities have accompanied the sci-
entific studies in order to promote public understanding of the
project and a sophisticated appreciation of its findings. A project
website will permit the public and professional managers access to
accumulated information, project status reports and analytic prod-
ucts.

GROWTH OF THE 1997/1998 YEAR CLASS OF THE EU-
ROPEAN C;REEN CRAB. CARCINUS MAENAS, IN OR-
EGON ESTUARIES. Sylvia Behrens Yamada. Chris Hunt,*
and Alex Kalin, Zoology Department. Oregon State University,
Corvallis, OR 97331-2914.

During the summer of 1998. a new year class of Carcinus
maenas appeared in Oregon estuaries as well as in Huinboldl Bay.
CA to the south and Willapa Bay. WA. Grays Harbor. WA and
Vancouver Island. B.C. to the noilh. This coast-wide colonization
is correlated with an El Nirio event of unusually strong northward
moving coastal currents from September 1997 to spring of 199S.
Crabs from the 1997/98 year class grew quickly, averaging 14 mm
in carapace width in June. 27 mm in July and 47 mm in September
1998. Growth ceased during the winter, resumed in May 1999 and
hv Ihc end of their second summer. lhe\ averaued 70 mm.

National Shcllfisheries Association. Seattle, Washinaton

Abstracts. 2000 Annual Meeting. March 19-23. 2000 635

Female Carciniis maenas from the 1997/98 year class carried
eggs in November and December 1998. Sexual maturity in Oregon
populations is thus reached within one year, while in the North Sea
and Maine it may take two to three years. A comparison of molt
increments of crabs from Oregon, the North Sea and Maine indi-
cates that growth per molt is constant regardless of geographic
location. Carcinus maenas in Oregon therefore molt more fre-
quently than in the North Sea or in Maine. The trade-off for this
faster growth rate is a shorter life span. While the life span for C.
maenas is 3-6 years in Maine, it may only be around 3 years in
Oregon.

While C. maenas reproduced in Oregon estuaries during the
winter of 1998/1999. the resulting offspring is not replacing the
parental year class. Recruitment during the summer of 1999 was
late and sparse. If recruitment in the next few years is also low,
then the resident C. maenas populations in Oregon estuaries will
die out until another coast-wide colonization event occurs with the
next El Nifio. This research was supported by Oregon Sea Grant.

PHYTOPLANKTON HARMFUL TO
SHELLFISH AND CONSUMERS

exposure) within a juvenile population ranges from 72-96% in
areas with no toxin history (e.g.. Lawrencetown River Estuary. LE,
Nova Scotia, and Mount Sinai Harbor. Long Island, New York), to
5 15% in areas with a long-term history of toxic blooms (Lepreau
Basin, LB. Bay of Fundy, New Brunswick). Differences of more
than an order of magnitude in nerve sensitivity to STX were ob-
served among individuals from LB and LE populations. Most LE
clams exhibited marked reduction of the nerve action potential at
10"'' g STX/ml, and were fully blocked within 20 sec at lO'l In
contrast, most LB clams displayed no effect even at lO"*^ and
required 3-5 niin. of exposure to induce full nerve block at lO"'' g
STX/ml. The effects of duration of toxification and depuration
were also tested: neither variable affected the nerve sensitivity of
LB clams. There was an apparent decrease in the sensitivity of LE
clams which survived prolonged (15-day) toxification. compared
to those exposed for 4-6 days, but this effect might be attributed
to selective mortality of the most sensitive LE phenotypes over
time. Relative merits of the toxin sensitivity indices used are dis-
cussed. We will attempt to identify adaptive mechanisms to toxins
at the biochemical and molecular level, by determining the pres-
ence of soluble toxin-binding saxiphilins in various clam tissues,
or genes encoding for these proteins, as well as by DNA sequenc-
ing of sodium channel STX receptor sites.

DOES THE HISTORY OF TOXIN EXPOSURE INFLU-
ENCE BIVALVE POPULATION RESPONSES TO PSP
TOXINS IN MYA ARENARIA?: I) BURROWING AND
NERVE RESPONSES. V. Monica Bricelj,* Institute for Marine
Biosciences. National Research Council (NRC). 1411 Oxford St..
Halifax. NS B3H 3Z1, Canada; Betty M. Twarog, Darling Marine
Center. Univ. of Maine. Walpole. ME 04573. USA; Scott P. Mac-
Quarrie and Pamela Chang, NRC. Halifax, and Vera L.
Trainer, Northwest Fisheries Science Center. Seattle. WA 981 12-
2097. USA.

Our ECOHAB (National Program on the Ecology and Ocean-
ography of Harmful Algal Blooms) study examines the magnitude
and causes of intraspecific variation in sensitivity to paralytic
shellfish poisoning (PSP) neurotoxins, and thus capacity for toxin
accumulation, in North American populations of softshell clams.
Mya arenaria. Our results suggest that Mya populations recur-
rently affected by toxic blooms may experience genetic or epige-
netic adaptation to PSP toxins via natural selection of more resis-
tant individuals. Individual sensitivity is here measured by inhibi-
tion of clam burrowing response after laboratory exposure to a
highly toxic dinoflagellate. Alexandrium tamarense (strain
PR 18b), and in vitro block of the action potential in isolated nerves
exposed to saxitoxin (STX). Burrowing inhibition was not induced
by a non-toxic strain of A. tamarense. The percentage of sensitive
clams (as determined by the burrowing index after 24 hrs. of toxin

HARMFUL ALGAL BLOOMS AND SHELLFISH TOXIC-
ITY IN WASHINGTON STATE. Rita A. Horner,* School of
Oceanography. Box 357940. University of Washington. Seattle,
WA 98195-7940; Frank H. Cox and Linda D. Hanson, Wash-
ington Department of Health/Shellfish Programs, P.O. Box 47824.
Olympia. WA 98304-7824.

Harmful algal blooms (HABs) and the toxins they produce are
an increasing threat to human health and fisheries resources around
the world. In western Washington marine waters, fewer than 20
phytoplankton species may produce marine toxins and are frequent
members of the phytoplankton community. Both the phytoplank-
ton species and the toxins they produce are most common from
April through October, but occur in all months of the year. Blooms
last a few days or several months, while the toxins in shellfish
usually last for weeks to months. Potentially harmful species may
be present, but produce little or no toxin or. conversely, only a few
cells may produce high levels of toxin. Environmental factors that
control the presence of harmful species and toxin production are
not well-known here and vary with the algal species, locality,
season, and year; population dynamics are poorly understood. The
current management tool is to close a fishery or area if toxins are
present in a product which means that broad geographic areas and
all shellfish species are involved when possibly only a small area
or a few species are affected.

636 Abstracts. 2000 Annual Meetine. March 19-23. 2000

National Shellfisheries Association, Seattle, Washington

IMPACT OF HARMFUL DINOFLAGELLATE HETERO-
CAPSA CIRCULARISQUAMA ON SHELLFISH AQUACUL-
TURE IN JAPAN. Yukihiko Matsuyama* and Takuji Uchida,

National Research Institute of Fisheries and Environment of Inland
Sea. Ohno. Hiroshima 739-0452, Japan; Tsuneo Honjo, Faculty of
Agriculture, Kyushu University, Hakozaki, Fukuoka 812-8581, Ja-
pan.

The novel dinotlagellate Heteiocapsa ciixukirisquama Horigu-
chi has been the causal agent of red tide on the Japanese coast
since 1988. The red tide due to H. circukirisqiuuna has destroyed
the shellfish aquaculture industries around the western part of Ja-
pan. Until 1998, 26 cases of//, circidarisquama red tide (including
15 incidences leading to fisheries damage) had been recorded in 14
locations of Vk'estern Japan. The red tide due to H. ciixularisqiiama
was associated with massive killing of commercially important
bivalve species: short-necked clam R philippinanmh Pacific oyster
Crassostrea gigas, pearl oyster Piiutciila fhcala. blue mussel Myti-
lus galloprovincialis edulis. etc. Economic losses of shellfish
aquaculture by direct killing of marketable products were esti-
mated about at least 10 billion-yen in the last decade The labora-
tory experiments demonstrated that H. circularisquama reduces
the clearance rate of bivalves at the density of 2-104 cells/I. and
kills them at 5-106 cells/1 in association with vigorous "clapping",
retraction of mantles and gills, valve closure, and alternation of
cardiac activities. Although the toxicity of H. circulah squama to
bivalves and gastropods is extraordinary, any fish killing, toxin
accumulation, and subsequent human illness have not been ob-
served during blooms of this species.

METHODS FOR DETECTING MARINE TOXINS. James
Hungerf'ord,* Ronald M. Manger, Sue Lee, Linda Leja,
Charles Kaysner, and Marleen Wekell, Seafood Products Re-
search Center, Pacific Regional Laboratory Northwest, USFDA,
Bothell, WA.

Detection of marine toxins is a crucial aspect of seafood safety.
Animal bioassays have for years been the mainstay of many moni-
toring programs. Alternative detection methods are now being em-
phasized, as replacement of animal bioassays is a goal for many
health agencies. Maintaining the present level of protection with
new detection methods and without detailed risk assessment data
implies the need to duplicate or at least parallel the observed
animal assay response. This complicates methods development,
since most marine toxins occur in several different forms. Toxin
multiplicily is observed in animal assays as a "response profile"
averaged over the toxin profile. Duplicating the response profiles
of animal bioassays is challenging. Chromatographic methods can
only accomplish (his by separating and detecting all toxins con-
Irihuling lo total potency. This often requires expensive and scarce
multiple loxin standards, hnmunoassays would seem ideal candi-
dates, and yet the need to conjugale hapien-scale toxins when

raising the antibodies can and often does change the desired re-
sponse profile. Most marine toxins are neurotoxins and many are
sodium channel active. For this reason there has been considerable
effort to develop assays for sodium channel toxins such as para-
lytic shellfish toxins, brevetoxins, and others by their toxic effects
or by sodium channel binding. These include direct detection of
membrane electrochemical effects, competitive binding assays us-
ing radiolabeled toxins with synaptosomes, and cytotoxicity assays
using cultured nerve cells. In our laboratory a cytotoxicity assay
was developed using a colored indicator of cell viability (mito-
chondrial dehydrogenase activity). This assay has been used to
detect both sodium channel blockers such as the paralytic shellfish
toxins and also sodium channel enhancers like the brevetoxins and
ciguatoxins. We have transferred this technology to several labo-
ratories and will soon run a small-scale validation study.

DOES THE HISTORY OF TOXIN EXPOSURE INFLU-
ENCE BIVALVE POPULATION RESPONSES TO PSP
TOXINS IN MY A ARENARIAl: II) FEEDING. SURVIVAL
AND TOXIN ACCUMULATION. Scott P. MacQuarrie* and
V. Monica Bricelj. National Research Council of Canada, Insti-
tute for Marine Biosciences, Halifax, Nova Scotia, Canada, B3H
3Z1.

The Bay of Fundy. Eastern Canada and the Gulf of Maine.
USA, experience annual, recurrent paralytic shellfish poisoning
(PSP) outbreaks, which negatively impact the extensive softshell
clam, Mya arenaria, fisheries in these regions. Two M. arenaria
populations, one with a history of recurrent, annual toxin events
(Lepreau Basin, LB, New Brunswick) and one with no history of
toxin exposure (Lawrencetown River Estuary, LE, Nova Scotia),
were compared in their responses to PSP toxins during laboratory
exposure io Alexandiiiim tamarense (strain PR 18b, ca. 60 pg. sax-
itoxin equivalents cell'). Repeated measurements of the same
individuals showed that signil'icant differences in feeding rates and
% burrowing between the two populations were maintained
throughout the experimental period, indicating thai these responses
do not acclimate with prolonged (two-week) toxin exposure.
Clearance rates were 4-8 times higher in M. arenaria from Lep-
reau Basin than in M. arenaria from Lawrencetown. and 54 to
88% of the LB clams were resistant (capable of burrowing)
whereas 86 to 98% of the LE clams were sensitive (unable to
burrow). Lawrencetown M. arenaria (non-burrowers) reached
mean peak toxicities of 5000 |jLg STXeq 100 g ' visceral mass
after 24 hrs of exposure and remained at thai level. The Lepreau
Basin population Iburrowers). however, continued to accumulate
toxins but ill a cyclic or tluctuating pattern, reaching to\icit> levels
up lo lOx those of the Lawrencetown populalion at 7 and 15 days
of toxification. Thus population differences in feeding and bur-
rowing during toxin exposure are renecled in their diffeiential
ahililv to accumuhitc loxiiis. Mosi imporlaiitly. LB clams exhibited
>9S'f sur\ ival v\hilc LE clams suffered cuniulati\'e mortalities of

National Shellfisheries Association. Seattle. Wasliinaton

Abstracts. 2000 Annual Meeting. March 19-23. 2000 637

32%. which started after one week of toxin exposure. A second
experiment was undertaken in which enrichment of rare pheno-
types (LB sensitive and LE resistant clams) allowed more detailed
investigation of inter- and especially intrapopulation variation.
Percent mortality varied greatly among the four groups, ranking as
follows: LE sensitive > LE resistant » LB sensitive > LB resis-
tant. In this presentation feeding and toxin uptake rates from this
experiment will be discussed. This study demonstrates that blooms
of PSP-producing dinoflagellates can cause both lethal and suble-
thal effects on Mya arenaria. but that these effects vary in their
expression both within and among populations.

DOMOIC ACID TOXICITY: PRACTICAL SOLUTIONS
FOR ORGANIZATIONS TO REDUCE THE IMPACT. John

S. Ramsdell, Marine Biotoxins Program. NOAA-National Ocean
Service. Charleston. SC 29412.

Domoic acid is a tricarboxylic acid produced by certain species
of the diatom genus Pseiid-nitzchia. It was identified as the caus-
ative agent of the amnesic shellfish poisoning in 1987 and since
that time the toxic algae has been determined in many regions of
the world. Substantial toxicological data have been generated since
1987 for domoic acid effects on mammals. This presentation will
summarize the major points about domoic acid toxicity, including
its toxicokinetics, adverse effects and mechanisms of susceptibil-
ity. This information will then be used to discuss practical ap-
proaches that can reduce the impact of domoic acid toxicity. These
approaches will include accurate communication of the hazards,
identification of high risk groups, and the prospects for biomoni-
toring.

APPLICATION OF DNA PROBES FOR DETECTION OF
HARMFUL ALGAE. Chris A. Scholin,* Monterey Bay
Aquarium Research Institute. 7700 Sandholdt Rd.. Moss Landing.
CA 95039.

Common problems associated with monitoring waters for
harmful algal bloom (HAB) species are distinguishing between
potentially toxic and non-toxic organisms, and quantifying the
potentially toxic species in discrete water samples routinely at
many locations. Toxin-producing diatoms of the genus Pseudo-
nitzschia are one group of organisms that exemplify these diffi-
culties. Toxic species are those that produce domoic acid (DA), the
causative agent of amnesic shellfish poisoning (ASP). At the genus
level, toxic and non-toxic species of Pseudo-nitzschia are readily
identifiable, but discriminating between different species can be
time consuming due to a need for detailed morphological analysis.
Species-specific DNA probes are now available for a number of
Pseudo-nitzschia species. These probes have been evaluated in a
variety of locations around the US and elsewhere in the world. In
New Zealand, the probes are used routinely in commercial shell-
fish growing areas as part of a DA risk assessment strategy. This

presentation will focus on the use of the probes as research tools,
the process by which they are being evaluated in field studies, and
their successful integration into monitoring programs like that in
New Zealand. Defining needs of the end-users of the probes will
be emphasized. Efforts to develop novel instrumentation for /;;
situ, autonomous detection of HAB species will be summarized.

HARMFUL ALGAL BLOOMS AND SHELLFISH AQUA-
CULTURE: IMPLICATIONS FOR THE FUTURE OF THE
INDUSTRY. Sandra E. Shumway,* Natural Science Division.
Southampton College of Long Island University. Southampton,
NY 11968.

Scientists and resource managers now generally agree that the
number and frequency of harmful algal blooms (HABS) are in-
creasing over time. Many blame (sometimes inaccurately) HAB
outbreaks for the loss of shellfish growing areas and impacts on
aquaculture operations. HABs occur throughout the world and, in
some regions, are commonplace and seasonal, while in other areas,
rare or unusual. HABs can have far-reaching effects on coastal
ecosystems, including ecosystem integrity, species interactions,
and aquatic animal health. They can also create significant impacts
on population growth, human health, local and regional econo-
mies, industry, and business. For many obvious reasons, algal
species associated with HABs that affect human health continue to
receive the most attention, with commercially important fish and
filter feeding shellfish being the primary organisms of concern.
However, these algal species are not the only ones of importance
when it comes to animal health, ecosystem condition, or socio-
economic factors, and many other fish species can also be im-
pacted. In many cases, the societal response to these outbreaks
focus on mitigation and control of these adverse effects. This
presentation will review current knowledge of HAB-shellfish in-
teractions woridwide and suggest ways in which shellfish aqua-
culture may be undertaken successfully in the face of potential
HAB outbreaks. The important interactions between science and
management will be emphasized, as well as ways in which shell-
fish aquaculture ventures may operate without imposing undue
ecological stress and operational expense.

BEHAVIORAL VARIABILITY OF THE TOXIC DI-
NOFLAGELLATE, PFIESTERIA PISCICIDA, WHEN IN-
TRODUCED TO LARVAL AND ADULT SHELLFISH. Jef-
frey Springer,*' Sandra E Shumway, ^'^ and JoAnn
Burkholder,' 'North Carolina State University-Center for Ap-
plied Aquatic Ecology, Raleigh, NC 27695 USA. ^Southampton
College of Long Island University, Southampton. NY 1 1968 USA.
and 'Bigelow Laboratory for Ocean Sciences. West Boothbay Har-
bor. ME 04575.

The toxic estuarine dinoflagellate, Pfiesteria piscicida. is a
causative agent of major fish kills in estuaries of the mid-Atlantic
and southeastern U.S. P. piscicida zoospores are unique among

638 Abstracts, 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle, Washington

most toxic dinoflagellates in that toxic strains exhibit directed
attack behavior towards live finfish, and produce toxin(s) which
strip epidermal tissue from finfish and impair the nervous system.
In this study we observed toxic zoospores kill Argopecten irradi-
ans and Crassoslrea virginica pediveligers within 60 seconds of
zoospore introduction, followed by active consumption of the
shellfish and encystment within the visceral cavity. At 25 psu. the
attack behavior was most pronounced toward larvae that had dis-
carded their velum, whereas larvae with active, extended vela ap-
peared to discourage zoospore attack and feeding behavior. How-
ever, at 15 psu zoospores of the same toxic isolate showed little
attraction or attack behavior toward oyster pediveligers. We also
tested the response of this clonal isolate to adult C. virginica.
Zoospores were actively cleared from suspension within a 24 hour
period with no evidence of narcosis as has been reported for other
toxic, clonal isolates off. piscicida. Examination of fecal material
indicated that the zoospores had formed temporary cysts, and had
not been adversely affected by their passage through the digestive
tract. Within 24 hours, 90% of the zoospores had excysted and
regained motility. The data indicate that P. piscicida zoospores can
show striking variability in response to shellfish, both at the spe-
cies level (in timing of response), and within a species depending
on the life cycle stage and the salinity.

DOMOIC ACID PRODUCTION BY PSEVDO-NITZSCHIA
PSEUDODELICATISSIMA OFF THE CENTRAL WASH-
INGTON COAST IS LINKED TO RECORD LEVELS OF
TOXIN IN RAZOR CLAMS. Vera L. Trainer,* Nicolaus G.
Adams, and John C. Wekell, National Marine Fisheries Service.
Northwest Fisheries Science Center. 2725 Montlake Blvd. E.. Se-
attle, WA 98112: Mitch Lesoing, Quileute Natural Resource,
Quileute Indian Tribe. 234 Front St., LaPush, WA 98350.

In the early fall of 1998, record levels of domoic acid were
measured in razor clams on the central WA coast within 18 days
of a nearly monospecific bloom of Pseiido-nitzschia pseiidodeti-
calissima. Field samples, consisting of 90-100% P. pseudodeti-
catissiina (up to 15 million cells/L seawater) were found by mass
spectroscopy to contain up to approximately 4 (xg domoic acid/L
seawater. Measurement of increasing levels of toxin in a cultured
isolate of this species using a receptor binding assay, showed that
this diatom is a domoic acid-producer in WA coastal waters. The
economic impacts of this single bloom in 1998 were over $15-20
million dollars due to the coastwide loss of razor clam harvest.
Because of the slow depuration of razor clams and the variability
of domoic acid levels measured in these bivalves, harvest closures
occurred also in the fall of 1999, again resulting in millions of
dollars in losses to the already economically-stressed coastal com-
munities. The I99S bloom of this pcnnatc diatom was preceded by
strong coastal upwclling in early September, indicated by high
levels of silicate and nitrate at a time of anomalously low raintall.
a typical phenomenon in post-El Niiio years. Subseijuoni wind

relaxation and reversal events are suggested to have resulted in the
Ekman transport of surface cell populations into nearshore waters
where nutrients were plentiful, providing optimal conditions for
bloom development.

THE DISTRIBUTION OF DOMOIC ACID CONCENTRA-
TIONS IN RAZOR CLAMS AS A FUNCTION OF ELEVA-
TION BETWEEN HIGH AND LOW TIDES AT KALA-
LOCH BEACH WASHINGTON. John C. Wekell* and Vera
Trainer, National Marine Fisheries Service. Northwest Fisheries
Science Center. 2725 Montlake Blvd. East. Seattle. WA 98112:
Dan Ayres and Doug Simons, Washington Department of Fish
and Wildlife. 48 Devonshire Rd.. Montesano. WA 98563.

Reported domoic acid levels in razor clams (Siliqiia patula) in
Washington State have been extremely variable and unpredictable,
resulting in emergency closures of harvest areas in 1991. 1998, and
1999. This may be due to locational differences in clam toxicity.
Information concerning variability in toxin levels relative to sam-
pling location is important in developing a reliable sampling plan
for managing domoic acid outbreaks. In November 1998. Kalaloch
Beach in Washington State reported record levels of domoic acid
in razor clams of about 300 ppm. Due to the relatively long re-
tention time of this toxin in these clams, a resource survey at
Kalaloch presented an opportunity for the study of domoic acid
levels as a function of tidal elevation. From July 28-31. 1999
(during the summer low tides) six "east-west" transects were
sampled at Kalaloch Beach, approximately 2 km apart. The eastern
terminus of each transect was approximately 50 ft below the high
tide mark and proceeded due west toward the water to the low tide
mark, a total distance of approximately 300 to 450 feet. Samples of
10 razor clams were taken at each 50 ft interval. Clams were
transported on ice to the Seattle laboratory, where they were in-
dividually measured, weighed, and shucked. The whole meats
were individually homogenized, placed in containers, and frozen
until analysis. Each clam was individually analyzed for domoic
acid. Data is presented on the distribution of domoic acid both
between transects (interspecific variability) and within (intraspe-
cific variability) each transect.

VARIANCE IN AMNESIC SHELLFISH POLSONING IN
GEOGRAPHICALLY DISCRETE POPULATIONS OF RA-
ZOR CLAMS {SILIQUA PATULA) IN BRITISH COLUM-
BIA. J. N. C. Whyte,* N. G. Ginther, and L. J. Keddy. Fisheries
and Oceans Canada. Pacific Biological Station. 3190 Hammond
Bay Road, Nanaimo. B.C., Canada, V9R 5K6: R. Chiang, Cana-
dian Food Inspection Agency, 2250 South Boundary Road,
Burnaby. B.C.. Canada. V5M 4L9.

Domoic acid (DA) the cause of Amnesic Shellfish Poisoning is
produced by Pseudo-niizschia spp. DA retention in razor clams is
significantly higher than in other Pacific bivalves, and provided a

National Shellfisheries Association. Seattle, Washington

Abstracis. 2000 Annual Meetine. March 19-23. 2000 639

means of diagnosing seasonal variance in DA producing blooms in
clam areas. Major populations of razor clams are limited to the
north coast of Graham Island (Mclntyre Bay), the Queen Charlotte
Islands, and Long Beach (Cox Bay) on the west coast of Vancou-
ver Island. DA in clams from Cox Bay over a 2 year period varied
from 3.3 ± 1.3-33.3 ± 6.9 |J.g/2. with higher toxicity following
storms, which suggested adxection of Pscnilo-nitzsclua from off-
shore. Toxicity in body tissues declined in the order of foot, si-
phon, gut (stomach and digestive system) and mantle (with adduc-
tor gill and gonad). However, the mantle always contained the
highest percentage of the total toxin accumulated. Data analysis
from Cox Bay clams indicated a clearance rate of 2.5 (j.g/g/d (r" =
0.7398). Toxicity in clams from Mclntyre Bay during the same
survey period ranged from 0.1-1.1 ± 0.3 (J-g/g. suggestive of in-
frequent formation of toxic Pseiido-nitzscl\ia blooms in the north
coast. Interestingly. 14 days after the conclusion of the 2 year
survey toxicity increased to 31.6 (xg/g. with the occurrence of a
major Pscuilo-iiirzschia bloom. Decline in toxicity over the next
year indicated a clearance rate of 1.9 (xg/g/d (r^ = 0.7202). A
significant increase in toxicity in whole and edible tissue of clams
from west to east along Mclntyre Bay was considered to reflect
increased on-shore catchment of Pseudo-nilzschici cells from the
counter-current oceanographic gyre impacting the spit that extends
into the eastern part of the Bay.

EXPERIMENTAL EXPOSURES OF BAY SCALLOPS TO
CULTURES OF SUSPECTED HARMFUL MICROALGAE.
Gary H. Wikfors,* Jennifer H. Alix. Milford Laboratory, North-
east Fisheries Science Center, NCAA Fisheries, Milford. CT
06460: Sandra E. Shumway, Sara Barcia. and Julie Cullum,
Southampton College. LIU. Southampton. NY 11968; Roxanna
M. Smolowitz, Marine Biological Laboratory. Woods Hole. MA
02543.

Widespread use of the term "Harmful Algal Bloom" begs the
question: Harmful to whom? Molluscan shellfish have been rec-
ognized as vectors of microalgal toxins to human consumers for
millennia, but detrimental effects of some microalgae upon the
mollusks themselves have received less attention. As part of a
larger study designed to investigate the role of grazing in the
bloom dynamics of microalgae for which there is some evidence of
grazing suppression, we conducted experimental exposures of bay
scallops, Argopecten irradians. at several life-history stages (em-
bryos, larvae, post-set, and juveniles) to a number of cultured
microalgal strains. Microalgae investigated included: 1 ) di-
noflagellates — two strains of Pmrocenlrum minimum, Cyrodinium
auieolum. and Gymnodinium splendens; 2) a raphidophyte —
Heterosigma carterae: and Prymnesiophytes — two strains of
Prymnesiiim pamim and one of P. patelliferum. Scallop response
variables measured included survival, growth, development, feed-
ing behavior, and histopathology. Effects ranging from subtle and
sublethal to acute toxicity were observed. The most dramatic, le-

thal effects were seen with a new strain of Prorocentnim minimum.
collected by Dr. Patricia Gilbert from a 1998 bloom in the York
River, MD, and with a new strain of Pnnincsium paniiiii. isolated
by Dr. Robert Guillard from Boothbay Harbor. ME. In addition to
limiting harvest of molluscan shellfish for human consumption,
clearly harmful algal blooms have the potential to affect the popu-
lation bioloav of molluscs themselves.

SHELLFISH BIOLOGY

THE BULBUS ARTERIOSUS OF THE CLAM MERCE-
NARIA MERCENARIA: ANATOMY AND PHARMACOL-
OGY. Lewis E. Deaton,* Bruce E. Felgenhauer, and Daniel W.
Duhon, Biology Department, University of Louisiana at Lafayette,
Lafayette, LA 70504.

In bivalves, the hemolymph is pumped by a heart comprised, in
general, of a ventricle and two auricles. Blood exits the heart via
one or two aortae. In the quahog. M. mercenaria. there is a large
swelling associated with the posterior aorta. This sac-like struc-
ture, the bulbus arteriosus, has a volume equal to that of the ven-
tricle. The function of this tissue is unknown. We have investi-
gated the anatomy, ultrastructure. and pharmacology of the bulbus.
The wall of the bulbus consists of a spongy matrix of connective
tissue interspersed with bundles of muscle. Neurons are also
present: the axons are gathered into bundles. We also observed
granulocytic hemocytes in the lumen and the wall of the bulbus.
Unlike that of the ventricle, the lumen of the bulbus is largely
devoid of trabeculae. The lumen of the bulbus is connected to that
of the ventricle by the posterior aorta. This vessel continues as a
tubular structure for at least half the length of the lumen of the
bulbus before the aorta empties into the bulbus. The isolated bul-
bus arteriosus contracts tonically in response to 5-hydroxytrypta-
mine. acetylcholine, and the molluscan neuropeptide FMRFamide.
The threshold for these effects is about 10"^ M. Bioassays of
acetone extracts of bulbus tissue on the ventricle of M. mercenaria
show that the bulbus contains acetylcholine and FMRFamide. We
injected ink into the lumen of the ventricle of M. mercenaria in
vivo to observe the flow of hemolymph through the anterior and
posterior aorta: the majority of the hemolymph ejected from the
ventricle enters the anterior circulation. We conclude that the bul-
bus is probably involved in the regulation of the relative volumes
of hemolymph delivered to the anterior and posterior aortae. In-
creases in the pressure of the hemolymph in the bulbus would
constrict or collapse the poterior aorta. In addition, the presence of
neurons in close association with the lumen of the bulbus suggests
that it may also function as a neurohemal site. We did not, how-
ever, see any release of products into the lumen of the bulbus from
neurons.

640 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

THE EFFECT OF LOW OXYGEN ON OYSTER SURVIVAL
DURING REEF RESTORATION EFFORTS IN BON
SECOUR BAY, ALABAMA. F. Scott Rikard* and Richard K.
Wallace, Auburn University Murine Extension and Research Cen-
ter. Mobile. AL 36615; David Rouse and Imad Saoud, Auburn
University, Department of Fisheries and Allied Aquaculture. Au-
burn. AL 36849.

Low dis.solved oxygen levels have been implicated in the de-
cline of once-productive oyster reefs in Bon Secour Bay. Alabama.
Since low dissolved oxygen often occurs near the bottom, it has
been suggested that successful reef restoration might begin by
increasing the height of the shell base above the existing bottom.
Experimental 1 m" plots constructed of PVC rings and filled with
oyster shell were established at Fish River Reef (depth 2.5 m) in
Bon Secour Bay at two levels (20 cm and 40 cm) above bottom
along with shell plots on bottom. Oysters held in mesh bags at
these three levels survived and grew well the first year but suffered
100% mortality between the June 8, 1999 and August 13, 1999
sample dates. Oyster shell cultch from the three levels was
sampled to analyze spat set and oyster growth. Shells sampled on
July 13. 1999 had live oysters attached: 0.83, 1.10, 1.07 oysters/
shell for the bottom. 20 cm and 40 cm levels, respectively. No live
oysters were found on shells sampled on August 18, 1999. Remote
water quality recording devices deployed continuously on Fish
River Reef, recorded three periods of extended low dissolved oxy-
gen (<0.5 mg/L) between the above sample dates. The longest
instance was from July 16-21. 1999 for a period of approximately
130 hours. Similar, periodic low oxygen events probably prevent
this and other reefs in the area from recovering to productive levels
even when cultch is used to raise reef elevations 20-40cm.

USE OF DNA MARKERS TO DETECT DIFFERENTIAL
LARVAL SETTLEMENT PATTERNS OF MYTILUS EDU-
LIS AND M. TROSSULUS. Ellen L. Kenchlngton* and Ken-
neth R. Freeman, Bedford Institute of Oceanography. PC Box
1006 Dartmouth. Nova Scotia, Canada B2Y 4A2; Scott P. Mac-
quarrie, Biology Department. Dalhousie University. Halifax.
Nova Scotia. Canada B3J 4J1; Shawn M. C. Robinson, St. .An-
drew's Biological Station. St. Andrew's, New Brunswick. Canada.
The mussel aquaculture industry on the east coast of Canada is
based on wild spal collection. The presence of Mylilus trossuliis
among commercially cultivated blue mussels {M. etliilis) limils
farm production, as the former has a lower yield and the thin shells
are prone to breakage in the sorting equipment. Reports of simul-
taneous spawning of the two species and larval periods of equal
duration combine to eliminate timing of collection as a means of
avoiding M. trossuliis at mixed-species farms. Field experiments
were performed in determine spawning time and depth preference
of the two species. Replicate polypropylene rt)pe spat collectors
were placed in the water during the last week of June and haulcti
and replaced al \\eckl> intervals until .August 7. and a fortnighlK

intervals thereafter through to October 16, encompassing the
spawning period. The nuclear internal transcribed spacer (ITS)
region of the ribosomal RNA gene array has been shown to dis-
tinguish these two species when digested with the restriction en-
zyme Hhal. Approximately 30 individual larvae were randomly
picked from the collectors from each of three depths (1 m. 3 m.
5 m) on replicate ropes for each collection time. Following DNA
extraction and PCR amplification of the ITS region and subsequent
enzymatic digestion, the larvae were identified to species against
known standards. The results indicate a clear statistically signifi-
cant settlement depth preference for the two species, with M. tros-
suliis favouring the shallower depths. By placing the collector
ropes below 5 m at this site, growers could have increased the
proportion of M. ediilis collected by 100% and reduced the pro-
portion of M. trossuliis by 50%.

REPRODUCTIVE BIOLOGY OF PACIFIC OYSTERS:
SOME ENIGMAS, Gretta 6'Sullivan* and Maire F. Mulcahy.

Department of Zoology and Animal Ecology. National University
of Ireland. Lee Maltings. Prospect Row, Cork, Ireland.

Crassostrea gigas. a non-native species in Ireland, is cultured
from hatchery produced spat, and was believed not to spawn under
Irish conditions. Steele in 1996 and 1997 found that C. gigas
spawned each year in Dungarvan Bay. but failed to spawn in Cork
harbour even though temperatures and chlorophyll a levels were
comparable at the two sites. She suggested that failure to spawn
might be due to an environmental contaminant such as TBT. Fur-
thermore Steele (1998) found only 0.002% hermaphrodites,
whereas Sato (pers comm.) found between 23-68% hermaphro-
dites in samples examined from May to August in Japan.

This 12-month study examined the gonadal development and
reproductive cycle of C. gigas in Dungarvan and Cork harbour,
together with temperatures and chlorophyll a levels. The number
of segments needed to establish accurately the gonadal variation
between male, female and hermaphrodite was examined and found
to be one. The possibility that TBT was responsible for the failure
of Cork harbour oysters to spawn was examined by looking at shell
and condition indices. It was found that oysters spawned again in
Dungarvan. but failed to spawn in Cork harbour, though condition
indices and gonadal maturatin were similar at both sites. However,
shell index was lower in Cork harbour, supporting the hypothesis
that TBT might be an inhibiting factor for spawning.

MOONLIGHT MADNESS AND LARVAL LAUNCH PADS:
TIDAL SYNCHRONIZATION OF MOUND FORMATION
AND HATCHIN(; B^ TANNER CRABS, CHIONOECETES
HAIRDI. Bradley (J, Stevens, NMFS. Kodiak Fisheries Research
Center. 301 Research Ct.. Kodiak. AK.

Using subniersibles and ROV's. we observed female Tanner
crabs forming dense aggregations of mounds during mating season
each spring from IWl to l')95. in 150 m depth in Chiniak Bay.

National Shelirisheries Association. Seattle. Washington

Abstracts. 2000 Annual Meetinc. March 19-23. 2000 641

Kodiak. Alaska. From mid- April to June 1999. we investigated the
relationship of mound formation to hatch timing and environmen-
tal factors on several fronts. A camera sled and ROV were used to
monitor aggregation behavior; crabs started forming mounds by 1-''
April, and continued until June 1. forming the largest mounds ever
seen. Female crabs brought into the lab, some captured from
mounds, released larvae from 1 May to 6 June. Individual crabs
required from 3 to 22 days (median 10) to release 14.000 to
226.000 larvae: the most released in a single day was 106.000.
Embryonic heartrate increased steadily until hatching, offering a
potential method for determining developmental stage. The me-
dian hatching date (17 May) coincided with the new moon, the
highest spring tide in May. and a monthly reversal of mean current
direction. There was no obvious correspondence between hatching
or mound formation and Secchi disk depth or water temperature.
Nor was there any significant difference in timing of hatching
between crabs maintained in filtered or unfiltered seawater. We
conclude that mound formation is associated with hatching, and is
timed to occur during a period of high tidal current flow, though
prior plankton blooms may be a partial cue. Mounds may serve as
"larval launch pads" to facilitate escapement from the silty bottom
and its boundary layer.

SHELLFISH HEALTH MANAGEMENT

ANTIMICROBIAL ACTIVITY IN CELL-FREE HEMO-
LYMPH OF OYSTERS AND MUSSELS. Robert S. Ander-
son* and Amy E. Beaven, Chesapeake Biological Laboratory.
University of Maryland Center for Environmental Science. P.O.
Box 38. Solomons. MD 20688.

The antimicrobial activity of sera from Crassostrea virginicci.
C. gigas. Mytilus ediilis. and Geukensia demisso was tested using
a laboratory propagated strain of the oyster parasite. Perkimiis
marinus, and a bacterial species. Bacillus megaterium. The growth
kinetics of P. marinus in the presence of bivalve sera were fol-
lowed turbidometrically. and the cidal effects of exposures to stan-
dardized serum protein levels determined. Bactericidal activity
was measured by determining the percent survivorship after serum
exposure by the MTS/PMS assay. Activity of sera were compared
after calculating EC^f, values (|xg serum protein per ml required to
inhibit/kill 50% of the test microbes). Sera from local and Maine
C. virginica had low. but detectable. anti-P. marinus activity (EC^,,
= 1-2 mg/ml), suggesting that exposure/infection was not a sole
determinant of activity. Sera from C. gigas had no anti-P. marinus
activity, although this species is reportedly less susceptible to this
parasite than C. virginica. Both M. edulis and G. demissa sera had
-100- to 200-fold greater anti-P. marinus activity than C. vir-

ginica. Anti-fi. megaterium activity was consistently recorded for
all the Crassostrea species tested (EC.;,, s 200 (jig/ml). as well as
for M. edulis (EC,;o = 45 |j,g/ml). No antibacterial activity was
measured in G. demissa serum. Hemocyte extracts of C. virginica
and the two mussels had higher anti-P. marinus specific activity
than the corresponding sera. Hemocyte extracts of C. virginica and
M. edulis had weaker anti-fl. megaterium activity than the corre-
sponding sera. Bivalve sera were fractionated by ultrafiltration to
determine the MW of anti-P. marinus proteins. Unlike the oysters.
M. edulis serum showed strong anti-f. marinus activity in the <10
kDa peptide fraction, suggesting the presence of defensin-like mol-
ecules. These data indicate antimicrobial agents show species-
specific patterns of expression and activity in bivalves, some may
be produced by hemocytes. and may partially determine resistance
to infectious disease.

MANAGEMENT OF JUVENILE OYSTER DISEASE (JOD)
IN MAINE. Bruce J. Barber,* Christopher V. Davis, Ryan B.
Carnegie, and Katherine J. Boettcher, School of Marine Sci-
ences. University of Maine. Orono. ME 04469.

Juvenile Oyster Disease (JOD) is a syndrome that affects ju-
venile oysters, Crassostrea virginica during the first growing sea-
son. Signs of JOD include reduced meat weight, uneven valve
growth, and characteristic conchiolin deposits on inner valve sur-
faces. Since 1988. JOD has been responsible for cumulative mor-
talities of up to 96% in the Damariscotta River, Maine. Efforts to
minimize the impact of this disease on commercial oyster produc-
tion have involved both short-term and long-term approaches. Ini-
tial research revealed that mortality caused by JOD was inversely
related to oyster size. Oysters with a mean shell height of 12.1 mm
had a cumulative mortality of 56.2% while larger oysters (25.9 mm
mean shell height) had a cumulative mortality of 13.6%. A sub-
sequent study determined that mortality caused by JOD was sea-
sonal in nature; cohorts placed in the river before June or after
mid-August had cumulative mortalities <20% while those de-
ployed between June and August had cumulative mortalities of
64-96%. Thus short term management strategies involve early
spawning and deployment to achieve maximal size prior to the
onset of disease. Longer term management has been accomplished
through genetic selection. Selected oysters (Flowers F,) had a
cumulative mortality of 1 1.2% compared to 95.7% for unselected
oysters. Ultimately, further management strategies will depend on
the identification of an etiological agent. Recent experiments
showed that oysters exposed to antibacterial agents had a lower
cumulative mortality (55%) than control groups (81%). Further,
bacteriological analysis revealed that a novel alpha-proteo-
bacterium is numerically dominant in oysters exhibiting signs of
JOD and not detected in healthy oysters. Challenge experiments
with this suspect pathogen are ongoing.

642 Abstracts. 2000 Annual Meeting, March 19-23. 2000

National Shelifisheries Association, Seattle. Washington

DESCRIPTION OF AN UNUSUAL PARASITE IN
PRAWNS. PANDALUS PLATYCEROS, IN BRITISH CO-
LUMBIA, CANADA. Susan M. Bower* and Gary R. Meyer,

Fisheries and Oceans Canada, Pacific Biological Station. Nan-
aimo. B.C. V9R 5K6. Canada.

A protozoa parasite, superficially similar to parasitic di-
noflagellates. with large plasniodia and numerous trophonts oc-
curred in up to 27% of the prawns from Malaspina Strait, British
Columbia. Infections in most prawns were cryptic but of sufficient
duration to affect secondary sexual characteristics and castrate the
host. Cryptic infections consisted of large plasmodia containing
numerous pleomorphic nuclei. Examination via electron micros-
copy revealed that in some areas of the Plasmodium, the outer
membrane was indistinct and the cytoplasm of the parasite ap-
peared to coalesce with the cytoplasm of lysed haemocytes. The
Plasmodia invaded the haemal sinuses of all tissues and then broke
up into trophonts with single nuclei. Prawns with gross evidence of
infection (body discolouration, lethargy and haemolymph milky
with a plethora of either spherical or discoid trophonts) rarely
exceeded a prevalence of 2% of an infected population fished with
traps. In a few prawns with mainly spherical trophonts, about 257r
of the trophonts were dividing. The ultrasiructure of nuclei con-
taining mitotic figures consisted of a few condensed chromosomes
attached by microtubules (spindle fibers) to centriole-like struc-
tures situated at a gap in the nuclear membrane. Nuclear division
of trophonts in binary fission was unlike that described for a para-
sitic dinoflagellate (Syndinium). Also, detailed morphological ex-
amination did not reveal features characteristic of parasitic di-
noflagellates (e.g., trichocysts in the cytoplasm and a flagellated
stage). Thus, the taxonomic affiliation of the parasite in P. plci-
tyceros must be addressed using tools additional to morphological
examination such as molecular analysis. Attempts to transmit the
infection between prawns in the laboratory were unsuccessful.

INSIDE THE SHELL OF AN INTERTIDAL OYSTER: LI-
ABILITIES AND BENEFITS? L. E. Burnett* and C. S. Mi-
lardu, Grice Marine Laboratory, University of Charleston, SC
29412.

When the oyster Cnissostrea \iri;iiiic(i is air exposed, it isolates
itself nearly cotiipletely from the outside environment. The envi-
ronment within the oyster shells change rapidly and dramatically.
The degree of change depends on the ambient lempcrature and the
microhabilat. An oyster in full sunlight becomes much holler than
those that arc shaded. Tissues become hypoxic, hut not anoxic, and
acidic. Hemolyniph O, pressure falls from .'^7 lorr in an oyster in
well-aerated water (l.S.S torr = air saturation) to 10 torr during
emersion. Po-, never lalls below 10 torr and the oyster depends
entirely on anaerobic metabolism. Hcmolymph pH falls as low as
6.0 at 35 "C due largely to the buildup of CO,. These changes are
similar to those that occur when an oyster is exposed to hypoxic
water. Liabilities: low (), and separately low pH depress ROI

production of oyster hemocytes. Although ROI production may
not be bactericidal in oysters /)er .sy' (Bramble & Anderson, 1999),
bactericidal activity of oyster hemocytes under these conditions
needs to be assessed. Benefits: the elevated CO, that occurs with
emersion stimulates the metabolism of the parasite Perkinsus
iiuiritiiis and this may benefit the oyster in that the parasite directs
more energy to respiration and less to growth and reproduction.
This explains why infections of PerkinsKs in intertidal oysters in
the southeast are rarely very intense. (SC Sea Grant R/ER-14)

DISEASE DIAGNOSIS BY PCR: FOOLPROOF OR FOOL-
HARDY? Eugene M. Burreson,* Virginia Institute of Marine
Science. College of William and Mary, Gloucester Point, VA
23062.

The polymerase chain reaction (PCR) is viewed by many as the
ultimate diagnostic tool because of its extreme sensitivity and
specificity. A positive PCR result is often interpreted as the un-
equivocal presence of a disease agent, and a negative PCR result
is often interpreted as the unequivocal absence of a disease agent.
However, the nature of PCR may result in false positives for
disease diagnoses. A positive PCR does not necessarily mean that
a viable disease agent is present because DNA may be isolated
from samples with lysed or non-viable organisms. Two kinds of
subsampling error potentially yield PCR false negatives —
subsampling of uninfected tissue from a host for DNA extraction
when infections are localized, and utilization of insufficient
amounts of extracted host/parasite genomic DNA for PCR analy-
ses. Replicate subsampling is recommended to minimize both
types of error. At present, PCR diagnosis should be used in con-
Junction with standard techniques where possible. Nonetheless,
PCR is extremely valuable for identifying known disease agents
for which a gene sequence has been determined, especially when
they occur in unexpected hosts. PCR is also extremely valuable
when followed by sequencing for determining the phylogenetic
position of undescribed parasites. More research is necessary com-
paring PCR and standard diagnostic techniques before PCR can be
recommended as the method of choice for disease diagnosis.

SHELLFISH HEALTH MANAGEMENT: A SYSTEM
LEVEL PERSPECTIVE FOR PERKINSUS MARINUS. David
Bushek.*' - .Jennifer Keesee,' Ben .lones.' Dave White." Matt
Ncet,' and Dwayne Porter,"'""" 'Barucli Inslitule. "Marine Sci-
ence Program and 'Department of Environmental Health Sciences.
University of South Carolina. Columbia. SC 2920S.

The oyster pathogen Perkinsus iinirimis has wreaked havoc on
natmal and cultured populalions olthe eastern oyster for more than
half a century. I-'ew management strategies have been devekiped to
minimize P. »;(//7/ii(.v-induced oyster mortality and none have been
effective. One reason may be a poor understanding of the pio-
cesses ihal ctmtrol parasite transmission. We present data from

National Shellfisheries Association. Seattle. Washinuton

Abstracts. 2000 Annual Meeting. March 19-23. 2000 643

three years of spatially intense seasonal monitoring of P. inariinis
infection intensities in two South Carolina estuaries. The data in-
clude El Nifio, La Nina and normal rainfall years and indicate that
physical processes related to transmission, namely water residence
time and flushing rates, are primary determinants of infection in-
tensity. Landscape-level anthropogenic impacts that alter these hy-
drological processes (eg., upland ditching and drainage, channel
dredging, jetty construction, etc) inay be more important factors in
exacerbating oyster mortality problems from P. mariinis than pol-
lutants commonly associated with development. Shellfish health
management can and should take advantage of these relationships
in three ways: 1 ) via site selection for planting, cultivating and
harvesting oysters. 2) for selecting sanctuaries and reserves, and 3)
to identify potential management regulations and mitigation efforts
for coastal development. To proceed, the principles of estuarine
oceanography need to be more widely incorporated into the man-
agement of Pcrkinsus marinus and most likely many other shell-
fish pathogens.

HIGH PERFORMANCE OF CRASSOSTREA ARIAKENSIS
IN CHESAPEAKE BAY. Gustavo W. Calvo,* Mark W. Luck-
enbach, and Eugene M. Burreson. School of Marine Science.
Virginia Institute of Marine Science. College of William and
Mary. Gloucester Point, VA 23062.

As native eastern oyster, Crassostrea virgiiiica. stocks have
declined throughout much of the mid-Atlantic seaboard of the
United States interest in the potential of non-native oyster species
to restore the fishery and ecological functions has grown. To ex-
amine the performance of triploid C. ariakensis in comparison
with that of diploid C. virginica. oysters (n = 300, age = 2 years,
mean shell height = 60-64 mm) were deployed in floating mesh
cages at each of two replicate sites within low, medium, and high
salinity regimes (respectively, <15%c, 15-25%o, >25%c) in Chesa-
peake Bay and the Atlantic Coast of Virginia. Over the 16 mo.
evaluation period, from May 1998 to September 1999, C. aria-
kensis exhibited higher disease resistance and superior survival
and growth than C. virginica. Final mean cumulative mortality was
>80'7f for C. virginica and <20% for C ariakensis. After 14 mo.
of deployment, mean shell height of C. ariakensis at low, moder-
ate, and high salinity sites, was respectively 96 mm, 125 mm, and
140 mm. In comparison, mean shell height of C. virginica was
respectively 72 mm, 85 mm, and 75 mm. Baseline samples re-
vealed no P. marinus and a 4% prevalence of H. nelsoni (MSX) in
C. virginica and 12% prevalence off. marinus and no MSX in C.
ariakensis. In all subsequent samples, collected in August and
October 1998, and in May, August and September 1999, preva-
lence and intensity of P. marinus infections were consistently
higher in C. virginica than in C. ariakensis. During the second
summer of disease exposure, prevalence in C. virginica was 100%
at all sites whereas prevalence in C. ariakensis ranged form
0-28%. Only light infections were present in C. ariakensis

whereas heavy infections were found in C. virginica. MSX was
absent in C. ariakensis and present in C. virginica. This study
demonstrated a high performance of adult C. ariakensis in the
lower Chesapeake Bay and in the Atlantic Coast of Virginia.

DEVELOPMENT OF A PCR ASSAY FOR DETECTION OF
BONAMIA OSTREAE IN FLAT OYSTERS, OSTREA EDU-
LIS. Ryan B. Carnegie,* Bruce J. Barber, and Daniel L. Distel.

School of Marine Sciences. University of Maine. Orono, ME
04469; Sarah C. Culloty, Department of Zoology and Animal
Ecology, University College, Cork, Ireland.

Rapid and sensitive methods for the detection of shellfish
pathogens are needed for effective disease management. Flat oys-
ters (Ostrea edulis) infected with the microcell parasite Bonamia
ostreae were used to develop a polymerase chain reaction (PCR)
assay that is faster and more sensitive than standard histology.
Genomic DNA was extracted from hemolymph of a Maine oyster
and the gill of an Irish oyster. Using the PCR and primers tuned to
protistan rDNA. a single, identical amplicon was obtained from
both samples. This product was determined by BLAST search to
closely resemble rDNA genes belonging to members of the Phy-
lum Haplosporidia. A PCR reaction specific for this sequence was
designed and used to assay hemolymph and gill tissue from 154
oysters scored for B. ostreae based on hemolymph smears (overall
B. ostreae prevalence was 44.8%). A product presumed to be the
B. ostreae sequence was generated in 100% of "heavily" infected
oysters; 100% of "moderately" infected oysters; 84.6% of "lightly"
infected oysters; 65.0% of "scarcely" infected oysters; and 61.2%
of those scored "uninfected". No PCR product was detected, how-
ever, in a negative control composed of 19 juvenile Crassostrea
virginica from Virginia. A positive PCR signal for B. o.?treae in a
high percentage of "uninfected" oysters does not necessarily rep-
resent spurious amplification. It is likely that most oysters in B.
ostreae-enzootic areas harbor parasites, but at levels too low to be
detected by standard cytological or histological methods. Indeed,
closer histopathological exainination of 26 of the above oysters
found 81.5% to harbor B. ostreae, including 93.3% that tested
positive for B. ostreae using PCR.

SEROLOGICAL AFFINITIES BETWEEN PERKINSUS
MARINUS AND SOME PARASITIC DINOFLAGELLATES.
Christopher F. Dungan* and Rosalee Hamilton, Cooperative
Oxford Laboratory. Maryland DNR. Oxford. MD 21654; David
Bushek, Jennifer Cardinal, and Alan Lewitus, Baruch Marine
Field Laboratory. University of South Carolina, Georgetown, SC
29442.

Nucleotide sequences of several genes from the apicomplexan
protozoan oyster pathogen Perkinsus marinus consistently suggest
that its strongest phylogenetic affinities are with dinoflagellate
taxa. To test for phenotypic evidence of this suggested taxonomic

644 Abstracts. 2000 Annual Meetins. March 19-23, 2000

National Shellfisheries Association. Seattle. Washington

linkage, antibodies to P. marimis known to have specificity for
Perkinsus species, but not other protozoa, were used to immuno-
stain free-living and parasitic dinoflagellate, and dinoflagellate-
like, organisms. Among tested dinoflagellates, 3/28 free-living
species (11%) and 6/7 parasitic species (86%) were labeled by
antibodies to P. marinus. These results indicate the presence of
shared antibody binding epitopes common to P. marinus and di-
noflagellates, in general. Antibodies to P. marinus consistently
labeled parasitic dinoflagellates infecting 6 crustacean hosts, but
failed to label a dinoflagellate-like parasite of Spot prawns. Pan-
dalus platyceros. from both Alaska. USA and British Colombia,
Canada. The reciprocity of this antibody crossreactivity was tested
by immunostaining the same suite of syndinean dinoflagellate
parasites, the dinoflagellate-like Spot prawn parasite, and P. mari-
nus. with antibodies against the Hematodinium sp. parasite of Nor-
way lobster, Nephrops norvegicus. \ni\-Hematodinium sp. anti-
bodies reciprocally labeled P. marinus and the 6 Hematodinium-
species infecting Crustacea, but also failed to label the
dinoflagellate-like Spot prawn parasite. Identical reciprocal cross-
reaction patterns for antibodies to P. marinus and Hematodinium
sp. from Norway lobsters, when each was tested against the same
suite of pathogens, confirms the presence of shared antigenic
epitopes among the apicomplexan oyster pathogen and parasitic
dinoflagellates infecting six crustacean species, and supports an
hypothesized phylogenetic affinity between these disparate proto-
zoan taxa. Diagnostic immunoassays to differentiate parasitic Per-
kinsus and Hematodinium species must control or eliminate the
identified crossreaction. The Spot prawn dinotlagellate-like para-
site is clearly distinct from both P. marinus and Hemalodiniiim-
species parasites of other crustacean hosts.

AN EXAMINATION OF ECOLOGICAL FACTORS GOV-
ERNING PLANKTONIC ABUNDANCE AND DISPERSAL
OF PERKINSUS MARINUS. Rebecca Ellin* and David
Busiiek, Belle W. Baruch Institute for Marine Biology and Coastal
Research, University of South Carolina, P.O. Box 1630, George-
town, SC 29442.

Direct transmission of Perkinsus marinus to (he Eastern oyster,
Crassostrea virginica. occurs via the water column. The processes
that infiuence planktonic transmission and dispersal remain poorly
understood because few studies have attempted to examine the
planktonic stages of P. marinus. For example, tidal infiuences on
planktonic P. marinus across an oyster reef have never been ex-
amined and only a few studies have addressed the seasonal pattern
of planktonic P. marinus. Wc hypo(hesi/e that tidal processes and
the abundance of oysters govern the dispersal and transmission of
P. marinus through the water column. To address these hypoth-
eses, we arc measuring changes in planktonic abundance of /'.
marinus across tidal cycles from the fronts and backs of four
interlidal creeks in North Inlet. South Carolina. Oysters had been
removed from two of these creeks enabling us to elucidalc ihc role

of oysters in the planktonic population dynamics of P. marinus.
Five hundred milliliter water samples have been collected monthly
from each location during neap tidal cycles since February 1999 (n
= 24 per site). Samples are filtered, incubated in RFTM, and
digested with NaOH to enumerate P. marinus. Preliminary exami-
nation of samples processed indicates an increase in P. marinus
cells at slack high and low tides for all creeks. Furthermore, creeks
with oysters exhibit higher P. marinus concentrations on flooding
tides when compared to creeks without oysters.

BACTERIAL PATHOGENS, DISEASES AND THEIR CON-
TROL IN BIVALVE SEED CULTURE. Ralph Elston,*

AquaTechnics/Pacific Shellfish Institute, PC Box 687, Carlsborg,
WA 98324 USA: Arthur Gee, Dept. Biology. Pacific Lutheran
University, Tacoma, WA 98447-0003; Russell P. Herwig, School
of Fisheries, University of Washington, 3707 Brooklyn Ave. N.E..
Seattle, WA 98105-6715.

Vibriosis is known as a disease of intensively cultured larval
shellfish but bacterial pathogens cause significant losses in nursery
cultures of juvenile bivalves. Typically, rod-shaped bacteria attach
to externally oriented periostracum and subsequently invade juve-
niles through the valve closure and along the internal shell surface.
Contact necrosis and sloughing of mantle epithelium results and,
when bacteria have invaded sufficiently far along the mantle, they
invade the still patent coelomic cavity of juvenile bivalves. A
chronic form of the disease occurs less frequently. Detailed studies
of invasive juvenile bacterial diseases are underway for the Pacific
oyster (Crassostrea gigas). Kumomoto oyster (Cra.fso.strea sika-
mea), geoduck clam (Panope abrupta), and other species.

Vibrio tubiashi. V. anguilllarum. V. tapetis and V. splendidiis
have previously been reported as causative or associated with lar-
val bivalve mortalities but there also appear to be significant un-
named vibrio-like pathogens of bivalve juveniles. Results of cur-
rent studies to characterize pathogenesis and link disease types to
bacterial species are underway, including identifying characteris-
tics of the causative agents by morphological, physiological,
nucleic acid and fatty acid analysis.

Bacterial pathogens enter culture .systems via sea water, brood
stock transport of in algal food cultures. They can be maintained
on system surfaces and their growth augmented by dissolved or-
ganic substrates generated by algal cultures, external algal blooms,
or metabolism of the cultured juveniles. Prevention and control
strategies must include routine sanitation of system surfaces, water
filtration, brood stock sanitation and maintenance of low dis.solved
organic levels. Antibiotics have been used in experimental settings
but are not routinely used on production scale systems due to cost
as well as risk of producing resistant strains. In the United States,
there are no antibiotics licensed for general use on molluscan
shellfish. A program to .select and test probiotic strains of bacteria,
as an alternative to antibiotic use, is underway and results to date
will he presented.

National Shellt'isheries Association. Seattle. Washinaton

Abxtmcts. 2000 Annual Meetin". March 19-23. 2000 645

LIFE HISTORY OF AN EXOTIC SABELLID POLY-
CHAETE. TEREBRASABELLA HETEROUNCINATA: IN-
FLUENCE OF TEMPERATURE AND FERTILIZATION
STRATEGY. Carl A. Finley* and Carolyn S. Friedman, Cali-
fornia Department of Fish and Game and Bodega Marine Labora-
tory. P.O. Box 247. Bodega Bay. CA 94923 USA.

The California abalone aquaculture industry has been strug-
gling to rid itself of an exotic sabeliid. Terebrasahella heteroiinci-
itata. following its accidental introduction from South Africa in the
late 1980s. The development of an effective management strategy
is dependent upon the better understanding of the life history of
this sabeliid. including its generation time and whether it is capable
of self-fertilization. In the present study, uninfested red abalone.
Halknis nifescens. were exposed over a 24 hr period to abalone
with heavy infestations at 11.2, 15.6 and 20.9 °C; temperatures
typically encountered in California. The larvae were subsequently
observed as they developed to specific life stages: initiation of
feeding, sexual maturation and the completion of their life history
or the production of a motile, infestive. larva. Approximately 50*^
of the sabellids examined at 1 1 .2. 15.6 and 20.9 °C had developed
the ability to feed by day 6. 5 and 4 (P < 0.001 ), became sexually
mature by day 83, 68 and 48 (P < 0.001 ) and had produced larvae
by day 298, 165 and 111 (P < 0.001). respectively. In a separate
study, uninfested abalone were exposed as above. Abalone with
single infestations were held in individual container at 18 °C
(single host and sabeliid per container). This first, parental, gen-
eration was held in isolation until individuals self-fertilized to
produce F, larvae. The F, larvae were subsequently isolated until
individuals again self-fertilized, producing a second-generation, F,
larvae. This research demonstrates that the life history and gen-
eration time of T. heteiouncinata are significantly temperature
dependent and that the products of self-fertilization are fully func-
tional organisms.

FIELD TRANSMISSION STUDIES OF HAPLOSPORIDIUM
NELSONI (MSX) USING SPECIFIC PRIMERS AND PCR
TECHNOLOGY. Susan E. Ford. Zhe Xu, and Gregory De-
brosse, Haskin Shellfish Research Laboratory. Rutgers University.
Port Norris, NJ 08349.

Management of shellfish populations subjected to disease re-
quires epizootiological data, including a knowledge of transmis-
sion. Incomplete understanding of transmission of the MSX dis-
ease agent. Haplosporidium iielsoni. is a critical barrier to man-
aging affected oysters. Using molecular techniques that
specifically detect H. nelsoni. we examined several aspects of the
transmission question during a preliminary study in Delaware Bay.
We monitored the presence of putative infective particles and
documented subsequent infections — or lack of them — in larvae
and juveniles in a land-based hatchery and nursery system, as well
as in native oysters in the Bay. Neither eyed larvae nor 1-mm spat
held in the hatchery in l-|j.m filtered. UV-treated water, became

infected, nor did juveniles held for an additional 5 weeks in the
same conditions. Those held during the same period in an on-land
nursery receiving raw bay water readily became infected. Positive
reactions in the feces/pseudofeces of patently uninfected oysters,
which we considered an indication of infective particles taken
from the water, were prevalent throughout the Bay in the spring
and early summer of 1999. They disappeared after midsummer as
Peikinsiis inariints (Dermo) infections appeared. The widespread
distribution of putative infective stages and positive PCR signals in
the tissues is in sharp contrast with the low prevalences of H.
nelsoni detected in Delaware Bay oysters with standard histology
during the past decade, suggesting that native oysters have become
highly resistant to H. nelsoni infection development.

"CANDIDATUS XENOHALIOTIS CALIFORNIENSIS." A
NEWLY DESCRIBED BACTERIAL PATHOGEN AND
ETIOLOGICAL AGENT OF ABALONE WITHERING SYN-
DROME. Carolyn S. Friedman,* Thea T. Robbins, and .lames
D. Moore, California Dept. of Fish & Game. Bodega Marine
Laboratory. P.O. Box 247, Bodega Bay, CA 94923; Jeffrey D.
Shields. Virginia Institute of Marine Science. Gloucester Point.
VA 23062: Karl B. Andree. Katherine A. Beauchamp, Dolores
B. Antonio, and Ronald P. Hedrick, Dept. of Medicine & Epi-
demiology. School of Veterinary Medicine, UC Davis. CA 95616.
Withering syndrome (WS) is a fatal disease of wild and cul-
tured abalone, Haliotis spp., that inhabit the west coast of North
America. Using a combination of morphological, serological, life
history and genomic (16S rDNA) characterization, we have iden-
tified a previously undescribed bacterium observed in abalone with
WS as a new member of the order Rickettsiales and propose the
provisional status of "Candidatus Xenohaliotis californiensis".
The Gram negative, pleomorphic bacterium is found within mem-
brane-bound cytoplasmic vacuoles of abalone gastrointestinal ep-
ithelial cells. The bacterium is not cultivable on synthetic media or
in fish cell lines and may be controlled by tetracyclines but not by
chloramphenicol, clarithromycin, or sarafioxicin. Phylogenetic
analysis based on the 16S rDNA of the bacterium places it in the
a-subclass of the class Proteobacteria. We tested the hypothesis
that this bacterium is the etiological agent of WS in two separate
trials in which asymptomatic red or black abalone ± WS were
administered a series of sham (3% saline) or OTC injections (21
mg/kg) over a 9 wk period. Both survival and feeding rates were
higher in treated abalone relative to control animals (p < 0.001,
p < 0.023 for red and black abalone, resp.). All red abalone and
-50% of the black abalone that received OTC survived, while
~40<7r of the red and 100% of the black abalone controls died
during this time. These studies indicate that WS is caused by
"Candidatiis Xenohaliotis californiensis" and that losses can be
minimized by administration of oxytetracycline. We have devel-
oped PCR and in situ hybridization tests for this bacterium and are
in the process of developing an oral therapeutant. These tools will
be useful in management of the disease and it's spread.

646 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

ISOLATION AND PRIMARY CULTURE OF EASTERN
OYSTER HEMOCYTES. Jerome F. La Peyre* and Yanli Li,

Depanment ot Veterinary Science, Louisiana State University. Ba-
ton Rouge. LA 70S03.

Most studies investigating the interactions of oyster hemocytes
with pathogens rely on short term assays lasting only a few hours.
The establishment of longer term hemocyte cultures (i.e.. days to
weeks) is needed to facilitate experimental manipulation. Such an
//! vitro system, for example, can be used to test the effects of
various chemicals in promoting killing of pathogens by hemocytes.
Our objectives for this initial study were to I ) compare five solu-
tions for their abilities to inhibit clumping of oyster hemocytes
during collection. 2) determine hemocyte sensitivity to antibiotics
to be used in decontamination solution and culture medium. 3)
optimize a basal medium to maintain hemocytes in primary culture
& 4) test the effects of various chemicals on hemocyte adhesion
either to promote the formation of hemocyte monolayers or to
maintain hemocyte in suspension cultures. Our in vitro system was
then evaluated by comparing Vilvio paraliacinolyticiis killing by
hemocytes immediately after sampling and after culture for one
week.

Using a variety of assays to measure oyster hemocyte mortality,
metabolic activity and number, we found that: I) Calcium and
Magnesium-free oyster saline containing 0.5% EDTA was optimal
for collecting hemocytes, 2) antifungal agents at concentrations
generally recommended for cell culture were highly toxic to
hemocytes, 3) increasing concentrations of carbohydrates, vita-
mins and amino acids were beneficial to hemocytes up to certain
concentrations, 4) several chemicals including concanavalin A in
basal medium and poly-D-lysine and fibronectin coated to culture
vessels promoted the formation of hemocyte monolayers, 5 ) aga-
rose coated to culture vessels was useful in maintaining hemocyte
in suspension cultures unattached to culture vessels. Finally.
Vibrio paraliaeinolyticiis killing by hemocytes immediately after
sampling and after culture for one week were comparable.

RETROVIRAL VECTOR-MEDIATED ONCOGENE
TRANSFER TO CREATE CRASSOSTREA VIRGINICA
CELL LINES. James D. Moore,* UC Davis Bodega Marine
Laboratory, Bodega Bay CA 94923; Viviane Boulo, UMR 219,
IFREMFR-CNRS, Univ. Montpelier 11, CP8(), 3409,^ Montpclier,
France; Jane C. Burns, Dcpt. of Pediatrics. UC San Diego. School
of Medicine. La .lolla CA 92()93-()S3(); Carolyn S. Friedman,
Dept. of Medicine and Epidemiology. UC Davis School of Vet-
erinary Medicine and Calil'ornia Dept. of Fish and Game, UC
Davis Bodega Marine Laboratory, Bodega Bay CA 94923.

Management of shellfish disease requires sensitive tools for
diagnosis and pathogen characleri/alion. While tools developed
for and applied in mammalian disease research are often quickly
adapted for use with shelHish. one conspicuous exception is the

failure to generate permanent cell lines froni marine shellfish tis-
sue. The absence of cell lines has particularly impeded research on
viruses and obligate intracellular bacteria. We are attempting to
create cell lines from tissues of the American oyster. Crassostrea
virginicu using a highly efficient method to integrate oncogenes
into the genome of cells in primary culture. Heart tissue primary
cultures were established in 24-well plates after enzymatic diges-
tion of aseptically dissected heart tissue. Cells were then infected
with replication-defective retroviral vector(s) containing viral pro-
moters driving expression of the gene(s) of interest. These retro-
viral vectors contain the envelope glycoprotein of vesicular sto-
matitis virus that binds to phospholipid components of the cell
membrane, thus allowing entry of the virus particle into a wide
range of cell types. Conditions for gene transfer and expression
were optimized using a vector construct containing the luciferase
reporter gene, and assaying luciferase activity of primary cultures
at 72 h post-infection with a scintillation counter in single photon
mode. Luciferase activity, as a measure of infection efficiency,
was linearly related to vector concentration from 5 x 10 to 8 x 10''
cfu/well. Addition of the vector at the lime of plating resulted in
higher activity than addition at later time periods. Luciferase ac-
tivity increased by centrifugation of plates (1000 g. 30 min). but
was reduced by addition of C virainica hemolymph to the medium
during infection. Heart primary cultures are now being infected
under optimal conditions with retroviral vectors encoding the
SV40 large T antigen and ras oncogenes. Cultures are being moni-
tored for morphological changes and replicative activity. Sup-
ported by the National Sea Grant College Gulf Oyster Industry
Program through California Sea Grant College Grant No.
NA86R60073.

DISTRIBUTION AND PREVALENCE OF BITTER CRAB
SYNDROME IN SNOW {CHIONOECETES OPILIO) AND
TANNER (C. BAIRDI) CRABS OF THE BERING SEA, 1988-
1996. J. Franli Morado,* National Marine Fisheries Service,
Alaska Fisheries Science Center. Resource Assessment & Conser-
vation Engineering Division, 7600 Sand Point Way NE, Seattle,
WA 9811.^-0070; Theodore R. Meyers, Alaska Department of
Fish & Game, Commercial Fisheries Management and De\elop-
menl Division, Fish Pathology Section, PO Box 2.'^.'i26. Juneau.
AK 91)802-2000; Robert S. Otto, National Marine Fisheries Ser-
\ ice. Alaska Fisheries Science Center. Kodiak Laboratory. PO Box
1638, Kodiak, AK 996 LS.

Bitter crab syndrome (BCS) is a fatal disease of crustaceans
that is caused by a parasitic dinotlagellate of the genus Hfiiuilo-
iliiiitiiii. Between 1988 and 1996, hemolymph samples from 14,359
liastern Bering Sea (F.BS) Tanner crabs, Chionoecclcs buirdi (N
= 5180) and C. oi>ilio (N = 9184) were examined for the pres-
ence of a parasitic dinoflagellate, Hemaiodiniiim sp.. the causati\e
agent of Bitter Crab Syndrome (BCS). For this lime period, total

National Shellfisheries Association. Seattle, Washinston

Abstracts. 2000 Annual Meeting. March 19-2.\ 2000 647

prevalences of BCS in C. bairdi and C opilio were 1.87% and
3.57%. respectively. In C bairdi. prevalences from yearly random
samples ranged from 0% in 1989 and 1994 to 5.68% in 1996.
Infections in both males and females were highest in 1996. reach-
ing 9.93% in females and 2.74% in males; however, overall He-
inatodinium prevalences were only slightly more elevated in fe-
males (1.93%) than males (1.65%). For C. opilio. yearly random
sample prevalences ranged from 0.30% in 1994 to 8.45% in 1988.
Highest Hematodiuium prevalences in C. opilio were observed
early in the survey; in 1988 during which male and female infec-
tion prevalences were 7.62% and 10.00%. respectively. Overall
parasitic prevalences in C. opilio were more elevated in females
(4.23%) than males (3.23%).

BCS infections in both C. opilio and C. bairdi were most com-
mon in the Bering Sea at latitudes above 57°N. In general, infec-
tion prevalences in C. opilio increased with increase in latitude
with prevalences of 50-80% common in Norton Sound and west of
St. Lawrence Island. Despite the fact that prevalences were gen-
erally lower in the Chukchi Sea than in Norton Sound and west of
St. Lawrence Island, a greater percentage of sampled stations were
positive for BCS in the Chukchi Sea. For C. bairdi. infections were
rare in the Eastern Bering Sea. and increased only slightly along
the shelf edge west and north of the Pribilof Islands.

For both C opilio and C. bairdi, infections were more common
in small crab less than 60 mm; after which, prevalences remained
low. In C. bairdi. the highest infection rates were observed in 20
mm crab attaining levels of 62.5% in males and 65% in females.
In C. opilio. highest prevalences were observed at 35 mm with
little difference in prevalence between males and females.

EFFECT OF A LYTIC PEPTIDE AND PROTEASE INHIBI-
TORS ON PERKINSUS MARINUS IN INFECTED
HEMOCYTES OF EASTERN OYSTERS. Amy D. Nickens*

and Terrence R. Tiersch, Aquaculture Research Station. Louisi-
ana State University Agricultural Center. Baton Rouge. LA 70820:
Jerome F. La Peyre, Department of Veterinary Science, Louisi-
ana State University. Baton Rouge, LA 70803.

In previous work, we found that a synthetic lytic peptide, phor-
21, killed P. marinus cells in vitro at concentrations that were not
lethal to oyster hemocytes. The efficacy of phor-2 1 was reduced
however by extracellular proteases of P. marinus. Further study
revealed that certain protease inhibitors, such as chymostatin and
potato chymotrypsin-1 (PCI-1 ), protected phor-2 1 against the pro-
teases of the parasite. In addition, each protease inhibitor was
found to suppress the propagation of P. marinas in vitro. There-
fore, we proposed that a combination of the lytic peptide and a
protease inhibitor may be effective in eliminating P. marinus from
eastern oysters. The objective of this study was to determine the
effects of the lytic peptide and protease inhibitors on P. marinus in
naturally infected hemocytes.

Hemocytes were collected from P. marinus infected oysters

and monolayers were established in basal medium. The hemocyte
monolayers were then exposed to phor-2 1, chymostatin, PCI-1 or
a combination of phor-2 1 and a protease inhibitor. Hemocytes in
basal medium alone or with DMSO (i.e., chymostatin solvent)
were used as control. The number of P. marinus in hemocytes at
the beginning of the experiment and after three days in culture was
determined by counting the number of hypnospores after incuba-
tion in modified Ray's fluid thioglycollate medium. Preliminary
results indicate that a combination of phor-2 1 and chymostatin,
chymostatin alone and a combination of phor-2 1 and PCI-1 were
most effective in reducing the number of parasites in our //; vitro
system.

MOLECULAR ANALYSES OF A PARASITE IN PRAWNS
{PANDALUS PLATYCEROS) FROM BRITISH COLUMBIA,
CANADA. Kimberly S. Reece* and Eugene M. Burreson, Vir-
ginia Institute of Marine Science, The College of William and
Mary, Gloucester Point, VA 23062; Susan M. Bower, Fisheries
and Oceans Canada, Pacific Biological Station, Nanaimo, B.C.
V9R 5K6, Canada; Christopher F. Dungan, Cooperative Oxford
Laboratory. Oxford. MD 21654.

A parasitic protozoan that appears morphologically similar to
dinoflagellates has been found in the northeast Pacific Spot prawn,
Pandalus platyceros. However, polyclonal antibodies raised
against the oyster parasite Perkinsus marinus that have been
shown to cross-react with several crustacean-parasitic dinoflagel-
lates did not bind to this parasite. Muscle and hemolymph tissue
samples from four infected prawns were obtained from Susan
Bower (Pacific Biological Station, British Columbia, Canada) and
DNA was extracted for nucleotide sequence analyses. Actin and
small subunit ribosomal RNA (SSU rRNA) gene sequences were
obtained from all the DNA isolations by amplification using "uni-
versal" primers in the polymerase chain reaction. Two distinct
classes of sequences were obtained for each gene. One class
showed high similarity to available crustacean sequences and was
presumed to be from host DNA. In BLAST searches of GenBank
with the second class of sequences, the highest match scores were
to protozoan sequences. Two different DNA probes for //; situ
hybridizations were designed based on unique regions of the SSU
rDNA sequence presumably from the parasite. Both probes hy-
bridized specifically to parasite cells in histological sections of
infected prawn tissue and did not hybridize to sections from other
invertebrates infected with parasitic dinoflagellates or haplospo-
ridians. Phylogenetic analyses based on the second class of se-
quences were done with data sets of more than 50 protozoan actin
and SSU rRNA gene sequences. Results of parsimony analyses
with both genes consistently grouped the prawn parasite with
members of the phylum Haplosporidia, not with the dinoflagel-
lates. These results confirm that two separate gene sequences from
the parasite have been amplified from infected prawn DNA ex-
tracts, and strongly suggest that this parasite has haplosporidian
affinities.

648 Abstracts. 2000 Anniuil Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

IDENTIFICATION OF A PROTOZOAN PARASITE IN
THE AMERICAN LOBSTER, HOMARUS AMERICANOS,
FROM LONG ISLAND SOUND. Spencer Russell,* Kristen
Hobbie, Tom Burrage, Claudia Koerting. Sylvain De Guise,
Salvatore Frasca Jr., and Ricliard A. Frencli. University of Con-
necticut, Dept. of Pathobiology. 61 North Eagleville Rd. U-89,
Storrs, CT 06269.

Mortalities of the American lobster, Homanis amerkanus, in
Long Island Sound have severely increased and as a result is
critically damaging the regional lobster industry. Necropsies were
performed on 75 individual lobsters collected from six different
locations in Long Island Sound. Gross observations observed in
'sick" lobsters included a pink discoloration to the ventral surface
of the abdomen (tail meat) and lethargic/limp behavior. An asso-
ciated coagulopathy of hemocytes is also observed in affected
lobster. Initial bacteriology findings include isolation of Vibrio
spp. and spirochetes. No Acracoccus have been isolated to date.
Histologic examination has been conducted on various tissues,
including heart, gill, hepatopancreas. antennary glands, intestine,
muscle, exoskeleton, eyes, antennae, and central nervous system.
The histopathology is consistent with a systemic inflammatory
disease affecting multiple tissues but primarily the nervous system.
Associated with lesions is a protozoan parasite morphologically
characterized as an amoeba, tentatively paramoeba sp.

COMPARATIVE EVALUATION OF THE MULTIPLEX
PCR WITH CONVENTIONAL DETECTION METHODS
FOR HAPLOSPORIDIUM NELSONI (MSX), HAPLOSPO-
RIDWM COSTALE (SSO), AND PERKINSUS MARINUS
(DERMO) IN THE EASTERN OYSTER, CRASSOSTREA
VIRGINICA. Spencer Russell, Soledad Penna, and Richard A.
French, University of Connecticut. Dept. of Pathobiology, 61
North Eagleville Rd, U-89, Storrs, CT 06269.

Presently, the monitoring of cultured oyster populations for
pathogens is infrequent due to the dependence on traditional, time
consuming diagnostic assays. A multiplex polymerase chain reac-
tion (MPCR) has been developed which rapidly detects the proto-
zoan parasites. Perkinsus inarinus (Dermo). Haplosiniricliiiin ncl-
soni (MSX) and Hai'losjxiriiliiiin ciistalc (SSO), which infect the
cultured oyster. Cra.ssoslrca virginica. Conventional diagnostic
methods (histopathology and Ray/Mackin fluid thioglycollate as-
say) for H. nelscmi. H. costulc and /'. nuiriiuis respectively we
compared and evaluated w ilh the MPCR. Ninety-one adult oysters
were collected from randomly selected beds in Westport. CT, (n =
37) and Mlltord. CT (n = .'>4) anil subjected to all three assays.
The Ray/Mackin assay detected /'. iiuiriiius infections in .59 of 91
(64%) oysters and MPCR revealed infections in 73 of 91 (80%)
oysters. Histological examination detected 37 of 91 (4()7r) oysters
infected with Haphispuridiuiu Plasmodia. The MPCR was able to
dilTcrentiate between the two lliiplosporidiiini plasmodia. delect-

ing 9 of 91 (10%) oysters infected only with H. nelsoni. 37 of 91
(40%) oysters with only H. costale. and 32 of 91 (35%) oysters
with mixed infections of H. nelsoni and H. costale. These results
indicate the MPCR is a more sensitive assay for the detection of P.
mariniis and is able to detect and differentiate between the two
Haplosporidium species. This would suggest that the MPCR can
be useful at low infection intensity by being able to detect patho-
gens, based on pathogen DNA concentrations as low a 10 fg., for
H. nelsoni and 1 pg. for both H. costale and P. mariniis.

RESULTS OF QPX FIELD STUDIES. Roxanna Smolowitz.*
Ernest Marks, and Chris Brothers, Marine Biological Labora-
tory, Woods Hole, MA; Dale Leavitt and Bruce Lancaster,

Woods Hole Oceanographic Institution, Woods Hole. MA.

Studies of QPX. (Quahog Parasite Unknown) begun in Octo-
ber, 1997, are presently being conducted in Provincetown and
Duxbury, MA. Spawn from three parentage groups were planted in
experimental plots, 10 ft x 10 ft. Parentage profiles of the three
hard clam (Mercenuria mercenaria) stains were: 20% wild/80%
notata (mixed parentage), 100% notata and 100% wild parentage.
Three types of management methods were used to attempt reduc-
tion of the disease occurrence and severity in the experimental
clams. Hard clams (mixed parentage only) deployed in Duxbury.
MA were planted in previously infected plots that had laid fallow
for one year before planting. Clams in Provincetown were planted
in three locations. One location was in a lease which contained
residual infected clams and represented a positive control plot. The
remaining two locations represented plots in which possible man-
agement methods would be tested. The first of these was located in
a heavily infected lease, adjacent to the positive control plot. How-
ever, this experimental plot's sediment was hydraulically turned
and limed one month before planting. The second of the two types
of managed plots was established in an area that had never been
aquacultured before (naive sediment).

As of the fall, 1999 sampling period, the percentage of mixed
parentage animals grossly positive in Duxbury was high (38%
contained mantle nodules), although no significant mortality was
noted. Based on previous experience, it is expected that mortality
will occur during the spring of 2000. As of fall, 1999, the mixed
parentage clams collected from Duxbury were significantly larger
than the mixed parentage Provincetown clams. It is therefore un-
likely that decreased food quantity/i.|uality of a bay or estuary is a
significant factor (stressor) in the positive development of the dis-
ease. Additionally, allowing land to lie fallow for a year before
planting does not appear to prevent the infection of subsequently
planted hard clams.

No nodules were noted grossly in any of the hard clam strains
sampled in Provincetown in the fall of 1999. The possibility of
strain resistance to QPX infections cannot be exaluated till Mibse-
tliienl samples are collected.

National Shellt'ishcries Association. Seattle, Washington

Abstracts. 2()()() Annual Mcetina. March 19-23. 2000 649

DNA-BASED MOLECULAR DIAGNOSTICS FOR THE
HARD CLAM PARASITE QPX (QUAHOG PARASITE UN-
KNOWN). Nancy A. Stokes,* Lisa M. Ragone Calvo, and Eu-
gene M. Burreson, Virginia Institute of Marine Science. College
of William and Mary, Gloucester Point, VA 23062.

The protistan parasite QPX (Quahog Parasite Unknown) has
been reported in populations of cultured hard clams Mercenaria
menenaria in New Brunswick and Prince Edward Island, Canada
and in Massachu.setts, New Jersey, and Virginia. USA. Aspects of
the life cycle, taxonomy, and epizootiology of QPX are poorly
understood. To date the sole diagnostic method for the pathogen is
histological examination of tissue sections. Development of addi-
tional diagnostic tools in the form of DNA probes for in situ
hybridization and primers for polymerase chain reaction (PCR)
amplification will enable researchers to better understand QPX and
its disease process.

Genomic DNA was isolated from QPX cultured cells and the
small subunit ribosomal DNA (SSU rDNA) was PCR amplified
and sequenced. The SSU rDNA sequences of QPX. M. merce-
naria. and several members of the phylum Labyrinthomorpha,
which is currently the tentative placement of QPX, were aligned
and regions specific for QPX were located. DNA-based diagnostic
tools were designed based on two of these specific regions. The
DNA probe specifically hybridized to QPX cells in tissue sections
of infected clams collected from Virginia and Massachusetts. The
PCR primers specifically amplified a 665 bp region of the QPX
SSU rDNA and were able to detect the QPX target sequence from
infected clam genomic DNA or from I fg of cloned QPX SSU
rDNA.

EFFECTS OF PERKINSOSIS ON THE CLAM RUDITAPES
DECUSSATUS INDUSTRY OF GALICIA (NW SPAIN). An-
tonio Villalba,* Sandra M. Casas, Maria J. Carballal. and Car-
men Lopez, Centre de Investigacions Mariiias, Aptdo. 13, Vil-
anova de Arousa 36620, Spain.

A research program is being developed to evaluate the potential
effect of perkinsosis in clam Ruditapes decussatits populations of
Galicia. Temporal patterns of variation of parasitization by Perk-
insus atlanlicus are being studied. Samples have been taken
monthly, since 1996, from a bed with high perkinsosis prevalence.
Clam size (age) and infection intensity were significantly corre-
lated. No infection was detected in clams smaller than 20 mm in
length. Every clam longer than 48 mm was infected. A seasonal
pattern of infection intensity variation was observed in the 4 study
years. High values were found in spring and late summer — early
autumn. The lowest values were recorded in winter.

Influence of the parasite on clam growth is being studied
through the effects on clam's energetic physiology. Experiments
were performed to estimate respiration and clearance rates of 50

clams, at 15 "C temperature and 35 ppt salinity. Then, clams were
processed for disease diagnosis. Both physiological rates were
lower only in heavy infections. New experiments will be per-
formed at warmer conditions. Consistently, a significant decrease
of condition was detected only in heavy infections, both in ripe and
resting gonad periods. Nevertheless condition decrease was not
severe in any case.

Influence of temperature and .salinity on zoosporulation and
viability of free life stages of P. atlanlicus was evaluated. Pre-
zoosporangia were exposed to different salinity conditions (2.5. 5.
10, 15, 20, 25 and 35 ppt), at 2 temperatures (19 and 28 "C). In
other experiment, prezoosporangia were held at different tempera-
tures (4, 10, 15, 22. 28 and 32 °C), at 30 ppt salinity. Zoosporu-
lation occurred in wide ranges of temperature (15-32 °C) and
salinity (10-35 ppt). The optimum values were 19-28 °C and
25-35 ppt. The highest temperatures in Galician rias (20-22 °C)
are within the optimum range for zoosporulation. Some prezoospo-
rangia survived up to 66 and 129 days at 4 and 10 °C, respectively.
Prezoosporangia that have been held at 4 and 10 °C for 16 and 67
days, respectively, and subsequently transferred to 22 and 28 °C,
gave rise to zoospores. Thus, prezoosporangia could overwinter
without zoosporulating. and would produce zoospores in favour-
able conditions. Zoospores survived for more than 20 days, at 28,
20. 15 and 10 °C in an experiment to test longevity.

DESIGN CRITERIA FOR MICROALGAL FEEDS PRO-
DUCTION SYSTEMS, AND THE GRAMPS EXPERIENCE.
Gary H. Wikfors* and Barry C. Smith, Milford Laboratory,
Northeast Fisheries Science Center. NCAA Fisheries. Milford, CT
06460; Loy Wilkinson, Coastal BioMarine, Bridgewater, CT
06752.

"We can't afford to feed them!" The "them" of this exclama-
tion are post-set molluscan shellfish, and the "we" are nearly every
shellfish farmer who needs to sell shellfish for more than it costs
to grow them. This universal paradigm drives the nursery strategy
of "raw-water" rearing of post-set shellfish seed. Consequences of
raw- water nursery culture are seasonal (temperature) constraints
on seed production; exposure of young animals to environmental
stresses, predators, and disease; and a loss of control over the
nutritional input to the animals. Land-based nursery culture of
molluscs under controlled conditions would solve most of these
limitations if the cost of producing feeds — cultured algae or pre-
pared diets — is lowered substantially. Current hatchery and nurs-
ery microalgal feed production systems incur costs in the range of
.$100-400 per dry kilogram of algal bioniass. At the high end of
this range, a farmer would need to sell bay scallop meats for about
$750 per pound; thus, a decrease in the range of two orders of
magnitude for microalgal feeds is needed for economical, con-
trolled shellfish rearing.

650 Ahstmcts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

Fundamental knowledge of microalgal culture requirements ex-
ists; therefore, design criteria for new microalgal feeds production
systems must focus further on economics. A simple, economic
analysis of using artificial light as the algal energy source indicates
that -99% of the cost is in electricity for the lights. Accordingly.
use of solar energy can accomplish a cost reduction of one order of
magnitude, but only if algal cultures can be sustained in the vary-
ing energy (light and temperature) field of natural solar cycles. A
new facility has been built at the Milford Laboratory — the Green-
house for Research on Algal Mass Production Systems
(CRAMPS) — to address the challenges of growing microalgal
aquaculture feeds in the undependable sunlight of the northeastern
US. Using familiar, simple culture containers (kalwall tubes and
open tanks), we are applying computer automation, process-
control loops, and other cross-field technologies from contempo-
rary industries, to microalgal culture on a pilot scale (ca. 20.000
liters production per day). CRAMPS successes and remaining
challenges will be described. An economic analysis of CRAMPS
operation indicates production costs in the range of $40 per dry
kilogram of algal biomass, with expectation of 50% savings with
optimization of control systems. This production cost appears to
enable extended, land-based nursery culture of shellfish seed, but
not yet grow-out of shellfish to market under controlled conditions.

TECHNOLOGICAL ADVANCES
IN MOLLUSCAN AQUACULTURE

THE USE OF PROBIOTIC TECHNIQUES FOR CONTROL-
LING BACTERIAL DISEASES IN MARINE INVERTE-
BRATE HATCHERIES. A. O. Alabi, Island Scallops Limited.
5552 West Island highway. Qualicum Beach. B.C. V9K 2C8.
Canada.

One of the difficulties encountered in commercial invertebrate
hatcheries has been the poor larval survivals attributed to attack by
opportunistic bacteria. A wide range of methods are therefore em-
ployed to limit and reduce the number of bacteria occurring in
hatchery water supplies and rearing systems: Chemotherapeutants
depend on a host having a higher tolerance threshold level to the
substance than the target organism. However, these differences are
often marginal and depend on the physiological state of the larvae.
Bacterial resistance has also been reported in response to wide-
spread and indiscriminate use of antibiotics. Other pre-treatment
methods frequently used are filtration, ultra-violet (UV) light ir-
radiation and ozonation. All these methods aim to reduce or elimi-
nate bacteria in the water. However, disinfection or partial steril-
ization of sea water appears to encourage the selective develop-
ment of bacterial communities which differ from those found in
natural sea water.

The onset of bacterial diseases has usuallv been altribulcd to

environmental changes which favor the development of excessive
levels of a particular pathogen.

Obtaining control of the microbial environment of larval rear-
ing systems should therefore permit increased manipulations of the
bacterial flora and lead to increa.sed larval survival. Such control
may be obtained by maintenance of balanced populations of bac-
teria and by the use of defined probiotics. This study details results
obtained in the successful commercial scale production of crusta-
ceans, bivalves and echinoderms using these techniques. Potential
shortcomings in the use of these methods are also discussed.

RECENT DEVELOPMENTS IN MOLLUSC HATCHERY

TECHNIQUES. John Bayes, Seasalter Shellfish Company.
Quercus. Willow Road. Whitstable. CT5 3DW. England.

Low impact and environmentally and economically viable al-
gae systems are described in detail including their installation,
operation and management in climates world-wide, and with par-
ticular reference to temperate regions such as are found in the West
coast of America and Canada. Diet selection and control including
reference to probiotic bacteria populations within the algae culture
systems are described. In addition, water management for larvae
and post-set juveniles to ensure maximum growth and survival is
discussed and will include cost benefit analyses of continuous flow
systems compared to batch cultivation.

RECIRCULATION SYSTEM DESIGN FOR SHELLFISH
WET STORAGE OR DEPURATION. S. Chen,* B. B. Saucier.
J. S. Zhu, and E. Durfey, Department of Biological Systems En-
gineering. Washington State University. Pullman. WA 99164
USA.

Research and development of new technologies that are appli-
cable to shellfish operations will strengthen the competitiveness
and enhance the sustainability of the shellfish industry. This pre-
sentation summarizes the findings of a study on optimal design of
recirculating systems for shellfish depuration and wet storage. The
project was funded by the Saltonstall-Kennedy Fisheries Research
and Development Grant Program and conducted with collabora-
tion from Taylor Resources. Inc. of Shelton. Washington. The use
of water recirculating technology to wet-storage and depuration
has the advantage of being cost effective, environmentally sound,
and location independent, while meeting sanitation and other regu-
latory requirements. Major research results to be presented cover
three major areas related to recirculating system design, including
waste excretion, biofiltration. and disinfection. The research ob-
tained quantitative information on waste generation from Manila
clams and indicated that temperature was a major factor determin-
ing the excretion rate of total ammonia, total Kjeldahl nitrogen,
and biochemical oxygen demand. Equations were developed for
estimating waste excretion under different conditions. The purpose
of biol'iltralion in a rccirculatini: svstcm is to convert ammonia to

National Slielltlsheries Association. Seattle. Washinaton

Abstracts. 2000 Annual Meeting. March 19-23. 2000 651

less toxic nitrate through biological nitrification. The biofiltration
study demonstrated that there was a very high nitrification poten-
tial for the biological filters even at a relatively low (10 °C) tem-
perature. A nitrification potential of 1000 nig of ammonia nitrogen
removal per square meter per day can be achieved for a total
ammonia concentration of 2 mg/1 in the water. The study also
demonstrated that UV disinfection units were very effective in
destroying coliform bacteria. The most important factors affecting
UV disinfection efficiency were the UV transmittance of the water
and the flow rate throush the UV unit.

TRIPLOID PRODUCTION OF MYT/LUS EDULIS IN
PRINCE EDWARD ISLAND. John W. Brake* and Jeffrey
Davidson, Atlantic Veterinary College. University of Prince Ed-
ward Island, and Jonathan Davis, Bay water. Inc. 15425 Smoland
Lane. Bainbridge Island. WA 981 10 USA.

The mussel aquaculture industry in Prince Edward Island (PEI).
Canada is a well established major contributor to the island
economy. PEI mussel production in 1998 was approximately
12.500 MT. with farm gate value exceeding $15.1 M (Can) and
export value exceeded $30 M (Can). The industry supports over
1250 full and part time jobs.

The production of non-reproducing harvestable triploid mus-
sels has been investigated for harvesting during the spawning sea-
son, when product quality and shipping are both problems, allow-
ing the marketing of a high quality product year round. Many
species have been produced and reared successfully as triploids
and the production of triploid Pacific oysters {Crassostrea gigas)
is currently extensively practiced in the Pacific Northwest. Iden-
tified methods of triploid induction (used at different levels or in
combinations) in shellfish include temperature and/or pressure
shocking and the use of chemicals such as caffeine, cytochalasin
B, or 6-dimethylaminopurine.

The mussel industry has recognized the potential of harvesting
triploid mussels during the spawning season. The objective of this
study is to elucidate the optimal triploid induction methods for
commercial use in PEI by the use of a matrix of previously iden-
tified triploidy induction methods. These combinations of methods
are ranked by % induction and % survivorship, as well as feasi-
bility in order to determine the best method. To date, inductions
trials with a '7c triploid induction of >90'7r have been obtained,
however, survival has been lower than expected. A new attempt to
investigate the exact treatment timing window is being investi-
gated in an attempt to improve survivorship. This method involves
epifluorescent microscopy and the timing of treatment based upon
the chromosome separation rather than polar body formation. The
current methodology being used for triploid induction optimization
and successful growth trials will be discussed along with some of
the results obtained to date.

APPLICATION OF A SHELLFISH SCIENCE CLUB
MODEL IN PUGET SOUND. WASHINGTON. Daniel P.

Cheney,* Pacific Shellfish Institute, 120 State Avenue NE #142.
Olympia, WA 98501; John L. Pitts, Bellwether Consulting, 3881
Leland Valley Rd. W.. Quilcene. WA 98376.

The Pacific Shellfish Institute (PSI) provided training and fa-
cility development for science and vocational teachers in schools
and tribal communities throughout the greater Puget Sound region
to apply a shellfish model developed for the Quilcene-Brinnon
Schools Shellfish Science Club. Quilcene. Washington. The pro-
gram and curriculum of this model were designed to teach high
school students how to farm shellfish, maintain water quality and
habitat, and utilize the scientific method in resource conservation.
Winner of the President's National Environmental Education
Award, the model combines community education with a "junior
achievement'", entrepreneurial incentive for students, using farmed
shellfish at local fairs and festivals. The introduction of water
quality education for K-12 students and the establishment of a
working relation,ship with local shellfish farmers moves the class-
room into the field. PSI initially extended the concept to schools
and tribes through a series of workshops. Two high schools were
identified with staff and facilities suitable for the program. Teach-
ers and students were introduced to a variety of shellfish culture
concepts. These included: a) classroom instruction; b) population
assessments and surveys on a commercial oyster culture site; c)
farm tours to view shellfish poly culture (clams, oysters, geo-
ducks); d) shellfish hatchery and processing plant field trips; and e)
geoduck farming techniques and research site sampling. PSI is
continuing to work with both schools to assist them with technical
information, shellfish bed management, and coordination with
shellfish farmer mentors. This project was supported by a grant
from the Puget Sound Water Quality Action Team, Public Involve-
ment and Education Fund.

MANIPULATION OF THE CEMENTING PROCESS OF
THE PURPLE-HINGE ROCK SCALLOP, CRASSADOMA
GIGANTEA. Carolynn S. Culver.* John B. Richards, and
Henry M. Page, Marine Science Institute. University of Califor-
nia. Santa Barbara. CA 93106.

Culture of the purple-hinge rock scallop. Crassadoma gigantea
(formerly Hinniles multirugosus) is of interest to many West Coast
aquaculturists. However, grow-out techniques used to culture other
bivalve species are not directly suitable for rock scallops. In con-
trast to other species, rock scallops end their free swimming phase
and usually attach permanently to hard substrata. Permanent at-
tachment is problematic for culturists. The shell becomes very
irregular during growth, conforming to the substrate topology.
This irregular shell shape, in addition to its firm attachment, causes
harvesting and marketing problems. Damage to the product and
culturing gear often results during removal of the attached scallop,
reducing product value and increasing production costs.

652 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

Because of the difficulties associated with scallop attachment,
economically feasible culture will depend on development of
methods for control or manipulation of the cementing process. We
have begun studies to identify biological and physical factors af-
fecting this process. Preliminary investigations suggest that once
competent to cement, rock scallops retain this ability throughout
their life. Size is an important biological factor affecting the ce-
menting process. However, size of attachment varies among habi-
tats, with some individuals never cementing. This indicates that
factors in addition to size are required for permanent attachment.
Evaluation of several physical characteristics of the attachment
substrate found that substrate type, texture and contour influenced
permanent attachment. Based on these findings, we have initiated
pilot field studies to evaluate various grow-out techniques, and to
assess the potential for manipulation of the cementing process of
C. gigantea.

PEI MUSSEL AQUACULTURE: CHANGING TECHNOLO-
GIES. T. Jeffrey Davidson,* Atlantic Veterinary College. Uni-
versity of PEL Charlottetown. PEI CIA due 4P3; Richard K.
Gallant, Department of Fisheries and Tourism, Government of
PEI, Charlottetown. PEI CIA 7N8.

Mussel aquaculture on PEI began in the late I970"s as an
experiment, progressed to a pilot project and is now in full com-
mercial production. At the onset of the industry, technology was
not well refined. Many new ideas and innovations had to be de-
veloped to adapt to the PEI estuarine environment and especially
to survive the harsh winter conditions. The industry's development
and sustainability was particularly due to the innovation of the
growers involved with the advancement of technologies. Because
prices for mussels have remained stable to growers for the past 10
years, the industry has had to find innovative ways to decrease
production costs and increase production. This presentation will
outline some of the developments and advancements in technology
that have taken place over the past 20 years in the culture, harvest,
transport and processing of mussels. They include specialized
aquaculture barges, hydraulic lifting equipment, innovative meth-
ods to decrease manual labour, improved cultiue equipment and
techniques to control predators.

DESIGN AND EVALUATION OF FLOATING UPWELLER
SYSTEMS FOR NURSERY CULTURE OF JUVENILE
CLAMS AND OYSTERS. Clirlstopher V. Davis.* Pemaquid
Oyster Company. P.O. Box 302. Waldoboro. ME 04.572; Dale F.
Leavitt, Southeastern Massachusetts Aquaculture Center, c/o Hur-
ley Library — Mass. Maritime Academy. 101 Academy Lane. Buz-
zards Bay. MA 02532; Joseph A. Mariano. Khuul Insiimie. 410
Main Street. Rockland. ML ()4S41.

Shellfish growers currently use various configuralions ol land-
baseil and floating upwcllcrs to expedite the nmsery phase of
commercial bi\al\e cultiue operations. Land-based upwellers s)s-

tems typically allow for ease of maintenance and security, but
depending on the elevation, tend to have high pumping costs. A
benefit of Floating Upweller System (FLUPSY) designs is greatly
reduced or zero pumping cost due to the minimal (2-10 cm) hy-
drostatic head required to force water through the upweller silos.
Although raft-based tidal-powered systems have no power require-
ments, adequate flow rates are intermittent and limited to sites with
suitable tidal tlow. Water pump-based FLUPSYs typically employ
centrifugal or axial tlow electric pumps to propel water through the
system. In contrast, low head airlift pumps are capable of moving
large volumes of water, are inherently simple to construct and
maintain, eliminate the risk of electrical shock, and have lower
operating costs compared to most electrically driven water pumps.
Pumping efficiency in airlift systems will be discussed as it relates
to the air supply, depth of air injection, lift, diameter of the edu-
cation pipe and air tlow. This presentation will describe perfor-
mance characteristics of FLUPSYs operated in New England wa-
ters and methods of optimizing the upweller design to both maxi-
mized growth rates and minimized operating costs.

THE USE OF A SCHIZOCHYTRIUM BASED DRY FEED
FOR JUVENILE REARING AND BROODSTOCK CONDI-
TIONING OF BIVALVE MOLLUSCS. Jonathan P. Davis*
and Clea R. Barenberg, Taylor Resources Bivalve Hatchery. 701
Broad Spit Road. Quilcene, WA 98376.

Cultured live algae represent a significant cost associated v\ ith
juvenile rearing of bivalves, including costs associated with brood
stock conditioning of adults. The availability of a low cost, lipid-
rich. heterotrophically grown Schizochytrutni sp. based dry formu-
lated feed (Sanders Brine Shrimp Company. Ogden. UT) has
stimulated research on its use as a supplement for use in feeding
larvae, post-set juveniles and adults in a variety of bivalves in-
cluding mussels (Mytihis fiaUoproviiuiaUs) and geoduck clams
(Panopea ahnipta).

Feeding trials were conducted using the Siluzocliyiiiniu based
feed at three supplement levels in juvenile mussels and geoducks
with differing results. Juvenile mussels maintained on the formu-
lated feed at 339r and 67'f and \0(Wc supplemental lesels. respec-
tively demonstrated similar increases in growth compared to seed
mussels grown on li\e algae only. Mussels fed at the lOO'/r supple-
mental level, for example, grew at 3.2% per day compared to 3.9%
per day for mussels fed live algae only. In addition, elevated levels
of long chain falty acids (primarily DHA) were obser\ed in mus-
sels fed the formulated feed compareil to mussels led li\e algae
only.

Juvenile geoduck clams demonsiraled (he opposite result as
clams maintained on the formulaled diet at 50'» and 75'r supple-
menial levels showed reduced growth compared to clams main-
tained on live algae only. In both cases, juvenile mussels and
geoducks were fed similar cell densities of live algae only, or
combinations ^1'i live aluae and disassociated Schizoclntriiim cells.

National Shellfisheries Association. Seattle, Washington

Abstracts. 2000 Annual Meeting, March 19-23. 2000 653

Additional feeding trials were conducted on adult brood stock
geoducks at 25 and 50% supplemental levels. Feeding rates were
maintained over a 4 week period and eggs from female clams
assessed for fatty acid content and subsequent larval performance.
These results, as well as the potential of utilizing dry formulated
feeds are discussed in terms of the comparative costs of rearing
live alsae at commercial bivalve facilities.

APPLIED TECHNOLOGICAL DEVELOPMENT FOR
HARD CLAM (MERCENARIA MERCENARIA) AQUACUL-
TURE IN NEW JERSEY. George E. Flimlin, Jr., Rutgers Co-
operative Extension. Toms River. NJ 08755.

The predominant form of shellfish culture in New Jersey
coastal bays is that of the Hard Clam or Northern Quahog. Mer-
cenaria mercenaria. Crop predation and biofouling of predator
control screens are significant contributors to mortalities through-
out the local industry. Inconsistent data on the exact numbers
planted and later harvested also affect the true accounting of suc-
cess.

Three small-scaled projects were initiated to address these
problems. The first was the field testing of a high frequency sound
blaster which was designed to repel blue crabs. CaUinectes sapi-
diis. from the planting area so that these crabs would not be en-
trained and thus covered over with the screen designed to exclude
them during the planting operation. The second was the develop-
ment of a device, which would clean the macroalgae from the
surface of the predator control screens, so that the unchecked algal
growth wouldn't suffocate the crop. Relying on a rotating brushes
attached to a slightly buoyant submersible mechanical device, this
machine may allow the grower to move away from the hand held
scraper normally used. And the third, was the development of a
seed counter that would allow the hatchery operator to better con-
trol the numbers of seed shipped or segregated for planting in
individual plots.

FLAT OYSTER CULTURE IN NOVA SCOTIA: STRATE-
GIES TO OPTIMIZE THE GROVVOUT OPERATION. A. L.
Mallet* and C. E. Carver, 4 Columbo Drive Dartmouth. Nova
Scotia. Canada, B2X 3H3.

This project was initiated to obtain strategic information on the
effect of different rearing systems and protocols on the perfor-
mance of European oysters from seed to market size. Several
strategies were being practiced by the Nova Scotia oyster industry.
but the lack of comparative data precluded an appropriate analysis
of the various methods. For example, there were little information
on the stocking densities for various oyster weight as well as the
handling frequencies for various grow-out systems. Our strategy
also need to take into account ice coverage during the winter
months.

In this study, the final analysis does evaluate the biological

performance of the oysters with the capital and operating costs for
the various growout systems. Overall, 1-y-old oysters did better in
lantern nets in terms of growth and survival. For 2-y-old oysters,
similar performance is obtained in lantern nets and off-bottom
tables whereas performance in 5-level suspended oyster bags was
3070^0% lower for the 1 -y-old and 2-y-old oysters. Overall losses
in tables were typically 40%. but certain operating conditions led
to a loss of 70%. Fouling levels were minimal on oyster tables but
was extensive on the suspended culture gear. Our calculations
suggest a 1-fold difference in labor cost between handling sus-
pended gear vs tables. The cost to produce an oyster, when handled
frequently, suggests that the oyster table is the preferred method
for final growout of flat oysters in Lunenburg.

CULTIVATION OF NATIVE OYSTERS AND NATIVE
MUSSELS ON RAFTS, TRAYS AND ON THE BOTTOM IN
MAINE, USA. Carter R. Newell,* Great Eastern Mussel Farms.
Inc.. P.O. Box 141. Tenants Harbor. ME 04860, Pemaquid Oyster
Company. Damariscotta, ME. USA.

This talk will show current technology in mussel and oyster
cultivation by two companies in Maine, USA. Oyster seed, after
growing in a small hatchery and in upwellers, are transferred to
floating trays where they grow to about 45 mm and are planted on
the bottom at densities of 75 to 100 per square meter. The trade-
mark name Pemaquid was developed early on and is held exclu-
sively by Pemaquid Oyster Company for identification of its oys-
ters in commerce. Rafts for overwintering small seed or holding
10.000 market-sized oysters are shown, utilizing trays from vinyl-
coated wire for holding oysters of different sizes.

Mussels are grown on the bottom using techniques first devel-
oped in the Netherlands, and recent improvements in seed spread-
ing will be discussed. New developments in mussel raft culture,
using technology adapted from Scotland. Spain, the U.S. and
Canada be demonstrated. Rafts of 30 ton capacity are seeded using
a biodegradable cotton wrap and 1 2 meter. 1/2 inch polysteel ropes
with polyethylene pegs and dropper weights. Final harvest yields
of 5 to 10 pounds of mussel per foot of rope, higher than longline
yields, is attributed to the use of pegs. Meat yields of over 50%-
have been achieved consistently in certain coastal Maine bays.
Technology is currently being transferred among members of a
mussel suspension culture working group of some 75 members.

A STUDY INVESTIGATING THE POTENTIAL OF AN AL-
TERNATIVE OYSTER SEED SOURCE FOR VIRGINIA
AQUACULTURISTS. Francis X. O'Beirn* and Mark W.
Luckenbach, Virginia Institute of Marine Science, Eastern Shore
Laboratory, College of William and Mary, Wachapreague, VA
23480.

In Virginia, a consequence of the reduced harvest of wild oyster
iCrassostrea yirginica) stocks has been the de\elopment of inten-
sive, hatchery-based oyster aquacullure. While this mdustry has

654 Abstracts. 2000 Annual Meeting, March 19-23. 2000

National Shellfisheries Association, Seattle, Washington

been growing steadily, one obstacle to its continued expansion is
the lack of a consistent supply of seed oysters both in terms of
quality and quantity. Aquaculturists have continually reiterated
that the biggest impediment to their expansion is the paucity of
seed oysters. East coast hatcheries are engaged in oyster seed
production, but demand and disease exposure considerations limit
availability. Consequently, we have investigated the feasibility of
utilizing the slowest growing oysters produced from the hatchery
(normally discarded) in a novel field nursery system. Four stocks
were deployed in Spring 1998 and three stocks in Fall 1998. at two
sites (Chincoteague and Wachapreague) on the Eastern Shore of
Virginia. Controls were grown in similar conditions to the runt
oysters. Growth and survival in each stock was monitored for 16
weeks. At the Chincoteague site, runt oysters performed equally as
well as the controls. At the Wachapreague site, the controls out-
grew the runt oy.sters. While there were some differences in the
performances of the stocks within a site, major differences were
apparent in growth and survival of stocks between the two growing
areas. The utilization of such "runt" oysters may be feasible, if the
aquaculturist is assured of good growing conditions. Otherwise the
risks associated with the practice may not warrant the investment
of time and resources.

ADVANCES IN THE CRYOPRESERVATION OF GA-
METES AND LARVAE OF THE EASTERN OYSTER. Car-
men G. Paniagua-Chavez,* John T. Buchanan, and Terrence
R. Tiersch, Aquaculture Research Station, Louisiana Agricultural
Experiment Station, Louisiana State University Agricultural Cen-
ter, Baton Rouge, LA 70820; John E. Supan, Office of Sea Grant
Development. Louisiana State University, Baton Rouge LA
70803.

Although the eastern oyster is considered to be an important
species in the United States, little attention has been directed to the
cryopreservation of gametes or larvae of this species. In this proj-
ect, we developed techniques for cryopreservation of gametes and
larvae, and we evaluated potential aquaculture applications to as-
sist hatchery production. Preliminary studies of cryopreservation
of oyster eggs were also performed. Dimethyl sulfoxide and pro-
pylene glycol plus sucrose were used to evaluate toxic effects of
cryoprotectants. Eggs were cooled at 1.5 °C or 2.5 °C/min or
plunged directly into liquid nitrogen. Dimethyl sulfoxide (0.88 M
and 1.75 M) and sucrose (0.12 M. and 0.25 M) were the least toxic
to eggs. The cooling rate yielding least damage to eggs was hi. 5
°C/min. however, only an average of 14 eggs (out of 200) were
stained with fluorescein diacetate and none were fertili/able.
Sperm or trochophorc larvae were fro/en at n2.5 ' C/min to h3() 'C
and plunged in liquid nitrogen. The optimum concentration of
cryoprolcclaiu was lO'/r propylene glycol for sperm and 10% or
15% propylene glycol for larvae. Frozen sperm and lar\ae were
transported to an oyster hatchery at (irand Isle. Louisiana. Thawed
sperm were used to ferlili/c Ircsli eggs. After 4 months. 1. ()()()

oysters from the control group. 230 oysters produced from thawed
sperm, 850 oysters from thawed larvae, and 57 oysters from natu-
ral spat fall were found. Oyster produced from thawed sperm and
larvae developed normally in the hatchery, demonstrating oppor-
tunities for their use in research and in industry.

A LOW COST, FLOATING AXIAL-FLOW UPWELLER
SHELLFISH NURSERY SYSTEM. Gregg Rivara,* Kim
Tetrault, and Michael Patricio, Cornell Cooperative Extension,
Suffolk County Marine Program, 3690 Cedar Beach Road,
Southold, NY 11971.

Originally used in 1996 in Southold, New York the Cornell
axial flow upweller has undergone some design changes making it
easier to operate while cheaper to construct. The current design is
based on a ten, fifty-five gallon silo module using a three-quarter
horsepower "pump" that can be expanded easily.

During 1999, two million hard clams were grown to an average
of 13 millimeters shell length in a four-module, forty-silo unit. The
system will be discussed in terms of construction, stocking, and
maintenance as well as building and operational costs. The pros
and cons of such a system, especially compared to other systems,
will also be discussed.

KUMAMATO OYSTER BROODSTOCK. Anja Robinson,

Coastal Oregon Marine Experiment Station, Hatfield Marine Sci-
ence Center, 2030 SE Marine Science Drive, Newport, OR 97366.

Kumamato oysters (Crassostrea sikamea) were brought to the
west coast of the United States in 1947. Experimental seed was
planted at various locations in Washington, Oregon and California.
Plantings were successful and this encouraged several oyster
growers to purchase commercial quantities of Kumamato seed
oysters yeariy. By 1953, a total of 3181 cases of seed were im-
ported and grown for the cocktail oyster market. Once the hatchery
technology was developed, Kumamato oyster seed was produced
in hatcheries on the west coast of the United States.

In the early seventies, in the first commercial oyster hatchery in
California. Kumamato and Pacific oysters were crossed in the hope
of producing a large, deep-cupped oyster with good meat quality.
However, the cross (called Gigamoto) grew up to be anything but
what was hoped for. Since commercial oyster growers had mixed
the cross with the rest of the Kumamato oyster seed, it was difficult
to separate true Kumamato oysters from the cross. It was not until
1990 that genetic technologies became available to distinguish
Kumamato oysters from Pacific oysters. Accordingly, the differ-
ences in the DNA pattern of true Kumanialo oysters have been
identified and commercial hatcheries ha\c been pro\ idcd with true
Kumamato hroodstock oys(ers so that they can produce pure Ku-
mamato seed.

National Shellfisheries Association. Seattle. Washington

Ahstracls. 2000 Annual Meeting. March 19-23. 2000 655

GROWTH OF JUVENILE CALICO SCALLOP. AR-
GOPECTEN GIBBUS, IN BERMUDA. AND ITS IMPLICA-
TIONS FOR AQUACULTURE. Saniia Sarkis.* Doerte Hors-
field. Greg Wells, Charles King, and Karen Smith, Bermuda
Biological Station for Research Inc.. 17 Biological lane. Ferry
Reach GEOl. Bermuda.

The calico scallop. Argopecren gibhiis. has been studied as a
candidate for aquaculture in Bermuda since 1996. Pediveligers
( 198.5 ± 16.0 |jLm) were set on day 1 1 after fertilisation on .^ mm
black polyethylene netting in I micron filtered seawater at a den-
sity of 23 larvae • ml"': temperature was initially inaintained at 22
°C ± I °C. and gradually decreased over a 1 2 day period to attain
ambient (19 °C ± I °C). Post-larvae were given 18 cells ■ jxl"' on
a daily basis; algal diet consisted of a mixture: Tetnuelmis clniii:
Thalassiosira pseudonana (clone 3H): Isochiysis galhaiui (clone:
TISO)— . On April 8. 1998. day 12 after setting, cultch with settled
spat was evenly distributed in 1 mm black polyethylene pouches
and transferred to longlines in the field; mean shell height at time
of transfer was <1 mm. Eight weeks later, spat were picked from
cultch. measured and counted: mean shell height was 1 1.4 ± 1.4
mm. A subsample of 2000 spat was distributed in 3 mm Japanese
pearl nets at an initial density of 150 spat -net"'. Density was
gradually reduced to 70 individuals • net"' in July, and to 40
individuals • net"' in September to allow for optimum growth.
Monthly growth rate was monitored in terms of shell height, mea-
sured from a subsample of 30 scallops from three pearl nets; sur-
vival rate and sea surface temperature were also recorded monthly.
Shell height increased to 48.0 ± 2.7 mm in 7 inonths; and was
measured to be 58.3 ± 4.5 mm for 21 months old scallops. Growth
rate ranged from 1.5 mm -month"' to 9.1 mm -month"': where
maximum rates were associated with high sea surface temperatures
(29.5 °C) during July and August. Survival rate was constant.
recorded as >90% per net per month. With a potential local market
value of SO. 80 per animal, and a 7 months growth to market size
following transfer to the field, the calico scallop is being further
considered for aquaculture purposes in Bermuda.

TETRAPLOID EASTERN OYSTERS: AN ARDUOUS EF-
FORT. John E. Supan.* Office of Sea Grant Development.
Louisiana State University, Baton Rouge. LA 70803: Standish
K. Allen. Jr., Virginia Institute of Marine Science. College of
William & Mary. Gloucester Point, VA 23062; Charles A.
Wilson, Coastal Fisheries Institute. Louisiana State University.
Baton Rouge. LA 70803.

A surviving brood of tetraploid eastern oysters. Cnissustrea
virginicci (Gmelin). was produced after eight attempts over four
years, using three separate triploid broodstocks. The theory and
technique were similar to those used for the production of tetra-
ploid C. gigas (Thunberg). Ripe triploid female oysters, dubbed
"blumoons"" due to their rare occurrence, were sexually identified

by a gonad smear and checked for ploidy by fiow cytometry
(FCM). Eggs were then stripped from the gonad, rinsed, and hy-
drated for 1 hour in filtered ambient seawater (FAS). After fertil-
ization with haploid sperm, tetraploidy was induced by inhibiting
the first polar body (PBl ) with 0.5 mg/L cytochalasin B; the eggs
were rinsed with 0.05% dimethyl sulfoxide in FAS when an un-
treated sub-sample of eggs exhibited 507c PB 1 . Larvae and result-
ing spat were reared using standard techniques. Earlier attempts
were stymied by the rarity of blumoons in the triploid broodstock.
or poor larval survival. In the three latest attempts. 1.600 putative
triploids were opened to verify .sex and ploidy. Between 0 and 10
blumoons were identified for tetraploid attempts in summers of
1997 and 1998. In June. 1999. the attempt was successful because
older triploids increased the female:male ratio and provided more
blumoons for tetraploid spawns. Approximately 500 juvenile oys-
ters survived from five larval broods of which 42% were tetra-
ploid. The seed are being maintained at the Sea Grant Oyster
Hatchery in Grand Isle. Louisiana and at the Virginia Institute of
Marine Science Aquaculture Genetics and Breeding Technology
Center. Preliminary FCM analyses of some individual oysters in-
dicate the occurrence of 2n/3n/4n mosaicism and 2n/4n individu-
als, probably due to 2n sperm. During indifferent or early game-
togenic development (Jan. -Feb.. 2000). individual tetraploid
adults will be identified by FCM prior to conducting 4n x 4n and
4n X 2n crosses to confirm the resulting production of tetraploid
and triploid generations, respectively.

WATER QUALITY AND
HARMFUL BACTERIA

USE OF HIGH HYDROSTATIC PRESSURE TO CONTROL
PATHOGENS IN RAW OYSTERS, Haejung An.* Hakan Ca-
lik. Haian He, Roger Adams, and Michael T. Morrissey, Oregon
State University-Seafood Lab, Astoria, OR 97103.

Raw oysters are the main vehicle of transmitting diseases in-
duced by Vibrio spp. In 1997. over 80 confirmed cases of Vibrio
parahaemolyticus (Vp) were reported in the Pacific Northwest,
and the cases has lingered in the last two years although the num-
bers diminished. The naturally occurring Vibrio spp. in oysters can
be controlled by use of a new technology, high hydrostatic pres-
sure (HHP) treatment. HHP is a nonthermal process which de-
stroys microorganisms with a minimal effect on overall appear-
ance, taste and texture of raw foods.

We are determining the effects of HHP treatment on Vp in
oysters to establish optimum HHP conditions for processing time
and pressure and evaluate the sensory changes related to HHP
treatment during storage. Different strains of Vp isolated from the

656 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle, Washington

commercial oysters and the clinically confirmed virulent Vp
strains obtained from FDA. Seattle. WA were tested for HHP
effects at different settings of pressure (35-50K psi) and time ( 10
sec- 1 5 min). The results showed that both environmental and
clinical Vp strains were equally susceptible to HHP treatment. The
optimum conditions for reducing pure culture of Vp from 10''
cfu/ml to 10' cfu/ml was achieved at 50K psi in 30 sec. while it
took 14.5 min at 35K psi. On the processing setting suitable for
commercial oysters, generally a 2-3 log reduction of total micro-
bial counts was observed. Sensory test showed HHP treated oys-
ters received the higher quality scores than the control group dur-
ing storage up to 22 days, demonstrating that HHP is a satisfactory
means to control pathogens in raw oysters.

CAN THE TILLAMOOK COUNTY PERFORMANCE
PARTNERSHIP RESTORE THE ECOLOGICAL BAL-
ANCE OF TILLAMOOK BAY? Deborah Cannon, Shellfish
Program Specialist, Food Safety Division, Oregon Department of
Agriculture, 635 Capitol St. NE. Salem, OR 97301; Kim Hatfield,
Scientific/Technical Program Coordinator, Tillamook County Per-
formance Partnership/Tillamook Bay National Estuary Project,
613 Commercial/PO Box 493, Garibaldi, OR 97118.

In 1999 the Tillamook Bay National Estuary Project completed
its Comprehensive Conservation and Management Plan for
Tillamook Bay, Oregon. The TBNEP, funded by the US Environ-
mental Protection Agency's National Estuary Program identified
four priority problems one of which is bacterial contamination and
other water quality issues, its effects on shellfish harvesting and
other human uses. The Tillamook County Performance Partner-
ship, which succeeds the TBNEP, has committed to implementing
sixty-three action plans from the CCMP by 2010. This paper dis-
cusses the challenges to meeting the stated goals: achieve water
quality standards for bacteria in rivers and Bay by 2010; document
at least a 25% reduction in bacteria loads to rivers, with appareiu
trends by 2005 and statistically significant results by 2010; and
achieve at least a 257i reduction every four years in the number of
days that rivers are not in compliance with water quality standards
for bacteria.

THE VARIOUS RELATIONSHIPS BETWEEN SHELLFISH
AND WATER QUALITY. William F. Dewey, Taylor Shellfish
Company. Inc.. 130 SE Lynch Road. Shelton. WA 9S5.S4.

Shellfish Growers dedicate considerable resources to protect
and restore clean w aicr lo produce wholesome shellfish which are
safe lo consume raw. This is the foundation of the National Shell-
fish Sanitation Program. Recogni/.cd as equally important is the
role that filter feeding molluscan shellfish play in cleansing water.
Touted as keystone species, significant efforts have been made lo

reintroduce oysters to New York Harbor and the Chesapeake Bay
to improve water quality in these systems. Aside from the valuable
function the oysters physically serve as habitat and refuge is their
ability to filter huge volumes of phytoplankton which, in heavy
blooms, can cause low dissolved oxygen problems when the
blooms die off as well as blocking critical sunlight for seagrasses
and macroalgae. In the Pacific Northwest a citizens group, orga-
nized in opposition to mussel farm expansion on the basis of the
aesthetic impact, is attempting to change the water-cleansing im-
age of shellfish into a water-polluting image. The group recently
filed suit in federal court claiming the feces, pseudofeces, mussel
shell debris and escaped mussel spat from propagating mussels are
a point source of pollution and require a National Pollution Dis-
charge Elimination System (NPDES) permit under the Clean Wa-
ter Act. This paper discusses the dichotomy between the views of
shellfish as polluters versus the view of shellfish as capable of
improving water quality and habitat.

SHELLFISH WATER QUALITY TRENDS AND THREATS
IN PUGET SOUND. Stuart D. Glasoe* and Duane Fagergren,

Office of the Governor, Puget Sound Water Quality Action Team,
P.O. Box 40900. Olympia. WA 98504-0900.

Puget Sound has some of the world's finest habitat for the
cultivation of clams, mussels and oysters. Commercially, these
products yield an annual farm-gate value of nearly $50 million. In
broader terms, shellfish harvesting is a cherished part of Puget
Sound's rich heritage and quality of life, and serves as a key
measure of the estuary's environmental health.

In the 1980s a number of the Sound's commercial shellfish
areas were downgraded primarily because of nonpoint source pol-
lution and additional monitoring information. This decline stabi-
lized in the 199()s as a result of targeted efforts to restore water
quality. A great success story, right? A broader review presents a
mixed picture and forecasts an uncertain future for the Sound's
shellfish tidelands, especially given the region's fast-growing
population. Upgrades over the past decade have generally been
offset by an equal number of downgrades. Some successful resto-
rations have been reversed by recurring problems. Other sites have
never recovered. And the harvesting classifications in most re-
stored areas are tenuous, requiring constant monitoring and follow-
up work.

Given the persistent nature of these water qualils threats, are
we using our tools and resources to achieve temporary fixes or to
make lasting changes' Do we have the \ ision and resolve to man-
age growth and control pollution in v\ays that will effectively
preserve our environmentally sensitive tidelands, or are we carry-
ing out a mission of haphazard restorations' Experiences in such
areas as Drayton Harbor. Burley Lagoon and Lower Hood Canal
pro\ itie some insight to these questions.

National Shellfisheries Association. Seattle. Washington

Abstracts. 2000 Annual Meeting. March 19-23. 2000 657

THE USE OF MULTIPLE ANTIBIOTIC RESISTANCE
AND MOLECULAR TECHNIQUES (PULSED FIELD GEL
ELECTROPHORESIS AND RIBOTYPING) FOR IDENTI-
FYING COLIFORM POLLUTION SOURCES. G. I. Scott,*
M. H. Fulton. B. C. Thompson, L. F. Webster. A. K. Leight,
E. F. VVirth, and J. Stewart, NOAA/NOS. CCEHBR. Charleston.
SC; G. P. Richards, Dept. of Agriculture. Delaware State Univ..
Do\er, DE; D. Chestnut, SC Dept. of Health and Env. Control.
Columbia. SC; R. F. Van Dolah, SC Dept. of Natural Resources,
Charleston. SC; S. Parveen, Univ. of Florida. Gainesville. PL.

Urbanization may result in significant inputs of contaminants
into salt marsh ecosystems. Significant discharges of bacterial pol-
lution from septic tanks and combined sewer overflows may occur,
often resulting in closure of shellfish harvesting waters due to the
presence of bacterial pollution. In addition, wildlife coliform pol-
lution sources may be present and contribute to shellfish closures.
There is a clear need to develop methods for differentiating human
versus wildlife coliform bacterial sources. Several novel methods
for differentiating human and wildlife coliform bacterial sources
were evaluated including Multiple Antibiotic Resistance (MAR),
Pulsed Field Gel Electrophoresis (PFGE). Ribotyping (RT) and
Fatty Acid Profiling (FAP). Surface water samples were collected
from several estuaries in SC as well as selected sewage treatment
plants and septic tanks. All samples were enumerated for fecal
coliform bacterial densities (MPNs). Samples were then analyzed
by API biotyping to isolate E. coli and were further analyzed by
MAR. PFGE. and RT. Results indicated that the % of E. coli
comprising the coliform group was increased with increasing ur-
banization and high MAR was found in areas adjoining sites with
septic tanks or influenced by sewer discharges. Wildlife areas had
negative MARs and a much lower % of E. coli. PFGE and RT
provided DNA differentiation of bacterial pollution sources in-
cluding septic tanks and domestic animal sources. These findings
indicate that these methods may be helpful in identifying different
sources of fecal coliform bacteria.

DISTRIBUTION OF VIBRIO PARAHAEMOLYTICUS IN
PUGET SOUND OYSTERS. WATER, AND SEDIMENTS
DURING SUMMER 1999. Russell P. Herwig* and Robyn M.
Estes, School of Fisheries. University of Washington. 3707 Brook-
lyn Ave NE, Seattle, WA 98105: Cindy L. Messey and Daniel P.
Cheney, Pacific Shellfish Institute. 120 State Ave NE, #142,
Olympia. WA 98501.

Vibrio parahaemolyticus (Vp) is a bacterium that is naturally
found in estuarine and marine waters around the worid. This or-
ganism can accumulate in filter-feeding organisms, such as oys-
ters, and be a problem with shellfish that are harvested during the
summer months. Selected strains of Vp may cause food borne

illness. In 1997 and 1998, several outbreaks of Vp gastroenteritis
were caused by the consumption of raw oysters harvested from the
Pacific Northwest. During the summer of 1999, oyster, sediment,
and water samples were collected from four sites in Puget Sound.
Washington. Levels of Vp were determined using a newly devel-
oped molecular Most Probable Number (MPN) method. Samples
were blended, diluted, and inoculated into a series of tubes con-
taining Alkaline Peptone Water (APW). Small aliquots of liquid
were removed from each incubated tube and used in a polymerase
chain reaction (PCR). This molecular method amplified a specific
fragment of DNA found in Vp. Using the new method, the enu-
meration of Vp was performed within 2 days, a much shorter time
compared to the FDA-approved method. Our results indicated that
levels of Vp in Puget Sound oysters, sediment, and water were not
detectable or very low in June 1999. increased at selected sites in
July, and remained elevated in August. Concentrations of Vp were
much higher in sediment compared to water samples. Although the
different Puget Sound sites had comparable water temperatures
and salinities during the summer, a site on Hood Canal had much
higher Vp levels in samples collected during July and August. The
reasons for the elevated levels of Vp at particular locations in Puget
Sound are not understood.

OUTBREAKS OF VIBRIO PARAHAEMOLYTICUS GAS-
TROENTERITIS FROM RAW OYSTER CONSUMPTION:
ASSESSING THE RISK OF CONSUMPTION AND GE-
NETIC METHODS FOR DETECTION OF PATHOGENIC
STRAINS. Charles A. Kaysner* and Angelo DePaola. Jr., Food
and Drug Administration. Bothell. WA 98021 and Dauphin Island.
AL 36528.

During the summers of 1997 and 1998. large outbreaks of V.
parahaemolxticiis gastroenteritis occurred from the consumption
of raw oysters in the US. The West Coast outbreak was the first to
have occurred in this country from the consumption of raw mol-
luscan shellfish; over 200 culture-confirmed cases were identified.
Over 400 cases were confirmed from oysters harvested from
Galveston Bay in Texas and 20 cases were confirmed in New York
and Connecticut from oysters originating from Long Island Sound.
Distinct serogroups of the pathogen were responsible for illnesses
on the West Coast in contrast to those on the East and Gulf Coasts.
Monitoring of shellfish samples by State and federal authorities
found low levels of V. parahaemolyticus in all implicated growing
areas, suggesting strains of low infectious dose. FDA has com-
pleted a risk assessment study for consumption of raw molluscan
shellfish as part of the Food Safety Initiative. Newly developed
genetic techniques were employed for the first time to determine
levels of V. parahaemolyticus in shellfish and detect the patho-
genic strains of the species.

658 Abstracts, 2000 Annual Meeting, March 19-23, 2000

National Shellfisheries Association, Seattle, Washington

DOES COMMUNITY INVOLVEMENT LEAD TO LONG-
TERM POLLUTION SOLUTIONS? T. L. King. Washington
Sea Grant Program, 1 1840 North Hwy 101, Shelton, WA 98584.
In Puget Sound, innovative education programs have helped
community volunteers to reduce pollution sources to the point of
reopening shellfish growing areas. Septic soldiers, fecal ferrets,
septic socials, and cranberry PIE programs have all helped to
educate and influence changes, but is this enough? Will it last?
Using case studies from Hood Canal and southern Puget Sound
communities, we will look at the impact these programs have had
two and three years later.

detectable levels thus reducing the risk of infection associated with
raw oyster consumption. In-shell oysters artificially inoculated
with V. vKhiificus and V. piiialiacinolyticits and live oysters natu-
rally contaminated with V. vidiiificus were heat treated with low
temperature pasteurization of 50 "C for up to 15 minutes. Samples
of processed and unprocessed oysters were enumerated for V.
vulnificus. V. parahaemolytkus. and aerobic spoilage bacteria dur-
ing a 0-14 day period. Low temperature pasteurization was effec-
tive in reducing these pathogens from >100,000 to nondetectable
levels in less than 10 minutes of processing. Spoilage bacteria were
reduced by 2-3 logs increasing the shelf-life in excess of 7 days
beyond live unprocessed oysters.

ISSC'S RESEARCH INITIATIVES. Ken B. Moore. Executive
Director, Interstate Shellfish Sanitation Conference 115 Atrium
Way, Suite 117, Columbia, SC 29223.

The Interstate Shellfish Sanitation Conference (ISSC) was or-
ganized in 1982 to address the safety and sanitation of molluscan
shellfish (oysters, clams, and whole and roe-on scallops). The
ISSC has a fonnal Memorandum of Understanding with the U.S.
Food and Drug Administration (FDA) to promote shellfish sani-
tation through the National Shellfish Sanitation Program (NSSP),
a tripartite cooperative program of Federal and State public health
officials and the shellfish industry working together to improve
shellfish safety. The ISSC recognizes that the NSSP is founded on
the premise that, through appropriate controls, molluscan shellfish
can be consumed raw by most people without reasonable risk. The
ISSC remains committed to that promise. In 1994, the ISSC es-
tablished an Executive Office and began to expand the activities of
the organization to more effectively coordinate and facilitate
implementation of the NSSP. The office acquired funding from
several federal agencies. A significant amount of these funds have
been directed to address research needs associated with the NSSP
implementations. To date approximately $400,000 has been
awarded to researchers to address issues associated with molluscan
shellfish public health. The presentation will focus on the organi-
zation, its research szoals and its research activities.

ELIMINATION OF VIBRIO CONTAMINATION IN RAW
IN-SHELL OYSTERS THROUGH LOW TEMPERATURE
PASTEURIZATION. D. L. Park.* Department of Food Science,

Louisiana Agricultural I^xperimcnt Station. Louisiana Slate Lhii-
versity. Baton Rouge, LA 70803; L. S. Andrews, Sugar Process-
ing Research Institute 1 100 Robert E. Lee Blvd., New Orleans, LA
70124; Y-P Chen. Department of Human Ecology, Tainan Col-
lege of Art and Technology, 1-2 52, Sheng-Li Rd., Tainan, Tai-
wan, 701.

Vihrii) viihiijuiis and V. pardlnicmdlylicns are natural inhahii-
ants of cstuarinc environments and may be iransmillcd lo humans
by ingestion of raw oysters. This study focused on llic use of low
temperature paslouri/alion. to reduce these Vibrio species lo non-

POSTERS

FREQUENCY OF SHELL REPAIRS IN COMMON CLAMS

FROM NEW JERSEY. Richard R. Alexander.* Dept Of Geo-
logical & Marine Science. Rider University, Lawrencevilie, NJ
08648; Gregory Diet!. Dept. of Zoology, North Carolina State

University, Raleigh. NC 27695.

Shells of Argopecteti irradians. Aiuulani civcilis. Anoiiiiii sim-
plex. Divaricella quadrisulacta. Dona.x variabilis. Ensis directiis.
Spisiila solidissinia. and Tagehis plebeius were collected from the
sandy tidal flat near Hereford Inlet, NJ (n = 5102). The salt marsh
and adjoining muddy tidal flat at Tuckerton NJ provided shells of
Crassostrea virginica. Ceukensia deinissa. Mercenaria inerce-
luiria. Mya arenaria. Mytihis edulis. and Petricola pholadiformis
(n = 4411). Astarte castanea was sampled from the beaches at
Belmar, NJ (n = 1000). Size-frequency distributions for shell
lengths (widths) at death versus lengths (widths) at inception of
shell repair reveal repair frequency (% of sample) and any size
refuge from sublethal breakage. Repair position was coded as dor-
sal, ventral, anterior, or posterior. Geometry of repaired breakage
was coded as scalloped, divoted, cleft, or embayed. D. cpiadrisid-
cala lacks shell repairs. Repairs in shells of D. variabilis {«\% of
sample) and A. castanea (1%) are usually localized ventrally as
scalloped margins. A. irradians bear mostly clefts (<29r) between
radial ribs. Repaired vahes in K photadifarmis {y/c) show poste-
riorly concentrated scalloped margins. Repaired shells of S. so-
lidissima (6%) include many embayed fractures on the ventral-
posterior. A si/c refuge from suhlclhal fracture exists. Shell repairs
in 7'. plebeius (59f ) are located anteriorly, ventrally, and posteri-
orly. Repairs in A. avails (8%) are concentrated ventrally, where
the foot protrudes. Shell repairs in G. demissa (lO'/r) are posteri-
orly concentrated divots mostly in smaller size-classes. All types
of shell repairs occur around the commissure of C. virginica
{ I I '» 1, but most are anterior scaliop'-"'-! margins. Shell repairs in M.
mercenaria ( 1 2'r ) are mostly posterior triangular divots that occur
over a wide range of size classes and are usually proximal to where
siphons prolruilcd. For M. edulis. shell repairs (19'*) of all four

National Shellt'isheries Association. Seattle. Washincton

Abstracts. 2000 Annual Meeting. March 19-23. 2000 659

types occur at all shell widths, but are located posteriorly almost
exclusively. Repairs in A. simplex (22%) occur at all sizes and are
concentrated ventrally. Shell repairs in E. directus (21%) occur at
all size classes as posteriorly scalloped margins near where si-
phons are incompletely withdrawn. Repairs in M. arenaria (317f )
occur over a wide range of shell widths at the anterior, posterior,
and ventral margins where the foot, siphons, and mantle, respec-
tively, are exposed when valves are closed.

SELECTION AND GROWTH OF CRASSOSTREA VIRGINI-
CIA BASED ON WATER QUALITY. Troy D. Alphin* and
Martin H. Posey, Depart. Biological Sciences. University of
North Carolina at Wilmington. Wilmington. NC 28403; David W.
Freshwater and Robert A. Yorlv, Center for Marine Science Re-
search, Wilmington, NC 28403.

The eastern oyster, Crassostrea virginica, has experienced
population declines over the last few decades throughout the At-
lantic and Gulf coasts, causing concern among both fishermen and
resource managers. This decline has been attributed to overharvest.
disease, and declining water quality and is currently the focus of
many studies. In North Carolina, as in other states, programs are in
place to help reverse this trend, through oyster relay programs.
placement of oyster shell to promote settlement, and experimental
breeding programs, Experimental breeding and outplant programs
ha\e had mixed success, especially when attempting to establish
populations in impacted systems. One contributing factor has been
suggested to be differences between populations having long-term
exposure to eutrophic and/or turbid conditions. This would suggest
the need for using differing parental stocks in certain outplant
programs. This project focuses on assessing the feasibility of using
Inter-SSR and RAPD methods for detecting possible population
structure and water quality specific markers in oysters. In addition
to genetic techniques, size distribution and density data will be
collected from the same oyster populations and correlated with
background water quality.

GAMMA IRRADIATION EFFECTS ON EARLY LIFE
STAGES OF THE EASTERN OYSTER. Ingrid Ardjosoe-
diro,* Nyanti Lee, John Supan, and Terrence R. Tiersch, Aqua-
culture Research Station. Louisiana Agricultural Experiment Sta-
tion. Louisiana State University Agricultural Center. Baton Rouge,
LA 70820; John E. Supan, Office of Sea Grant Development,
Louisiana State University, Baton Rouge, LA 70803.

The application of radiation has been used in invertebrate stud-
ies to investigate growth increases, and to induce sterility, such as
the control of agricultural pest species. The possibility of creating
a sterile oyster is of importance for the production of higher meat
yields during the spawning season and for the use of transgenic
oysters and exotic species. Preliminary studies were performed
during May to July of 1998 to evaluate the effects of gamma

irradiation on larvae of the eastern oyster. Larvae were irradiated
at 24 h or 48 h after fertilization, using a cobalt-60 gamma irra-
diator (Shepherd model) at doses of 0, 3, 5, 10, 15, 20, or 25 krad,
at a rate of 1528 rad/min. There were significant differences in
mortality between 0 and 5 krad and between 3 and 5 krad in the
48-h larvae at 24 h after irradiation. At 36 h after irradiation, these
larvae showed no significant differences in mortality among treat-
ments. These preliminary results indicate that the effects of gamma
radiation are dependent on larval stage and time after irradiation.
This project was continued during the summer of 1999 when
the larval stages trochophore, D-stage, and umbo were exposed to
gamma radiation at doses of 0, 5, 10, 15, or 20 krad. Growth and
survival was monitored at 2 d, 4 d, and 6 d after radiation. It was
observed that resistance to radiation increased with the develop-
mental stage of the larvae. These studies provide an initial step in
evaluation of the utility of irradiation in oyster production. Further
studies will address the reproductive ability of irradiated oysters.

FEEDING SELECTIVITY OF NATIVE FRESHWATER
MUSSELS (UNIONIDAE) AND COMPETITION WITH ZE-
BRA MUSSELS. Shirley M. Baker* and Jeffrey S. Levinton,

Dept. Ecology and Evolution, State University of New York,
Stony Brook. 11790.

The invasion of the Hudson River by the zebra mussel {Dre-
isseiia polymorpha) has resulted in a decline of cyanobacteria.
Microcystis in particular, and a rise to dominance by diatoms.
Since the invasion, the density of native mussels (Family Unioni-
dae) has declined 30-90%. Our objective was to examine feeding
selectivity in unionid mussels and determine the potential compe-
tition for food between native mussels and zebra mussels. We
examined feeding rate and feeding selectivity of several species of
native mussels, using flow cytometry. We found that, like zebra
mussels, unionids are capable of efficiently sorting and rejecting
particles. As in zebra mussels, the cyanobacterium Microcystis
was preferentially ingested by unionids over almost all other par-
ticle types tested. One exception, however, was the unionid
Amblema plicata, which preferentially ingested a diatom, Cyclo-
tella. over Microcystis. Amblema has been less severely affected
by zebra mussels than have many other unionid species. Like zebra
mussels, unionids vary their clearance rates according to the com-
position of the suspension. Unionid clearance rates were signifi-
cantly lower in the presence of typically rejected particles such as
the large green alga, Scenedesmus. Although Microcystis was a
preferred particle type, unionid clearance rates were not stimulated
by its presence to the same degree as zebra mussel clearance rates
are. Our selectivity and clearance rate data suggest that varying
degrees of competition with zebra mussels for particular, and lim-
ited, food types may be an underlying factor in the decline of
native mussel abundance and diversity.

660 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

VIABLE BUT NON-CULTURABLE RESPONSE FOR
PHASE VARIANTS OF VIBRIO VULNIFICUS IN CLAMS.
Wafa Birbari, Anita Wright,* and Gary Rodrick, Food Science
and Human Nutrition Dept.. University of Florida. Gainesville. FL
32611.

Vibrio vulnificus is the leading cause of human mortalities from
bacterial infections associated with the consumption of seafood,
particularly raw oysters. Decreased disease prevalence correlates
with colder water temperatures that have been shown to induce a
viable but non-culturable (VBNC) state. Resuscitation to the cul-
turable state is achieved by elevating incubation temperature prior
to cultivation. V. vulnificus also exhibits phase variation between
opaque (O) and translucent (T) colony morphologies that corre-
lates with capsule expression. In microcosms, both phase variants
were shown to exhibit similar kinetics for induction and resusci-
tation of VBNC cells, and morphotypes remained stable. VBNC
cells could be resuscitated from clams after 48 h. Uptake of V.
vulnificus in clams induced a conversion of O to T morphotype but
not in the other direction. Studies are ongoing using mutants with
genetically defined disruptions of the CPS locus.

PERKINSUS MARINUS POPULATION DYNAMICS IN
NORTH INLET, SOUTH CAROLINA— AN ECOSYSTEM
MODEL. Jodi Brewster* and Dave Bushek, Baruch Marine
Field Laboratory. Baruch Institute for Marine Biology and Coastal
Research, Georgetown. SC 29442; Richard Dame, Department of
Marine Science, Coastal Carolina University. Conway. SC 29528.
Perkinsus marinus, a protozoan parasite of the eastern oyster
(Crassostrea virginica), influences oyster population dynamics in
estuarine ecosystems on the east and Gulf coasts of the United
States. It is the causative agent of "dermo" disease that has con-
tributed to the collapse of eastern oyster fisheries. The parasite can.
however, exist within an estuary without causing extensive mor-
talities. The mechanisms that regulate P. marinus populations at
the ecosystem level, and thus its impact on oyster populations,
remain unclear. The parasite is transmitted through the water col-
umn, encountering host oysters as they filter the water. Oysters can
remove a portion of the filtered parasites, but many will invade
tissues and proliferate within the oysters. Those factors that deter-
mine the fate of parasites within the oysters and during planktonic
transmission are key to understanding the mechanisms that affect
P. marinus epizootics within the oyster population. A box model
was constructed using Stella 5.1 software to simulate the popula-
tions dynamics of P. marinus in North Inlet Estuary, South Caro-
lina. Our objective is to develop a model that can be used to test
new concepts and hypotheses, and to evaluate consequences of
climate change, estuarine variability, and anthropogenic impacts
on the P. marinus populations. The information obtained should
help define better oyster reef management strategies.

PERKINSUS DISEASE PROGRESSION IN FIELD OYS-
TERS: A MODELING STUDY. Diane J. Brousseau, Fairfield
University. Fairfield, CT 06430: Jenny A. Baglivo. Boston Col-
lege. Chestnut Hill. MA 02467.

During 1997, oysters from six locations in Connecticut (Black
Rock Harbor. Bridgeport; Saugatuck River. Westport; Thames
River, Waterford; Mystic River, Stonington). New York (Oyster
Bay) and Massachusetts (Cotuit) were analyzed for the presence of
Perkinsus marinus and water temperatures were monitored at each
site. Median values of disease prevalence were between 96% and
100% for adult oysters and between 30% and 68% for juveniles.
Infection levels in oyster parasites began climbing when water
temperatures reached 1 3- 1 6 °C at the four Connecticut sites. These
results substantiate earlier observations made for the Bridgeport
site and suggest a different pattern of infection development from
that reported in oysters from locations further south, where tem-
peratures >20 °C are required. A temperature-disease course
model was developed to predict the effect of seasonal water tem-
perature changes on disease progression of P. marinus in field
populations of Crassostrea virginica in Long Island Sound. This
model allows the grower/manager to predict Dermo intensity in
shellfish beds if field water temperature patterns are known. Such
information can be used to select oyster growout beds and deter-
mine optimal time to harvest.

IDENTIFICATION OF A SERINE PROTEASE GENE IN
PERKINSUS MARINUS. Gwynne D. Brown* and Kimberly S.
Reece, Virginia Institute of Marine Science. The College of Wil-
liam and Mary, Gloucester Point, VA 23062.

Perkinsus marinus was identified as the causative agent of
Dermo disease in the eastern oyster. Crassostrea virginica, in the
late I940"s. Fifty years later P. marinus has expanded its geo-
graphic range, heavily impacting previously unaffected oyster
populations of the Northeast, yet little is known regarding the
pathogenic mechanisms of this parasite. Identification of proteins
whose activities and gene expression levels correlate with viru-
lence will promote an understanding of disease mechanisms and
facilitate the development of more effective disease management
strategies. Proteases have been found to play a key role in patho-
genesis of several parasitic protozoans. We have identified a serine
protease gene from P. marinus using "universal" degenerate prim-
ers in the polymerase chain reaction to amplify a 475 bp fragment.
This gene appears to be closely related to the subtilisin gene family
of serine proteases. Using digoxigenin to label the amplified frag-
ment we have screened a P. marinus \ phage genomic library.
DNA from hybridizing phage has been isolated and subjected to
Southern blot analysis. At least two different types of recombinant
clones have been identified. We are cunently in the process of
subcloning and sequencing the DNA fragments to characterize the
complete serine protease genes.

National Shellfisheries Association. Seattle. Wasiiinizton

Abstracls. 2000 Annual Meetin". March 19-23, 2000 661

RESEARCH-SCALE CULTURE OF OYSTER LARVAE.
John T. Buchanan, °^' Department of Oceanography and Coastal
Sciences. Louisiana State University. Baton Rouge. LA 70803;
Carmen G. Paniagua and Terrence R. Tiersch, Aquaculture
Research Station. Louisiana State University Agricultural Center.
Baton Rouge. LA 70820; Richard K. Cooper, Department of
Veterinary Science. Louisiana State University. Baton Rouge. LA
70803.

The eastern oyster, Crassostrea virginica. comprises an impor-
tant national fishery. Research on gene transfer and cryopreserva-
tion of gametes and larvae can lead to improvement in this indus-
try, although it was necessary to develop research-scale techniques
for the holding of oysters and the culture of larvae. Along with
consistent production of high quality gametes and larvae, consid-
eration must be given to experimental replication, avoidance of
contamination, and containment of genetically-modified organ-
isms. All of our work was done with artificial seawater in recir-
culating systems over 100 km from the nearest coastal area. We
examined the effect of several variables on the production of ga-
metes and larvae of the eastern oyster. First, we developed proto-
cols for holding broodstock in the laboratory, acclimation to labo-
ratory conditions, and collection of gametes. We developed meth-
ods to assay gamete quality and for cold storage of gametes.
Second, we optimized methods for the small-scale production of
oyster larvae by artificial fertilization by examining the effect of
container volume, aeration, and artificial water source on larval
survival. Using these techniques, we successfully transferred the
gene for red-shifted green fluorescent protein (rsGFP) into oyster
sperm, embryos and adults. These techniques have also been used
in experiments to cryopreserve eastern oyster sperm and tro-
chophore larvae. A program such as this would be useful for the
siTiall-scale production and culture of shellfish larvae for a variety
of experimental purposes.

THE EFFECT OF OYSTER REMOVAL ON INTENSITIES
OF PERKINSLS MARINUS INFECTIONS IN NATIVE OYS-
TER POPULATIONS. Emily Butsic* and Richard Dame, De-
partment of Marine Science. Coastal Carolina University. Conway.
SC 29526; David Bushek, Baruch Marine Field Laboratory. Uni-
versity of South Carolina. Georgetown. SC 29442.

The parasitic oyster pathogen Perkinsiis marimis has caused
many problems for the east and Gulf Coast oyster industries. Rec-
ommended management strategies include fallowing beds after
removing infected oysters and timing the planting and harvesting
of oysters around seasonal infection cycles. To examine the effec-
tiveness of the fallowing strategy, we measured P. marinus infec-
tion levels before and after the removal of native oysters. Oysters
from three to five locations along each of eight intertidal creeks in

North Inlet Estuary. South Carolina were processed by Ray's fluid
thioglycollate medium tissue assay for P. marinus. Infection in-
tensities were similar among all eight creeks in July of 1997.
During January-February 1998. oysters were removed from four
of the eight creeks and subsequently allowed to repopulate via
natural recruitment. In early August 1 999. the creeks from which
the oysters had been removed showed lower levels of P. marinus
than control creeks. These observations appear to indicate that
fallowing oyster beds may help control P. marinus infections and
minimize subsequent oyster mortality. Other studies, however,
have shown that P. nuirinus-free oysters placed in these same eight
creeks contracted infections at the same rates. The lower intensities
observed in August 1999, in the creeks where oysters had been
removed, was an artifact of age. Oysters in these creeks were
younger and therefore had less time to develop infections than the
older oysters in the control creeks. Thus, early harvesting may be
may still be required to avoid mortalities by P. marinus.

DEVELOPMENT AND VERIFICATION OF A SIMPLE
MODEL FOR PERKINSUS MARINUS ABUNDANCE IN
CHESAPEAKE BAY OYSTERS. Lisa M. Ragone Calvo* and
Eugene M. Burreson, Virginia Institute of Marine Science, Col-
lege of William and Mary, Gloucester Point, VA 23062.

A simulation model was developed to investigate the popula-
tion dynamics of the protistan parasite. Perkinsus marinus. within
its host, the eastern oyster, Crassostrea virginica. The main ob-
jective was to evaluate the relationship between P. marinus popu-
lation dynamics and environmental conditions in order to predict
the onset and termination of P. marinus epizootics in Chesapeake
Bay oyster populations. The model was calibrated using data de-
rived from laboratory experiments and from field observations of
P. marinus prevalence and intensity in the James River, Virginia
for the years 1990-1993. The relatively simple, individual based
model is driven by temperature and salinity and tracks in vivo
parasite density through time at a daily time step. The model was
verified with five years ( 1994-1998) of monthly field observations
of parasite abundance at three oyster bars located along a salinity
gradient in the James River. Five year simulations, initiated on
Julian day 1 with a single parasite input corresponding to the actual
observed abundance for that month, significantly correlated (p <
0.001. r = 0.439 to 0.729) with observed abundances of P. mari-
nus at the three James River oyster bars. Predicted parasite abun-
dances were stabilized during the 5 year simulation for all three
oyster populations without the additional input of transmission
events, suggesting that a single transmission event can result in P.
marinus becoming enzootic in an area for a long period of time.

662 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association. Seattle. Washington

POTENTIAL TRIPLOID PRODUCTION OF OYSTERS US-
ING SECOND METAPHASE OOCYTES. Gregory M.
Coates,* Aquaculture Research Station. Louisiana State Univer-
sity Agricultural Center. Louisiana Agricultural Experiment Sta-
tion. Baton Rouge, LA 70820; John E. Supan, Office of Sea
Grant Development, Louisiana State University, Baton Rouge. LA
70803.

Due to the fact that tetraploid eastern oysters Crassostrea vir-
ginica are not yet available to spawn with diploids to produce
all-triploid populations (interploid triploidsl. successful triploid
production of C. virginicu is dependent on chemical induction
usually with cytochalasin B (CB). Treatment efficiency with CB
usually varies significantly due to asynchronous meiotic matura-
tion of the oocytes dissected from ripe adult oysters. The neuro-
hormone serotonin (5-hydroxytriptamine. 5-HT) and alkaline sea-
water (prepared with 0.1 M NaOH). used previously on other
molluscan species to re-initiate meiosis. were used separately to
treat unfertilized oocytes prior to fertilization. Dissected oocytes
that normally arrest at either the first prophase (germinal vesicle.
GV) or first metaphase (germinal vesicle breakdown, GVBD) of
meiosis, prior to fertilization, were exposed to 5-HT and alkaline
seawater to examine the effect on oocyte development.

Oocytes from ripe adult oysters were removed, rinsed, and
exposed to various 5-HT concentrations ( 1 |xM, 10 jxM, and 100
(jlM) and four different pH concentrations (7.0. 8.0. 9.3. 10.0) in
filtered seawater at 25 °C. Exposure to 5-HT caused a significant
increase (P < 0.05) in meiotic maturation rate when compared to
the control. Oocytes matured to second metaphase (first polar body
stage) in all 5-HT treatments and the control. Significant increases
(P < 0.05) in the rate and stage of meiotic maturation occurred in
the alkaline treatments (pH 9.3 and 10.0). Alkaline seawater acti-
vated the oocytes not only to first polar body stage, but also to
second polar body stage and cleavage. Oocytes that reached first
polar body stage could be fertilized and treated with CB immedi-
ately after fertilization to inhibit the second polar body and pro-
duce triploids. Using activated oocytes may improve triploid in-
duction techniques by reducing variation in the stage of meiotic
development.

ENERGY RESERVES IN PERKINSUS MARINUS IN-
FECTED AND UNINFECTED OYSTERS. V. G. Encomio,* S.
Stickler, and F. L. Chu, Virginia Institute of Marine Science.
College of William and Mary. Gloucester Point, VA 23062.

Energy reserves are important lor fueling gametogenesis and
providing energy during stress. Variation in energy reserves be-
tween oyster populations is primarily attributed to environmental
differences. Additionally, genetically based intraspecific variation
in reproductive timing of oysters has been found in previous stud-
ies. By that same notion, intraspecific variation in seasonal pat-
terns of energy storage may also possess some degree of genetic
influence. We are examining intraspecillc variation in biochemical

composition and the effects of parasitism on energy reserves in
eastern oysters (Crassostrea virginica) and the role of energy re-
serves in tolerance to Dermo disease. Comparisons will be made
between hatchery raised progeny from presumably genetically dis-
tinct oyster populations. These oysters represent geographically
disparate populations (Gulf of Mexico and Chesapeake Bay) and
populations (Gulf — Oyster Bayou. Hackberry Bay. Grande Terre;
Chesapeake — Tangier Sound, Choptank River, Lower Rappahan-
nock; Hatchery strain — CrosBred) exhibiting variation in tolerance
to the protozoan parasite Perkiiisus mariiuis. These oyster stocks
have been deployed at two sites within the Chesapeake Bay where
Dermo disease, but not MSX, is known to occur. Glycogen, lipid
(total lipids, polar and neutral lipids), and protein contents, will be
measured in tissues of individual oysters and correlated with
changes in shell height, condition index, and Perkinsus mariitiis
body burden. Preliminary measurements of recently deployed oys-
ter stocks show similar glycogen contents between various oyster
stocks (p = 0.13). However, as intensity off. luurinns enzootic
periods increases, we expect to see both between and within popu-
lation variation in disease response. Other preliminary measure-
ments of adult oysters showed that total lipid contents in heavily
infected oysters decreased compared to non-infected, light and
moderately infected oysters.

SPECIES DESIGNATION AMONG SYMPATRIC OYS-
TERS CRASSOSTREA ARIAKENSIS, C. GIGAS, AND C.
SIKAMAEA. Elizabeth A. Francis,* Kimberly S. Reece, and

Standish K. Allen, Virginia Institute of Marine Science. College
of William and Mary. Gloucester Point. VA. 23062; Patrick M.
Gaffney, College of Marine Studies. University of Delaware.
Lewes. DE 19958.

Little has been published about the distribution or population
genetic structure of the Asian oyster Crassostrea ariakensis. This
species, found sympatrically with C. gigas and C. sikamaea. is
believed to be distributed throughout the warm coastal waters of
Pakistan. India. China and Japan. Howe\er. morphological plas-
ticity and possible hybridization with congeneric species make
identifying C. ariakensis difficult. Collection of C. ariakensis from
its presumed distribution is underway for studies aimed at resolv-
ing this confusion. Initial sampling efforts have resulted in speci-
mens collected from Japan and se\eral locations in China. Species
identification of these samples using two interspecific typing keys
yielded conflicting results. Restriction fragment polymorphism
(RFLP) data using nuclear and mitochondrial loci suggested that
the putative C. ariakensis samples actually contained all three
sympatric species. Another species identification key based on
RFI.P analysis of the 16S locus did not support these results. Many
individuals typing as C. gigas using the first key were identified as
C. ariakensis with the second key. To provide greater resolution
among relationsliips. ITS-1 sequence data lor individuals from
each site were compared to sequences of "known" C. ariakensis

National Shellt'isheries Association. Seattle. Washington

Ahslrocrs. 2000 Annual Meeting. March 19-23. 2000 663

and C. gigas. Phylogenetic analyses using both parsimony and
distance indicate the presence of two ciades. one including all the
samples collected from China and Japan, and another consisting of
the "known" C. gigas.

EFFECT OF CLAY SUSPENSIONS ON CLEARANCE
RATE IN THREE SPECIES OF BENTHIC INVERTE-
BRATES. Dane Frank,'* Lisa Evvert," Sandra Shumway," and
J. Evan Ward,' 'Department of Marine Sciences. University of
Connecticut. Groton. CT 06340; "Southampton College. Long Is-
land University. Southampton. NY 11968.

Harmful algal blooms pose a threat to areas where fisheries
products are a vital part of the economy. Recent attempts are being
made, especially in Asia, to displace harmful algal blooms by
spraying fine particulate mineral suspensions (e.g. "china clay")
over the surface of affected coastal waters. In practice, the particles
adsorb onto the surface of the algal cells, promoting coagulation
and displacement to the bottom. Very little is known, however,
about the impact of this technique on benthic communities and
processes. To examine the effects of differing concentrations of
china clay, and kaolin on clearance rate, short-term laboratory
experiments were performed on three benthic species: the bay
scallop {Argopeaen irradians). the eastern oyster (Crassostrea
virgiuica). and the tunicate (Molgula manhattemis). Colleagues in
Korea furnished us with the china clay used in their field trials.
Depletion rate assays were performed using solutions of 10. 100.
1000. or 10.000 mg/1 of either china clay or kaolin suspended with
Rhodomonas lens (I x lO*" cells/ml) in 0.4 ixm filtered seawater.
Particle concentrations and size distributions were determined be-
fore and after experiments, using a Coulter Multisizer. Results
showed a decrease in weight specific clearance rates with increas-
ing concentrations of clay or kaolin in all species. Both the oyster
and the scallop showed a slight increase in particle clearance at
100 mg/l. and the lunicates showed a similar increase at 100 mg/1.
These results strongly suggest that further studies are required to
determine the full effects of this clay-coagulation practice on the
benthos.

OPEN OCEAN, SUBMERGED LONGLINE CULTURE OF
THE BLUE MUSSEL IN NEW ENGLAND: A FIRST-YEAR
PROGRESS REPORT. Ray Grizzle. Jackson Estuarine Labora-
tory. University of New Hampshire. Durham. NH 03824; Ricliard
Langan, The Cooperative Institute for Coastal and Estuarine En-
vironmental Technology. University of New Hampshire. Durham.
NH 03824.

The blue mussel. Mytihis edidis. has supported a substantial
aquaculture industry in New England for over 20 years. The pre-
dominant method is bottom culture in shallow, nearshore waters.

The present project is aimed at assessing the development of sus-
pension culture techniques in open ocean waters of New England,
and involves spat/seed collection and growout. Spat collection
experiments conducted in 1998 and 1999 showed wide spatial and
temporal variability in set densities, but adequate numbers were
caught in spring/eariy summer both years near the mouth of the
Piscataqua River. NH. Major problems encountered were over-
growth of the collectors by tubularian hydroids and apparent stunt-
ing of the seed at high mussel densities. Sufficient seed to stock
>700 m of socking material were obtained in 1998. These mussels
were deployed to the submerged longline, which is constructed of
2.8 cm diameter "polysteel" rope, on 2 July 1999. The longline is
located 10 km offshore from Portsmouth Harbor. NH in about 60
m of water. Mussel size and environmental conditions at the site
have been monitored monthly since July. Overall, the mussels
averaged 1 .0 mm shell growth/wk from 2 July through 9 Novem-
ber. Based on growth data thus far. a total time for spat set to
harvest could be substantially less than 2 yr.

HOW HAVE THE WARNING LABELS AND NEGATIVE
PUBLICITY ASSOCIATED WITH VIBRIO VULNIFICUS
IMPACTED DEMAND FOR GULF OF MEXICO PRO-
DUCED OYSTERS? Walter R. Keithly Jr.* and Hamady
Diop, Center for Coastal, Energy, and Environmental Resources,
Louisiana State University, Baton Rouge, LA 70803.

Vibrio vubuficiis. a naturally growing bacteria that thrives in
the warmer waters of the Gulf of Mexico, has been linked to
numerous illnesses and deaths from the consumption of raw shell-
fish harvested from these waters. While ingestion of this bacteria
is harmless to the vast majority of the shellfish consuming public,
consequences can be extreme for those individuals with compro-
mised immune systems. California, in response to eight confirmed
vilirio related cases between 1985 and August 1990. five of them
fatal, mandated as of 1 March 1991. that all restaurants and stores
selling raw Gulf of Mexico oyster product post warning signs that
would advise consumers of the potential adverse health effects
associated with consumption of the raw Gulf of Mexico oyster
product. This state's action, the first of its kind in the nation, was
followed shortly thereafter by other states, including Louisiana and
Florida. The purpose of this paper is to examine whether the warn-
ing labels and associated negative publicity surrounding the issue
impacted the demand for the Gulf of Mexico produced product. To
accomplish this objective, the Gulf of Mexico dockside oyster
price was estimated using standard econometric techniques for the
1981-97 period based on quarteriy data. The results suggest that
the dockside price was significantly impacted with the overall
extent of the impact depending on season.

664 Abstracts. 2000 Annual Meeting March 19-23. 2000

National Shelifisheries Association, Seattle, Washington

DEVELOPMENT OF A DEFINED MEDIUM FOR CELLS
OF THE EASTERN OYSTER CRASSOSTREA VIRGINICA.
Yanii Li and Jerome F. La Peyre,* Department of Veterinary
Science, Louisiana State University. Baton Rouge. LA 70803.
USA.

A variety of commercial culture media supplemented with fetal
bovine serum have been used to maintain oyster cells in primary
cultures. Although certain ingredients found in oyster plasma are
often added to these commercial media there have been limited
attempts to evaluate the benefits of these ingredients to oyster cells
and to optimize their concentrations. Moreover, no culture medium
has yet been developed specifically for cells of oysters or other
bivalve molluscs. This is surprising since it is well known that
formulation of a culture medium based on the biochemical com-
position of insect plasma in 1956, was a major development that
facilitated the establishment of numerous insect cell lines. The
objective of this study was therefore to formulate and optimize a
defined culture medium for oyster cells.

Using a basal medium optimized in a previous study, the effects
of more than 30 ingredients on primary ventricle cell cultures were
first evaluated individually over a broad range of concentrations.
Evaluation was done by comparing cellular metabolic activity and
by observing the morphology and contractility of cultured cells. In
a second set of experiments, the combined effects of selected
ingredients were determined using a statistical optimization ap-
proach based on a Plackett-Burmann statistical design. A defined
medium (LA-2) was then formulated by supplementing our basal
medium with all beneficial ingredients and the merit of LA-2 was
determined by comparing it to commercial L-15 medium (Leibo-
vitz) which has most been used to culture oyster cells. Results
indicated that LA-2 was far superior to L-15 for maintaining oyster
cells in primary cultures. We are now testing a number of supple-
ments to further improve a medium for culluring oyster cells.

VEINED RAPA WHELKS (RAFANA VENOSA) IN THE
CHESAPF;AKE BAY: current STATUS AND PRELIMI-
NARY REPORTS ON LARVAL GROWTH AND DEVEL-
OPMENT. Roger Mann and Juliana M. Harding, Depart
ment of Fisheries Science, Virginia Institute of Marine Science,
Gloucester Point, VA 2.3062.

Since the initial discovery of the Veined Rapa Whelk (Rapuim
venosit) in the Lower Chesapeake Bay in June 1999, over 650 adult
specimens have been donated to the Virginia Institute of Marine
Science (VIMS) Rapa Whelk research program. Continuing efforts
to map the whelk's distribution in the Lower Chesapeake Bay
indicate no new range exlensioiis and a relatively constant popu-
lation in the lower .lames Ri\cr. Hampton Bar. and Ocean View/
Little Creek regions. During the summer of 1999. VIMS' brood-
stock animals laid o\er 500 egg masses with egg laying activity

beginning in May and continuing through August. Egg masses and
the resulting larvae were successfully cultured through metamor-
phosis. Recently settled young Rapana have a wide range of di-
etary capabilities and will feed on local fauna including barnacles,
oyster spat, mussels, and Mcicoma. Growth post settlement can be
quite rapid. The oldest juveniles from the 1999 larval cultures
reached total shell lengths in excess of 20 mm within 4 months of
metamorphosis and settlement.

RELEASE OF MUCOPOLYSACCHARIDES BY BI-
VALVED MOLLUSCS AND THEIR CONTRIBUTION TO
THE PRODUCTION OF TRANSPARENT EXOPOLYMER
PARTICLES (TEP) IN NEAR SHORE WATERS. Michael P.
McKee,* J. Evan Ward, and Lisa M. Milke, Department of
Marine Sciences, University of Connecticut, Groton, CT 06340;
Bruce A. MacDonald, Department of Biology, University of New
Brunswick, Saint John, N.B., Canada, E2L 4L5.

In the marine environment, the presence of large, discrete,
transparent exopolymer particles (TEP) has been directly corre-
lated with the flocculation of phytoplankton, detritus, and other
particles into aggregates known as marine snow. Phytoplankton
and bacteria have both been shown to contribute to the production
of TEP, via the exudation of precursor sticky inucopolysaccha-
rides. Little is known, however, about other potential sources of
mucins that could lead to TEP forination. The purpose of this
research is to identify other potential sources of TEP.

Many benthic suspension feeders utilize mucus-coated struc-
tures to capture and transport food particles. Recent endoscopic
studies have shown that these suspension feeding processes are
accompanied by changes in the cohesive nature of mucins. Hydra-
tion of mucins may occur when inucus-coaled feeding structures
are exposed to ambient waters. The solubilized mucopolysaccha-
rides could then be transported out of the pallial cavity and into
open waters. In addition, rejection of pseudofeces, a mucous par-
ticle inatrix, from the mantle cavity may also serve as a source of
additional TEP production. The working hypothesis of our re-
search is that bivalves and other suspension feeders release sig-
nificant amounts of mucins into the surrounding water, and that
tliis material enhances TEP production through physico-chemical
processes, resulting in increased flocculation of particles. Concen-
trations of TEP in both the laboratory and field setting are deter-
nuned using an Alcian Blue staining technique and quantified
using a spectrophotometer. Preliminary field data from the Long
Island Sound and Bermuda suggest that the presence of TEP,
above background levels, is coirelalcd with proximity to blue mus-
sel (Mytilus cilidis) beds and other suspension feeders, respec-
tively. Additional laboratory experiments are being conducted in
order to t|uaiitily the production of TEP by bivalves under con-
trolled conditions.

National Shellfisheries Association. Seattle. Washinuton

Abstracts. 2()()() Annual Meetinu. March 19-23, 2000 665

IMPROVED PROCEDURE TO COUNT PERKINSUS MARl-
NUS IN EASTERN OYSTER HEMOLYMPH. Amy D. Nick-
ens,* Aquacullure Research Station. Louisiana State University
Agricultural Center. Baton Rouge. LA 70820; Eric Wagner and
Jerome F. La Peyre, Department of Veterinary Science. Louisi-
ana State University, Baton Rouge. LA 70803.

Perkiiisus mariniis infection intensity in Crassostrea virginica
can be quantified without sacrificing the oyster by determining the
density of the parasite in hemolyniph samples. The hemolymph
assay involves several steps. ( 1 ) the enlargement of the parasites in
Ray"s fluid thioglyeollate medium (RFTM), (2) their subsequent
isolation from blood cell debris and (3) their quantification after
staining with Lugol's solution. However, each step has yet to be
optimized. The objective of this study was to improve the proce-
dure for counting P. mariniis in oyster hemolymph. We examined
changes in the number and size (diameter) of isolated parasites as
a result of ( 1 ) adding different volumes of RFTM (0.2 ml. 1 ml. 5
ml and 25 ml). (2) adding supplements (lipid and oyster extracts)
to RFTM. (3) adding five types of FTM (e.g.. with and without
agar or beef extract) (4) adding different numbers of hemocytes
(10"'^. 10* and 10^ cells). We also evaluated the use of sodium
hydroxide (NaOH) to digest cellular debris and facilitate cell
counting.

Our most significant finding was that incubation in RFTM
supplemented with lipid (code liver oil) caused parasites to reach
a significantly larger size (26 jjim) than did incubation in RFTM
supplemented with oyster extract (17 mm) or saline control (11
mm). We also found that the absence of agar from fluid thiogly-
eollate medium greatly simplified sample processing without af-
fecting the number or size of parasites. It was clear from this study
that simple modifications of the standard hemolymph procedure
could be made to improve counting of P. mariniis in oyster
hemolymph.

TIDAL FLUCTUATION IN PHYTOPIGMENT CONCEN-
TRATIONS AND SEDIMENT LOAD AT A MANILA
CLAM, RUDITAPES PHILIPPINARUM, FARMING
GROUND. Katsuyulvi Numaguchi,* National Research Institute
of Fisheries Science. 6-31 Nagai. Yokosuka. Kanagawa 238-0316,
Japan.

Fluctuations of the quantity of fractionated particles of sus-
pended solid and phytopigment (chlorophyll a and phaeo-
pigments) concentrations in the bottom sea water and sediment
with the tidal rhythm was investigated at the Kikuchi River estuary
of a Manila clam. Ruditapes pliitippinanim. farming ground. Chlo-
rophyll a in the bottom seawater increased at flood and high tide,
and decreased at ebb and low tide. Fine particles in the bottom
seawater had a high ratio of chlorophyll a ( 10-100 p-m; 59%) and
phaeo-pigments (1.2-10 p.m: 59%). Chlorophyll a in the sediment
increased from flood to high tide, and decreased from ebb to low
tide. Fine particles in the sediment had a high ratio of chlorophyll

a ( 1.2-50 |j.m; M'/c) and phaeo-pigments ( 1.2-50 |xm; 66%). Phy-
topigment contents contained in the dige.stive diverticula of Manila
clams increased from the flood to the high tide, and phytopigments
contained in the digestive diverticula of Manila clam were almost
all phaeo-pigments (78-98%).

These results suggest that phytoplankton are supplied from off-
shore to the estuary with the tide during flood to high tide periods.
And during these periods, phytopigments in the Manila clam di-
gestive diverticula increased rapidly. These results indicate that
most of algal diet to Manila clams may be supplied from offshore
to the estuarine Manila clam farming ground. Further, there is a
high quantity of fine particles chlorophyll a and phaeo-pigment
contained in the bottoiri seawater and sediment at the estuary of the
Manila clam farming ground.

1999 OREGON STATE UNIVERSITY STUDIES CON-
DUCTED ON THE EUROPEAN GREEN CRAB, CARCINUS
MAENAS, SUPPORTED BY OREGON SEA GRANT. Kelly
Falacios,* Department of Marine Resource Management: Sylvia
Yamada, Laura Hauck, and Alex Kalin, Zoology Department;
Chris Hunt,* Environmental Science Department, Oregon State
University, Corvallis, OR 97331-2914.

Since the arrival of the European Green crab, Caniniis inaenas,
in San Francisco Bay in 1989, there has been widespread concern
for the potential impact on Western Pacific estuarine communities
and shellfish industries. With these concerns in mind we set out to
determine a few of the limits, impacts, affects and differences in
invasion history to this aquatic nuisance specie. With the arrival of
this crab predator also came the anival of a new prey handling
technique to the Littorines of Oregon, supported by lab studies and
field studies using tethered lines. Lab studies were also conducted
on preference, handling and consumption on a number of bivalves,
including commercially important species. Trapping and lab pre-
dation studies suggest that C. maenas may be limited by adult
native Red Rock crab. Cancer procliutiis. Measures of molt rates
and frequency suggest that C. maenas may reach its terminal molt
stage at a younger age in Oregon estuaries than it has throughout
it's native range, or in previous invasions. Each of these efforts
were supported by Oregon Sea Grant.

THE REPRODUCTIVE CYCLE OF CAPTIVE FEMALE
GOLDEN KING CRAB, LITHODES AEQUISPINUS. A. J.
Paul* and J. M. Paul, University of Alaska, Institute of Marine
Science, Seward Marine Center, Seward, AK 99664.

The predicted increase in carapace length (CD for adult fe-
males (CL 104 to 157 mm) was expressed by the equation; New
CL (mm) = Initial CL (0.97) + 10.21; (r" = 0,91). Increases in
CL averaged 5% (SD = 2%) for egg bearing females. The small-
est and largest egg bearing females in our collections were typi-
cally 120 mm and 150 mm respectively. Using the CL growth

666 Abstracts. 2000 Annual Meeting. March 19-23. 2000

National Shellfisheries Association, Seattle. Washington

equation a 120 mm female would require 5 molts to reach 150 mm
and produce one clutch with each molt if she survived that long.
The egg clutches hatched asynchronously with some hatching
during every month of the year. An average of 3 1 days passed
between the time the first and last larvae in a clutch hatched. After
a female's eggs hatched she did not molt until an average of 194
days had passed. Soft shell females extruded eggs about 2 to 3 days
later. The incubation period averaged 302 days. There was con-
siderable variability in the duration of each of these growth and
reproductive events because they could occur at any time of year
with different thermal conditions. Typically females required 530
days, or 1.4 years, to complete a reproductive cycle in the labo-
ratory.

OYSTER RESTORATION IN CHESAPEAKE BAY: EF-
FECTS OF OYSTER DENSITY ON THE ASSOCIATED
BENTHIC COMMUNITY. K. T. Paynter, Chesapeake Biologi
cal Laboratory and Department of Biology. University of Mary-
land, College Park, MD 20742 USA.

Restoration of shellfish beds has at least two obvious applica-
tions: commercial and ecological. While the commercial value of
restored areas to the shellfish industry may be obvious, the eco-
logical value of restored shellfish beds has been difficult to quan-
tify. Oyster bar restoration in Chesapeake Bay has only recently
begun in earnest. In 1995 and 1996 10 acres of oyster "bottom"
was restored with 1 million hatchery produced oyster spat per acre.
In 1997 and 1998. additional areas were restored in the Choptank.
Patuxent and Chester Rivers. Underwater videography has been
employed to examine the differences in the benthic communities
associated with high (2 million/acre) and low (250.000/acre) den-
sity oyster plantings. Initial results show that high density planting
results in significantly higher numbers of individuals and species
associated with the reef. Anemones, barnacles, hooked mussels,
gobies and blennies are among the inhabitants of recently restored
reefs. In contrast, low density plantings or shells alone harbored
relatively few individuals and fewer species as well. Oyster density
appears to have a substantial and important effect on the reef
community that develops within and around restored reefs.

CARDIOREGULATORY NERVES ARE NOT THE
SOURCE OF TEMPERATURE-INDUCED HEART RATE
MODULATION IN THE AMERICAN LOBSTER
{HOMARUS AMERICANUS). S. M. C. Schreiber* and W. H.
Watson IH, Dcpl. of Zoology, University of New Hampshire.
Durham. NH 03824.

Lobsters are known to express behavioral responses to lem-
peralure. including bolh temporalurc avoidance and a preferred
temperature range. lloue\er. the ph\siological effects of tempera-
ture ihal may inlluence these behaviors are not well understood.
Previous research on the effects ol temperature on the heart of the

lobster indicates that some source of temperature-induced heart
rate modulation exists in intact animals. The cardioregulatory
nerves, which control many of the heart responses, had been as-
sumed to be the source of this modulation. However, as tempera-
ture responses are often long-term and neurotransmitters are usu-
ally associated with short-term responses, a different source for
this modulation was hypothesized. To eliminate the possibility that
the cardioregulatory nerves were controlling the heart rate re-
sponse to temperature, the effects of temperature on the heart rates
of lobsters with lesioned cardioregulatory nerves were examined.
Lobsters were exposed to a series of 5 °C temperature increases
from 5-25 °C. The responses of lobsters with lesioned cardioregu-
latory nerves were compared to responses of intact lobsters. Over
the range of temperatures examined, no significant differences
were observed in the responses of lesioned and intact animals.
Therefore, it can be concluded that the cardioregulatory nerves are
not responsible for the temperature-induced modulation of lobster
heart rate.

DERMOWATCH: A NEW TOOL FOR MANAGING PER-
KINSVS MARINUS DISEASE IN EASTERN OYSTERS,
CRASSOSTREA VIRGINICA. Thomas M. Soniat,* Department
of Biological Sciences, Nicholls State University, Thibodaux, LA
70310: Enrique V. Kortright, Kortright Corporation. 102 Allen-
dale Dr. Thibodaux, LA 70301; Sammy M. Ray, Department of
Marine Biology, Texas A&M University at Galveston, Galveston,
TX 77553.

A website called DermoWatch has been established (inrir.
hlueblee.com/denno) to track the progression of Perkinsiis iiniri-
nus ( = Dermocystidium marinum) in Galveston Bay and allow
users from other locations to also calculate a time to a critical level
of disease (\.c,n)- Data on water temperature (T) and salinity (S),
initial level of disease and oyster length are entered. An embedded
model converts measured weighted incidence (Wli values and the
critical Wl to parasite number (a WI of 1.5 is considered critical),
calculates a rate of change (r) of the parasite population using
measured values of T and S, and solves for t^-^,, by simulation.
Estimates of I, ,„ from Galveston Bay and other areas should sup-
port decisions concerning transplanting infected oysters to lower
salinity areas, harvesting heavily-infected populations early, and
diverting freshwater into high-salinity estuaries.

GROWTH. MORTALITY, AND DEFENSE AGAINST PER-
klNSUS MARINUS IN EASTERN OYSTERS. CRASSOS-
TREA VIRGINICA. S. M. Stickler,* V. G. Encomio. F.-L. Chu,
and S. K. Allen, Jr., Virginia Institute of Marine Science. College
of William and Mary. Gloucester Point. VA 23062.

The restoration of Eastern Oyster {Crassostreii vir^inica) popu-
lations can be accelerated with the development of strains resistant
(o Dermo disease, caused by the proto/oan parasite. Perkinsiis

National Shellfisheries Association. Seattle. Washiinizton

Abstracts. 2000 Annual Meeting. March 19-23. 2000 667

maiiiiiis. To date, this has meant the slow, methodical approach of
selectively breeding oysters that have survived repeated exposure
to the parasite. By identifying effective defense mechanisms in
surviving individuals or populations, however, we can develop
markers that will expedite the production of resistant strains. This
study uses a suite of assays to measure potential cellular and hu-
moral defense mechanisms within and between distinct geographic
oyster populations. Wild oysters believed to be resistant to Dermo
were harvested from both the Gulf of Mexico (Louisiana) and
Chesapeake Bay. Hatchery spawned and raised progeny of these
oysters have been placed in floats at two sites in both Chesapeake
Bay and the Gulf. We are currently comparing growth and mor-
tality and sampling animals to assess variation in defense capabil-
ity between stocks. Assays include: hemocyte counts and
hemocyte killing of P. marimis cells, and protein levels,
hemolymph lysosomal enzyme levels, and protease inhibitor lev-
els. All assays are correlated against an optimized body burden
fluid thioglycollate media assay for P. marinus prevalence and
intensity for each individual oyster. We are also examining physi-
ological fitness of animals grown out in Chesapeake Bay to de-
termine the effects of parasitism on energy reserves in oysters and
the role of energy reserves in tolerance to Dermo infection.

WESTERN REGIONAL AQUACULTURE INDUSTRY
SITUATION AND OUTLOOK REPORT: A SHELLFISH
PERSPECTIVE. Derrick R. Toba* and Kenneth K. Chew,

Western Regional Aquaculture Center. School of Fisheries Box
357980, University of Washington, Seattle, WA 98195-7980.

A survey to estimate the aquaculture production in the twelve
western states was conducted for the Western Regional Aquacul-
ture Center (WRAC). The western region includes Alaska. Ari-
zona. California. Colorado. Idaho. Montana. Nevada. New
Mexico. Oregon. Utah, Washington and Wyoming. This study was
designed to 1 ) provide production estimates for all species cul-
tured, and identify the states in which they are produced: 2) pro-
\ide information on the current status of each industry and esti-
mate future (year 2002) production: and 3) determine product
forms sold by the producers and provide the average selling price.
Information on finfish (salmon, trout, cattish, tilapia and others).
shellfish (oysters, mussels and others), aquatic plants and non-
foodfish were collected for the survey.

Total aquaculture production in 1997 for the western region
was 140.000 pounds, which was valued at $161,000. The three
leading states in 1997 aquaculture production. Washington (58.3
million pounds). Idaho (43.5 million pounds) and California (30.5
million pounds), combined to produce over 94.57^ of the total
weight. California ($70.6 million). Washington ($46.7 million)
and Idaho ($33.7 million) combined to account for 93.7% of the
total value of 1997 aquaculture production in the western region.

Of the total 1997 western region aquaculture production. 55.1
million pounds (39.4%) and valued at $38.5 million (,23.9%) were

shellfish. Five states reported commercial shellfish aquaculture
harvests and sales: Alaska. Arizona. California. Oregon, and
Washington. In each of the coastal states, oysters comprised the
majority of shellfish production. Other species harvested included
mussels, clams, scallops, abalone, crawfish, and shrimp. Trends
over the past ten years were analyzed for each state and species.

APPLICATION OF UNDERWATER TIME-LAPSED
VIDEO TECHNOLOGY TO OBSERVE KING AND TAN-
NER CRAB BEHAVIOR IN AND AROUND COMMERCIAL
CRAB POTS. Donn Tracy, Alaska Department of Fish and
Game. Commercial Fisheries Division. 211 Mission Rd. Kodiak.
AK 99615.

Observations of crab behavior in and around crab pots actively
tlshed for extended soak periods have recently been made possible
by the advent of an autonomous underwater video recording sys-
tem. A built-in microprocessor allows time-lapse video event pro-
gramming for observations over an unlimited time span. During a
pilot study in 1998. red king crabs in Bristol Bay. Alaska were
observed entering and egressing five pots over soak periods rang-
ing between twenty-four and seventy-two hours. In the 1999 Ber-
ing Sea snow crab fishery observations spanning similar soak pe-
riods were made in four commercially fished pots. Future appli-
cation of this prototype system holds promise for gaining insight
into numerous aspects of crab behavior and the performance of
commercial pot gear.

THE GENETIC ASSESSMENT OF AN "ENHANCED" BAY
SCALLOP POPULATION: DO HATCHERY SCALLOPS
PRODUCE SUCCESSFUL RECRUITS? Ami E. Wilbur,* De-
partment of Biological Sciences, University of North Carolina-
Wilmington, Wilmington, NC 28403: William S. Arnold and
Theresa M. Bert, Florida Marine Research Institute. 100 8th Ave
S.E.. St. Petersburg, FL 33701.

Restoration and enhancement of shellfish populations have be-
come increasingly more common as overfishing, habitat degrada-
tion and disease decimate wild populations. Numerous techniques
have been employed (ie. open seeding of juveniles, spawner sanc-
tuaries, habitat rehabilitation) to mitigate for these losses but the
relative success of such techniques is often difficult to evaluate. As
part of Florida's ongoing effort to enhance bay scallop populations
on the Gulf Coast, we have implemented an extensive genetic
monitoring program to assess the impact of the planting of hatch-
ery stocks on local recruitment. We have developed an anay of
genetic markers (mtDNA. microsatellites and introns) that can
function as a "genetic tag" for the enhancement stocks, and allow
unambiguous identification of the progeny of the outplanted scal-
lops. Preliminary analysis of post-enhancement recruitment, as-
sessed using spat collectors indicates no substantial numerical im-

668 Abstracts. 2000 Annual Meeting, March 19-23, 2000

National Shellfisheries Association. Seattle, Washington

provement over pre-enhancement recruitment rates. Genetic evalu-
ation of the post-enhancement population is ongoing. Restriction
fragment length polymorphism (RFLP) data based on a 833bp
mtDNA fragment reveals a slight increase (3%) in the wild popu-
lation of a rare haplotype that was abundant in the hatchery stocks,
suggesting some contribution of hatchery scallops to the post-
enhancement wild population.

TRACKING FECAL SOURCES IN DRAYTON HARBOR.
Rob Zisette,' Walter T. Trial,' and Mansour Samadpour,'

'Herrera Environmental Consultants. 2200 Sixth Avenue, Suite
601, Seattle. WA. USA 98121. ^Dept. of Environmental Health,
University of Washington, PO Box 357234, Seattle. WA, USA
98195.

Drayton Harbor, located at the Washington/British Columbia
border, was closed in 1995 to commercial oyster farming and
recreational shellfish harvesting due to the presence of high levels
of fecal coliform bacteria. Sources of fecal contamination were
believed to include discharge of municipal sewage in urban areas,
failing septic systems, poor animal management practices in rural

areas, and discharges from seafood processors and live-aboards in
local marinas. A genetic fingerprinting technique was employed in
1998 to determine which sources were most significant and should
be the focus of watershed management efforts. Escherichia coli
were isolated from oyster tissue samples and from water samples
collected on four occasions from two urban streams, one rural
stream, and four locations in the harbor.

Using the polymerase chain reaction (PCR) technique, DNA
from these E. coli were compared to E. coli DNA from known
fecal sources. Percent matches between known and unknown fecal
sources were used to assess the relative contribution of the various
fecal sources to streams, marine waters, and oysters in the Drayton
Harbor watershed. Human fecal sources were only identified in the
rural stream, and livestock were the primary fecal source to this
stream. Pets and waterfowl were the only fecal sources identified
for the urban streams. Identified sources to marine waters and
oysters included waterfowl, livestock, seals, and seafood process-
ing wastewater. Watershed management recommendations in-
cluded improvement of livestock management practices, correc-
tion of septic system failures, treatment of seafood processing
wastewater, and public education.

Joiinuil of Shellfish Resetnch. Vol. 19, No. 1, 669-680. 2000.

ABSTRACTS OF TECHNICAL PAPERS

Presented at the

INTERNATIONAL SYMPOSIUM ON LOBSTER HEALTH MANAGEMENT

Adelaide, Australia
September 19-22, 1999

Convenors: Louis H. Evans
J. Brian Jones

669

International Symposium on Lobster Health Management Abstracts. September 19-22. 1999 671

CONTENTS

Robert C. Bayer. Deanna L. Prince. Maya A. Crosby, Ben Tall, Seynabou Fall and Michael B. Loiighlin

Health management of the American lobster 673

Benjamin K. Diggles

Diseases in spiny lobster holding in New Zealand 673

Knut E. Jorstad, 0ivind Bergh and Kari Andersen

Health aspects in Norwegian lobster stock enhancement: Principles and practice 674

Kenneth Soderhdll

Re\ iew of crustacean immunity 674

Brian Jones

Cellular response to injury in spiny lobsters 674

Jeff Jago, Bob Dunstan, Japo Jiissila and Louis H. Evans

Techniques for emuneration and morphology of hemocytes in western rock lobster {Pimtilinis cygniis George) 674

Andrew G. Jeffs

Can compromised condition explain early mortalities in spiny lobster culture? 675

Elena Tsvetnenko, Jeremy Brown and Louis H. Evans

Measures of condition in dietary studies on western rock lobster post-pueruli 675

Ruth E. Renter, Michael Geddes and Louis H. Evans

Tail rot in southern rock lobsters (Jasus edwardsii) 675

Judith Handlinger, Jeremy Carson, Arthur Ritar and Bradley Crear

A study of diseases in cultured phyllosonia larvae and juveniles of southern rock lobster (Jasus edwardsii) 676

Glen W. Davidson, Patrick T. Spanoghe and Brian D. Paterson

Indicators of stress in the hemolymph of the western rock lobster {Pamdirus cygniis George) 676

Patrick T. Spanoghe and Philip K. Bourne

Physiological profiles and vigour index of lobsters (Paiuilinis cygiuis) delivered to processing facilities 676

Richard J. B. Musgrove

The use of hemolymph chemistry in condition assessment of the southern rock lobster {Jasus edwardsii) 677

Japo Jussila, Elena Tsvetnenko, Jeff Jago and Louis H. Evans

Post-harvest handling stress in western rock lobster: Hemocytes" point of view 677

Richard J. Cawthorn

Coldwater lobster health: A North American perspective 677

Louis H. Evans, Seema Fotedar, Japo Jussila, Shannon McBride and Elena Tsvetnenko

Immunological measures of stress in spiny lobsters [Paiuilirus cygiuis George) 678

John H. Norton, Naomi Levy and Kelly Field

A preliminary evaluation of three hemolymph tests to assess health status in tropical rock lobsters

{Pamdirus ornatus) "'o

Manel Dias-Wanigasekera, Jean Pierre Dufour and Philip V. Mladenov

Responses of spiny lobster {Jasus edwardsii) fed L-carnitine as a dietary supplement under temperature and

starvation stress "78

Stephen Hood

Post-harvest handling in the western rock lobster fishery 679

Brian D. Paterson, Patrick T. Spanoghe and Glen W. Davidson

Identifying indicators of stress during post-harvest handling of western rock lobsters {Pamdirus cygiuis) 679

Michelle M. Pritchard and H. Harry Taylor

The effects of temperature on the respiratory function of the New Zealand lobster {Jasus edwardsii) in air and water.. 679
Brian D. Paterson, Glen W. Davidson and Patrick T. Spanoghe

Determination of total protein in hemolymph of the western rock lobster {Panutirus cygnus George) by refractometry . 680
Seema Fotedar, Anne Barnes, Louis Evans, Mike Geddes and Ruth Renter

Investigations of bacteremia in spiny rock lobsters 680

Intemalioiial Symposium on Lobster Health Management

Ahstnicis. September 19-22, 1999 673

HEALTH MANAGEMENT OF THE AMERICAN LOB-
STER. Robert C. Bayer, Deanna L. Prince, and Maya A.
Crosby, University of Maine, 22 Coburn Hall. Orono. Maine
04469-0014, USA: Ben Tall and Seynabou Fall, JIFSAN,
USFDA, Washington DC 20204 USA; Michael B. Loughlin, De-
partment of Bio-systems Science and Engineering, University of
Maine, Orono, Maine 04469, USA.

The American lobster, Homarus americaiuts, is subject to sev-
eral health problems that appear during post-harvest storage and
transport. Major sources of post-harvest losses include gaffkemia
ordered tail", ciliated protozoan disease, shell disease, and vibriosis
and other types of Gram-negative bacterial infections.

Catastrophic losses of lobsters have been most consistently
associated with gaffkemia. Infection results when the bacterium
Aerococcus viridans breaches the integument through wounds. A
fatal sepsis is the ultimate outcome of infection, with the onset of
mortality dictated by temperature. Gaffkemia is presently moni-
tored by individuals in the lobster industry, with a simple
hemolymph culture technique that uses syringes pre-loaded with a
selective medium. Lobsters in storage can be treated for gaffkemia
with a feed that contains oxytetracyline. Industry use of this feed
has greatly reduced associated mortalities.

Ciliated protozoan disease is also associated with some lobster
mortality in storage. The causative organism is Anophryoides hae-
mophita. which invades lobster tissues and hemal spaces through
perforations of the integument. .Acute infections are typically char-
acterized by the presence of large numbers of ciliates freely swim-
ming in the hemolymph, and are readily detected by microscopy.
Mortality may be due to tissue destruction and loss of hemocytes
produced by invading ciliates, or may be due to secondary invad-
ers.

Shell disease also contributes to some market losses in long-
term storage facilities. Erosion and necrosis of the exoskeleton not
only make lobsters unattractive to the consumer, but also prone to
weakness and mortality. Causative factors of shell disease are not
conclusively established, but bacteriological examinations indicate
that shell lesions are principally associated with bacteria of the
genera Vibrio. Pseitdomonas. and Aeromonas. There is also a
strong relationship between shell disease and lobster source, and a
possible link to lobster nutrition.

Lobster health problems related to Viljiio spp. and other Gram-
negative bacterial pathogens are apparently increasing in signifi-
cance. Recent, high mortalities in some Maine lobster pounds have
been associated with a strain of Gram-negative bacteria identified
as Vibrio fluvialis. A simple hemolymph culture test may be useful
in screening for infections of this type. Environmental or other
etiological factors may also be important in this type of infection.
Lobsters with this syndrome are weak and lethargic. A recent study
to characterize biochemically and genetically 19 different isolates
obtained from diseased lobsters indicated that the isolates were
highly susceptible to a variety of antibiotics tested. However, re-
sistance to erythromycin was observed in 6 of the strains. These
organisms have a strict temperature growth requirement and are

halophilic. Analysis by pulsed field gel electrophoresis revealed 5
highly related subgroups; one strain could not be typed. Strains
were found to possess multiple plasmids suggesting that plasmid
carriage is found in these strains. However, the role of plasmids in
pathogenesis is unknown. Further analysis of the crude preparation
showed that it was not a cell-associated protease; indicating the
expression of putative adherence factors. A tissue culture assay
showed that polymyxin B ly.sates obtained from cells could cause
elongation of Chinese hamster ovary cells, implying the presence
of a putative enterotoxin. Small, irregularly distributed, spike-like,
electron-dense deposits were observed on individual cells analyzed
with an Alcian blue-lysine electron microscopy staining method.
These same cells were observed to form clusters of various sizes,
held together by similar spike-like structures, interdigitating be-
tween the cells. In contrast, unstained, control cells either were
barren of all surface structures or displayed a continuous, fine,
lace-like coating of extracellular material. These results suggest
expression of either a capsule or an array of surface glycoprotein
structures.

DISEASES IN SPINY LOBSTER HOLDING IN NEW

ZEALAND. Benjamin K. Diggles, National Institute of Water
and Atmospheric Research Ltd, PC Box 14-901 Kilbimie, Well-
ington, New Zealand.

In recent years a significant amount of research has been con-
ducted in New Zealand investigating methods of culturing two
species of spiny lobsters, Jasits edwardsii and Jasiis verreauxi.
This research and recent legislative changes have lead to the es-
tablishment of a small scale commercial mariculture industry
based on grow-out of 7. edwardsii pueruli collected from the wild.
Various disease agents have contributed to morbidity and mortality
in each of these species, especially during the early stages of
development and refinement of rearing techniques. Mortalities of
puerulus and juvenile J. edwardsii in experimental holding facili-
ties were due to the invasive fungus Haliplithoros cf. milfordensis
and secondary vibriosis. Fouling of gills of J. edwardsii juveniles
with a thin, septate fungi, a filamentous Leucothrix-Vike bacteria,
free living nematodes and ectocommensal ciliates were probably
due to poor water quality and system design in lobster rearing
systems utilising recirculated seawater. Chronic, low level mor-
talities of adult J. edwardsii in experimental holding tanks were
associated with symptoms of swelling, a condition termed Turgid
Lobster Syndrome (TLS). Bacteria isolated from lobsters display-
ing TLS included Vibrio luin'eyi and V. splendidus I, however the
aetiology of TLS remains undetermined at present. A small num-
ber of moribund adult J. edwardsii in a dietary experiment pre-
sented pathological symptoms reminiscent of necrotizing hepato-
pancreatitis. The crustacean pathogen Vilnio han'eyi was isolated
from moribund phyllosomas of / verreauxi exhibiting luminous
vibriosis during an acute mortality event in an experimental culture
facility. The gross signs of each disease and some suggestions for
their prevention and control are described.

674 Abstracts. September 19-22, 1999

International Symposium on Lobster Health Management

HEALTH ASPECTS IN NORWEGIAN LOBSTER STOCK
ENHANCEMENT: PRINCIPLES AND PRACTICE. Knut E.
Jorstad, 0ivind Bergh, and Karl Andersen. Institute of Marine
Research. Bergen. Norway. C/- CSIRO Marine Laboratory. GPO
Box 1538. Hobart. Tasmania 7001.

Unless special precautions are taken, large scale releases into
the environment of artificially propagated organisms can represent
increased risks of spreading diseases in wild populations. In the
government funded research program (PUSH) on sea ranching and
stock enhancement conducted in Norway from 1990 to 1998. dis-
ease testing of broodstock and juveniles was required before per-
mission for release was given (salmon). In the case of the enhance-
ment project of a local stock of European lobster. Homarus gam-
marus, at the Kvits0y islands, all information available was
associated with the lobster disease Gaffkaemia caused by the
pathogen Aerococciis viridans. This disease is commonly known
in American lobster, Homarus americanus, but had spread to Eu-
rope possibly through commercial import of live specimens. In
Norway it was first reported in imported American lobster in 1976,
and extensive investigation in Norwegian wild populations in
1981-1984 concluded that the pathogen was not endemic in Nor-
wegian waters. Several cases of the disease were reported in the
1990s at Kvits0y. but analyses of the berried animals used as
broodstock in the enhancement project revealed no pathogens and
the activities were conducted according to schedule. The fish dis-
ease problems in the Norwegian aquaculture industry have re-
quired a more comprehensive legislation and a new law was es-
tablished from 1998 and regulated all kinds of diseases in farmed
and wild organisms including the marine environment. All new
plans for establishing lobster hatcheries for stock enhancement and
farming will be carefully evaluated by veterinary authorities.

Cellular immune reactions are important in defence, and two
communicating proteins have been isolated and cloned: beta-1,3-
glucan and peroxinectin. Both of these proteins are associated with
the proPO-system and of great surprise was the finding that per-
oxinectin had a functional peroxidase domain but peroxidase ac-
tivity was not involved in the cell adhesion activity of peroxinectin
and instead a KGD motif was found to be of importance. Recently
antibacterial peptides have been characterised in shrimp and they
have been named penaeidins.

CELLULAR RESPONSE TO INJURY IN SPINY LOB-
STERS. Brian Jones, Fisheries WA, 3 Baron-Hay Court, South
Perth, Western Australia, 6151.

This paper presents a review of the cellular defense mecha-
nisms of spiny lobsters. These mechanisms can be divided, for
convenience, into three broad groupings: maintenance of exoskel-
eton integrity; foreign agent recognition, inactivation and elimina-
tion from the internal organs; and repair of damage by toxins.
Cellular defense mechanisms are dependent on circulating
hemocytes and phagocytes, fixed phagocytes and tlbrocytes. The
process or processes by which these cell types are generated and
mature in the animal have not yet been adequately described. In
addition, attention has only recently focused on the way in which
cellular defence responses are influenced by environmental stress
and by the nutritional and moult status of the lobster. These are
areas of critical importance to animal husbandry and production in
aquaculture. While rapid advances are being made in the under-
standing of humoral defense mechanisms of crustaceans there are
still large gaps in our understanding of the cellular components of
the system in spiny lobsters.

REVIEW OF CRUSTACEAN IMMUNITY. Kenneth Soder-
hall. Department of Comparative Physiology, Evolutionary Biol-
ogy Center, University of Uppsala, Villavagen 6. 752 36 Uppsala.
Sweden.

Crustaceans lack immune memory and have therefore to rely
on innate immune reactions. One such reaction is the clotting
process which is very efficient and rapid and consists of a clotting
protein present in plasma and a transglutaminase in the blood cells.
The clotting protein has been cloned and belongs to the vitelloge-
nin superfamily of proteins. Clotting proteins have also been pu-
rified from shrimp and they are very similar in properties to that of
crayfish. Another innate immune defence process is the so called
proPO-system which is a non-self recognition system and which
upon activation by microbial products generates several factors
which will aid in the elimination of foreign particles or parasites.
This system has been studied in greatest detail in freshwater cray-
fish and most of the proPO-componcnts have been purified and
cloned. Recently, we have also been able to clone some proPO-
components from a shrimp. I'cucinis iiionodan.

TECHNIQUES FOR EMUNERATION AND MORPHOL-
OGY OF HEMOCYTES IN WESTERN ROCK LOBSTER
(PANULIRUS CYGNUS GEORGE). JelT Jago and Bob Dun-
stan. School of Biomedical Sciences. Curtin University. GPO Box
U1987, Perth. Western Australia 6845; Japo Jussila, Institute of
Applied Biotechnology, Kuopio University, Kuopio, Suomi-
Finland; Louis H. Evans, Aquatic Science Research Unit, Muresk
Institute of Agriculture, Curtin University, GPO Box U1987,
Perth, Western Australia 6845.

A review of anticoagulant strategies for the enumeration and
differentiation of total hemocytes counts (THC) in western rock
lobster samples collected in various field conditions will be pre-
sented. Anticoagulant criteria required preservation of morpho-
logical characteristics and arresting of clotting and cell adhesion
processes for a minimum period of 24 hours will be described as
will the characteristic features of hemocyte types found in Panii-
liriis cygniis.

International Symposium on Lobster Health Management

Ahstractx. September 19-22. 1999 675

CAN COMPROMISED CONDITION EXPLAIN EARLY
MORTALITIES IN SPINY LOBSTER CULTURE? Andrew

G. Jeffs. National Institute of Water and Atmospheric Research
Ltd, 269 Khyber Pass Road. Newmarket. Auckland. New Zealand.
The lit'ecycle of spiny lobsters involves a long larval period that
can last for over two years in some species. Planktonic lobster
larvae metamorphose to nektonic pueruli. which make their way
into shallow waters to settle and later moult to become benthic
juveniles. The distance travelled by the pueruli of many species is
estimated to be in the order of tens of kilometres, a journey taking
up to several weeks. Interestingly, the puerulus in many species
appears to be non-feeding, relying entirely on reserves accumu-
lated during the larval phase. Recent research has indicated that
stored lipid is critical for fueling the onshore movement and sub-
sequent moulting of the puerulus. Furthermore, some of this re-
search suggests that a proportion of puerulus may be bereft of
energy stores upon settlement. This may greatly affect their sub-
sequent chances of survival by preventing development to the
moult or exposing them to increased disease risks as a conse-
quence of a lowered immune response. This possibility was inves-
tigated at a commercial aquaculture facility where high mortalities
were experienced among pueruli taken from the wild as seed stock.
Biochemical techniques previously developed for assessing con-
dition in pueruli were used for samples of live, moribund and dead
lobsters sampled from the aquaculture facility. The results of this
study confirm the importance of lipids to the post-settlement de-
velopment of puerulus and suggest that the mortalities experienced
at the facility were not related to depleted lipid reserves.

MEASURES OF CONDITION IN DIETARY STUDIES ON
WESTERN ROCK LOBSTER POST-PUERULI. Elena Tsvet-
nenko, Jeremy Brown, and Louis H. Evans. Aquatic Science
Research Unit. Muresk Institute of Agriculture. Curtin University
of Technology. GPO Box U1987, Perth. Western Australia 6845.

A nutritional study has been conducted for nine weeks on west-
em rock lobster post-pueruli fed either fresh mussel diet (Dl) or
one of four artificial diets, two in moist (D2 and D3) and two in dry
(D4 and D5) pelleted form. Artificial diets were designed in
CSIRO Division of Marine Research, Queensland. The formula-
tion and chemical composition of the diets was proprietary infor-
mation and not supplied.

Growth rates and condition indices were determined for all
treatment groups. Growth rates were expressed as average daily
gain and specific growth rate. At the commencement of the ex-
periment, moisture content of digestive gland and tail muscle, and
hepatosomatic and muscle-somatic wet and dry indices were de-
termined. At the end of the experiment animals from each treat-

ment were examined for the same parameters. In addition, at the
end of the experiment, hemolymph was extracted from animals fed
Dl and D2. Total number of hemocytes and percent of granular
cells were determined in hemolymph samples.

Lobsters fed the natural mussel diet grew significantly faster
than those fed the artificial diets. Changes in digestive gland and
muscle indices indicated deterioration of animals' condition during
the course of the trial in all treatments. This deterioration was more
pronounced in lobsters fed artificial diets compared to lobsters fed
mussel diet. While there were no significant differences between
growth rates in animals fed artificial diets, significant differences
were observed in several of the condition indices. The hemolymph
parameters, total hemocyte count and proportion of granular cells
were in agreement with tissue indices. The use of condition indices
for assessing lobster nutritional and health status will be discussed.

TAIL ROT IN SOUTHERN ROCK LOBSTERS UASUS ED-
WARDSIt). Ruth E. Renter, Veterinary Pathology Services. PC
Box 445. Glenside. South Australia 5065: Michael Geddes, De-
partment of Environmental Biology. University of Adelaide, Ade-
laide, South Australia 5005; Louis H. Evans, Aquatic Science
Research Unit, Muresk Institute of Agriculture, Curtin University
of Technology, GPO Box U1987, Perth, Western Australia 6845.
Tail rot has been identified as a continuing problem in captive
lobsters kept in holding facilities to maximise return on invest-
ment. Chitin-destroying bacteria have been identified overseas in
lobsters held in groups over winter. In South Australia the condi-
tion has been seen during the summer period from December to
April. As part of another study on nutrition, samples were col-
lected in March 1999 from five groups of lobsters being fed dif-
ferent diets and held in varying locations on the South coast.
Lesions on the tail and/or claw were identified in 1 1 animals.
Histopathology done on 7 samples showed inflammation often
associated with cracks and fissures in the overlying chitin. Of four
samples cultured. Vibrio alginolyricus was cultured from all
samples, while Plesiomonas shigelloides also obtained from one of
the samples. V. alginolyticus and Aeromonas hydrophila were cul-
tured from similar lesions in lob,sters in holding cages in March
1998. These organisms are commonly present in marine and es-
tuarine environments. However they have been associated with
skin damage, ulcers, anemia, and tail and fin rot in finfish. and
mortality after handling in eels. In the situation described, handling
and holding of the lobsters, in association with elevated water
temperatures, could be predisposing to invasion of damaged tissue
by organisms such as V. alginolyticus.

676 Abstracts. September 19-22, 1999

International Symposium on Lobster Health Management

A STUDY OF DISEASES IN CULTURED PHYLLOSOMA
LARVAE AND JUVENILES OF SOUTHERN ROCK LOB-
STER UASVS EDWARDSII). Judith Handlinger, Jeremy Car-
son, Arthur Ritar, and Bradley Crear, Tasmanian Aquaculture
and Fisheries Institute. University of Tasmania. Hobart. Tasmania.
Cultured phyllosoma larvae and juveniles of southern rock lob-
ster Uasiis edwardsii) were monitored for disease for almost two
years. No major disease outbreaks and no specific pathogens have
been seen in either age group, though relatively few were exam-
ined. A variety of external fouling organisms and occasional
deeper invasions were seen, which have contributed to either spo-
radic losses or to background low level mortality. Fouling consis-
tently involved adhered Leucothrix-W/x bacteria that provided a
habitat for a complex microbiota consisting of clumps of smaller
bacteria, stalked peritrich ciliates. Chilodonella-Wke flagellates.
amoebae, and occasional fungi. Bacteria isolated from animals
held in culture, and possibly associated with focal degeneration
and adhesion of the exoskeleton. particularly in appendages, in-
cluded Flavobacteium species and mixed Vibrio species including
V. anguillarum, V. alginolyticus and V. tubiashii, all recognised
pathogens of several aquatic animals. Histological examination
suggested these bacteria were largely in small granulomas in ap-
pendages (gills in juveniles), or in the hepatopancreas tubules. V.
harveyi was once isolated from newly collected small juveniles
with digestive tubule degeneration and occasional granulomas
with visible bacteria. Isolation of bacteria from hemolymph of
juveniles was rare, and there was no histological evidence of bac-
teremia in either age group. Heavy fouling and gill tip necrosis
reflected water quality (high ammonia, low dissolved oxygen) and
was largely controlled by manipulation of environmental condi-
tions.

INDICATORS OF STRESS IN THE HEMOLYMPH OF THE
WESTERN ROCK LOBSTER iPANUURUS CYGNUS
GEORGE). Glen W. Davidson and Patrick T. Spanoghe, Centre
for Food Technology. Queensland Department of Primary Indus-
tries, C/- School of Biomedical Sciences, Curtin University of
Technology, GPO Box U1987. Perth. Western Australia 684.^:
Brian D. Paterson, Centre for Food Technology, Queensland De-
partment of Primary Industries, Queensland .19 Hercules street,
Hamilton, Queensland 4007.

The western rock lobster Painilirus cygniis supports a large,
sustainable commercial fishery in Western Australia. This species
is wholly sub-tidal, but experiences repeated episodes of exposure
to air during post-harvest. The effect of air exposure on the lobsters
is confounded by handling/disturbance and temperature fluctua-
tion. The cumulative effect of these stressors is observed as a
reduction in the muscle tone, vigour and responsiveness of the
lobsters. However, depending on the severity and duration of the
stressful encounter, these symptoms may reflect anything from
temporary exhaustion to permanent damage resulluiL' in imniincm

death. Traditional methods of grading lobsters for various product
forms depend on visual assessments of animals and, for the above
reasons, can be misleading. The purpose of the present work was
to identify physiological indicators of stress that are associated
with future morbidity and mortality during post-harvest handling.
After implementing practices which maximise survivorship, useful
physiological indicators can be used to assess the sublethal effects
of alternative handling practices. Focusing on hemolymph con-
stituents, a range of metabolites, electrolytes, enzyme activities,
proteins, and hemolymph blood gas and acid-base parameters were
measured in lobsters undergoing actual or simulated post-harvest
handling treatments. These factory-based experiments were sup-
ported by closely controlled laboratory experiments. Baseline lev-
els of parameters of interest were determined in free-ranging lob-
sters in the field by divers equipped with SCUBA, and also in
acclimation studies in the laboratory. These studies also provide
information regarding how the parameters vary with moulting ac-
tivity. In this paper we discuss normal ranges for potential stress
indicators in P. cygnus. The response dynamics of hemolymph
variables to specific stressors are also presented along with some
interpretation of their physiological significance.

PHYSIOLOGICAL PROFILES AND VIGOUR INDEX OF
LOBSTERS IPANULIRUS CYGNUS) DELIVERED TO PRO-
CESSING FACILITIES. Patrick T. Spanoghe, Centre for Food
Technology. Queensland Department of Primary Industries, C/-
School of Biomedical Sciences. Curtin University of Technology,
GPO Box U1987, Perth, Western Australia 6845; Philip K.
Bourne, School of Biomedical Sciences, Curtin University of
Technology, GPO Box UI987, Perth, Western Australia 6845.

In Western Australia, lobsters delivered to processing premises
are routinely subjected to a grading procedure, aiming at selecting
from the catch the animals best suited for live export marketing.
This assessment is essentially based on a range of criteria referring
to the general morphological appearance of the animals such as the
size, the colour of the shell, the number of missing appendages but
also to the behavioural appearance of the animals, that is essen-
tially the intensity of their somatic responses to physical stimula-
tion. This paper presents and discusses the results of a study com-
paring the physiological profiles of a sample of lobsters assessed
and graded for vigour, with reference to a selection of behavioural
observations. It presents the results of an analysis aimed at iden-
tifying the physiological variables best correlated to a vigour in-
dex.

International Symposium on Lobster Health Management

Abstracts. September 19-22. 1999 677

THE USE OF HEMOLYMPH CHEMISTRY IN CONDI-
TION ASSESSMENT OF THE SOUTHERN ROCK LOB-
STER UASUS EDWARDSII). Richard J. B. Musgrove, SARDI

Aquatic Sciences. 2 Hamra Ave. West Beach . South Australia
5024.

A study was carried out to develop a condition index for the
characterisation of temporal and spatial changes in condition and
growth for the southern rock lobster. Jasiis edwardsii.

Serum protein concentration has been accepted as a coarse
indicator of condition, because of its correlation with muscle mass,
particularly percent muscle mass, which has been shown to decline
on starvation and to change with moult stage. The problem has
been in the integration of the moult cycle-dependent body com-
position with the measurement of condition. The mouh cycle and
condition are inextricably linked. Simply measuring tissue or
hemolymph composition is of doubtful utility if the moult stage is
not accurately known. This is made especially difficult if inter-
moult (Cj) is long, as is the case with many lobsters. There is no
way of distinguishing between those at the beginning and those at
the end of this stage. This is particularly important as muscle and
storage tissue accumulation continue from ecdysis until late pre-
moult.

In this paper I present and discuss a method for condition
assessment of commercial-sized lobsters based on the serum pro-
tein: tissue relationship and blood pigmentation. The latter may be
broken down into eight stages, four of which occur during inter-
moult. It is shown that both percent dry tissue and total dry tissue
may be predicted using a combination of the two measurements.
The use of blood lipid in prediction of moult increment is also
discussed.

POST-HARVEST HANDLING STRESS IN WESTERN
ROCK LOBSTER: HEMOCYTES' POINT OF VIEW. Japo
Jussila, Institute of Applied Biotechnology. Kuopio University.
Kuopio, Suomi-Finland; Elena Tsvetnenko and Louis H. Evans,

Aquatic Science Research Unit, Muresk Institute of Agriculture,
Curtin University. GPO Box U1987. Perth. Western Australia
6845; Jeff Jago. School of Biomedical Sciences. Curtin Univer-
sity, GPO Box U1987, Perth, Western Australia 6845,

The hemocyte response in western rock lobster (Paiudiriis cyi>-
mis) to conditions of post-harvest handling stress was investigated
in a series of studies carried out either under practical conditions or
in a research laboratory. Total hemocyte counts (THCs) and dif-
ferential hemocyte counts (DHCs) were studied along with other
indicators of stress. Lobsters were sampled in different stages of
post-harvest handling or as part of specially planned experiments,
with the hemocyte sample (200 jxLj collected from either ventral

or pericardial sinus into precooled Na-Cacodylate anticoagulant
and later analysed for THCs and DHCs. The minimum sample
number required to give significant differences between treatment
groups was observed to be 10, while statistical methods suggested
a sample size of 18. The results suggested that THCs and DHCs
could be used as stress or condition indicators under various post-
harvest handling conditions. THCs were indicative of physical
disturbance, loss of condition and starvation, while air exposure as
sole stressors seemed to cause less changes in the numbers of
circulating hemocytes. The changes in DHCs were more prone to
indicate changes in the lobsters' physical condition, and the de-
creasing hyalinocytes proportion seemed to correlate with bacter-
emia. It was concluded that between 4 and 8 x 10* cells/mL of
THCs could be considered to be an undisturbed background level,
while THCs lower than that could indicate worsening condition
and point of mortality and levels higher than that could indicate
different levels of stress. The indications of stress or change in the
lobsters' physical condition could be seen both in THCs and
DHCs. These could be used as tools in measuring the effects of
post-harvest handling on western rock lobsters, especially in con-
junction with other stress parameters.

COLDWATER LOBSTER HEALTH: A NORTH AMERI-
CAN PERSPECTIVE. Richard J. Cawthorn, Lobster Health
Research Centre. Atlantic Veterinary College, University of Prince
Edwards Island, Charlottetown, Prince Edwards Island, Canada
CIA 4P3.

In North America, one of the largest traditional surviving fish-
eries involves the coldwater clawed Amercian lobster Homanis
ainericanus. However, post-harvest losses are conservatively esti-
mated at 10-15%. representing an economic impact of $50-75
million annually. The mandate of the Lobster Health Research
Centre is to apply the principles of veterinary medicine to the
post-harvest sector of crustacean fisheries and to crustacean aqua-
culture. The primary task is to define what constitutes a healthy
lobster, and subsequently to maintain or enhance the health status
of lobsters. Important infectious diseases in confinement situations
include "bumber car" disease caused by the ciliate Anophn-oides
haemophiki. gaffkemia caused by the bacterium Aerococcits viri-
daiis. and shell disease associated with bacterial species of Aero-
inonas. Pseudomonas and Vibrio. Additional factors reducing lob-
ster health are improper handling, exposure to adverse weather,
inappropriate bait, inadequate nutrition and environmental stress-
ors. Lobster health surveillance requires knowledge of ecosystem
health, development of lobster databanks, and interaction at all
levels of the fishery to enhance lobster health management.

678 Ahstnwts, September 19-22. 1999

International Symposium on Lobster Health Management

IMMUNOLOGICAL MEASURES OF STRESS IN SPINY
LOBSTERS (PANULIRUS CYGNUS GEORGE). Louis H.
Evans, Seema Fotedar, Japo Jussila, Shannon McBride, and
Elena Tsvetnenko, Aquatic Science Research Unit, Muresk Insti-
tute of Agriculture. Curtin University of Technology, GPO Box
U1987, Perth, Western Australia 6845.

Methodology for six different assays of immunological stress
parameters, total hemocyte counts (THC), differential hemocyte
counts or % granular cells (%gran), antibacterial factor (ABF),
phagocytic capacity (phag). clotting time (clot) and hemolymph
bacterial colony counts (CFU/ml; bact) were developed and ap-
plied in studies of stress responses in the spiny lobster Pamdirus
cygnits. An investigation of the influence of handling procedures
and other minor disturbances on the levels of immune parameters
in lobsters held in the laboratory showed that THC increased &
phag decreased within minutes of exposure to a handling stressor
(placement in a foam box and shaken for 1 min and every subse-
quent 30 min for 2 h). clot decreased two hr after stressor exposure
while ABF and '/rgran were unaffected by minor stressors. Bac-
terial levels in hemolymph in the test lobsters were high at the
beginning of the experiment and showed no significant variation
following stressor exposure.

In two simulated transport and live shipment trials (November
1998 and March 1999) in which lobsters were held either in water
(submerged), in air (humid air) or in a spray system (spray) at
ambient temperature (November 22 °C; March 26 °C) for 6 h in
five enclosed compartments fitted with either tlowthrough (flow)
or recirculating (recirc) water systems (flow submerged, recirc
submerged, humid air. How spray and recirc spray) all parameters
in hemolymph taken at the completion of the 6 h exposure period
studied showed a consistent pattern of variation between treatment
groups with the exception of THC. Mean values of ABF, bact and
clot in lobsters held in humid air or in spray systems were higher
and mean '/r gran was lower than values observed in the submerged
lobsters. The patterns of variation in ABF, bact, clot and 7fgran in
the five different treatments mirrored the pattern of survival of
lobsters following tank storage and simulated shipment with sur-
viving lobsters showing lower ABF, bact and clot and higher
%gran than lobsters which died or were weak at packout or fol-
lowing simulated live transport. Hemolymph did not clot in a small
proporlit)n (.^.9-l().2Vf ) of lobsters from all five treatments in the
first trial and in the How submerged, recirc submerged and humid
air treatments in the second trial. The percentage of lobsters with
hemolymph which didn't clot was significantly higher in the tlow
spray and recirc spray treatments in the March trial (2.S..'iVr and
20.8% respectively). Autopsies performed on weak lobsters re-
moved from factory tanks within one to several days after the
simulated transport showed that the mortalities were likely to have
been caused by bacterial infections in the bladder and the aniennal
glands. The results suggest that THC and phagocytic capacity
show rapid responses to postharvest handling stressors, clotting
time initially decreases and then increases while bacteremia and

the levels of ABF increase following stressor exposure. The ap-
plication of these variations in immune parameters to the determi-
nation of the stress status or health status of postharvest lobsters
will be discussed.

A PRELIMINARY EVALUATION OF THREE
HEMOLYMPH TESTS TO ASSESS HEALTH STATUS IN
TROPICAL ROCK LOBSTERS {PANULIRUS ORNATUS).
John H. Norton, Naomi Levy* and Kelly Field, Oonoonba Vet-
erinary Laboratories, Queensland Department of Primary Indus-
tries, PC Box 1085, Townsville, Queensland 4810.

Three hemolymph tests were evaluated on small numbers of
tropical rock lobsters Pamtlinis oiiialiis as possible indicators of
health status. These included a phenoloxidase test, a red blood cell
(RBC) agglutination test and an antibacterial test. They were con-
ducted on both clinically normal and sick adult lobsters. Highly
significant differences (P < 0.01 ) were obtained for the phenoloxi-
dase and RBC agglutination tests. Although no significant differ-
ence was obtained for the antibacterial test, further work with
larger numbers of lobsters may prove otherwise. These pilot ex-
periments strongly suggest that further experimentation with these
three tests on both clinically normal and sick adult rock lobster
hemolymph would be productive.

RESPONSES OF SPINY LOBSTER UASUS EDWARDSII)
FED L-CARNITINE AS A DIETARY SUPPLEMENT UN-
DER TEMPERATURE AND STARVATION STRESS. Manel
Dias-Wanigasekera, Jean Pierre Dufour, and Philip V. Mlad-
enov. Department of Marine Science. University of Otago. PC
Box 56. Dunedin. New Zealand.

The effects of 1-carnitine supplemented in a squid based diet
formulated for juvenile Jastis eihvttrdsii were investigated in three
feeding experiments. Growth and survival responses were collec-
tively expressed as the normalized biomass index (NBI). L-
carnitine was included at a level of .^00 mg/kg. The NBI was 6.7
for lobsters fed a diet supplemented with 1-carnitine and fatty
acids, in comparison with lobsters fed an unsupplemented diet
(NBI = 4.07) or mussel (6.06) as food. Survival was positively
correlated to the NBI (r2 = 0.64). After starvation for two weeks,
98'/f of the lobster |u\eniles fed 1-carnitine sur\ ived, whereas a
survival of 70% was obtained for lobsters fed other diets. In a
subsequent experiment, exposure to high temperature stress
(32 °C/3h), caused an immediate mortality of 17% in 1-carnitine
fed animals and 48% in Juvenile lobsters fed an unsupplemented
diet. Fxposure to low temperature stress (0 C/(ih) produced an
imrnedialc mortality of 45% in juvenile lobsters fed mussel and
l2'/f to 18'/( in animals fed various unsupplemented feeds. All
1-carnitine fed animals survived. Independent of the diet, all ani-
mals showed growth retardation following temperature shock.
Growth promoting effects of l-carniline were seen immediately

International Symposium on Lobster Health Management

Ahslrcuts. September 19-22. 1999 679

after administration via feed, whereby the animals fed this com-
pound displayed an acceleration of the moulting process. Supple-
mentation of diets with regulated amounts of l-carnitine over short
periods improves growth on a short-term basis, and this improve-
ment could be used to advantage in starter diets. L-carnitine also
improves physiological resistance of lobsters to stressful condi-
tions often met with during long-term holding, aquaculture and
live transport. The biological activity of l-carnitine in rock lobsters
seems to be between that of a growth promoter and a vitamin
compound. However, care needs to be exerted so that the period of
feeding and level of supplementation are carefully monitored.

POST-HARVEST HANDLING IN THE WESTERN ROCK
LOBSTER FISHERY. Stephen Hood, MG Kailis Group of
Companies. 50 Mews Road. Fremantle WA 6160.

The western rock lobster, Panulirus cygmis. forms the basis for
one of the world's largest lobster fisheries with a catch value in
excess of A$250 million annually. The fishery supports over 600
catcher vessels along almost 1000 km of coastline and offshore to
a depth of 250 m. This large geographical fishing area and the
highly seasonal nature of the industry creates unique post-harvest
handling considerations for both the wild capture and processing
sectors of the industry. Currently, only some 30% of the total catch
is exported as live product, largely as a consequence of the sea-
sonal and geographical distribution of the catch. This presentation
deals with post-harvest handling considerations from capture
through to final export as a live product, as well as addressing
some of the other processing techniques. The presentation also
deals with potential and actual problem areas in the post-harvest
sector, applied research that has been undertaken to overcome
these problems, and the potential for future research to further
enhance the value of the catch.

IDENTIFYING INDICATORS OF STRESS DURING POST-
HARVEST HANDLING OF WESTERN ROCK LOBSTERS
(PANULIRUS CYGNUS). Brian D. Paterson. Centre for Food
Technology. Queensland Department of Primary Industries,
Queensland. 19 Hercules street. Hamilton, Queensland 4007; Pat-
rick T. Spanoghe and Glen W. Davidson, Centre for Food Tech-
nology, Queensland Department of Primary Industries, CI- School
of Biomedical Sciences, Curtin University of Technology, GPO
Box U1987, Perth. Western Australia 6845.

Lobsters are stressed when a factor, sometimes called a stress-
or, causes their internal physiology to deviate from normal. Cur-
rently western rock lobsters. PanuUrus cygmis. are graded a num-
ber of times during post-harvest handling to remove injured or
damaged lobsters as well as removing individuals that are consid-
ered to be "weak' on the basis of their posture or responsiveness.
These stressed lobsters are singled out because experience shows

they are unlikely to survive for long in the factory. Simply know-
ing that these lobsters are stressed may not be of much practical
use. We want to know what it is about particular lobsters that mean
that they survive a handling treatment but die subsequently. To
find these indicators we subjected large numbers of lobsters to a
controlled period of stress, then sampled their blood immediately
to measure a number of physiological parameters. The lobsters
were tagged and stored in a factory and their fate recorded. The
group of lobsters that survived differed statistically from the group
that didn't survive in a number of parameters. But examining the
frequency distributions of selected parameters highlights how dif-
ficult it is to apply one parameter ineaningfully to individual lob-
sters. The distributions of the survivor and non-survivor samples
can overlap considerably. However, discriminant analysis, a multi-
variate technique that uses combinations of parameters to describe
the differences between groups can be used to identify the param-
eters contributing to these discriminant functions and thus provide
the stress indicator we seek. Dead lobsters are easy to count, but
knowing the key physiological characteristics of lobsters that have
been stressed too much and 'exhausted' by a handling treatment
provides important feedback on how to change that treatment to
minimise losses.

THE EFFECTS OF TEMPERATURE ON THE RESPIRA-
TORY FUNCTION OF THE NEW ZEALAND LOBSTER
UASUS EDWARDSm IN AIR AND WATER. Michelle M.
Prltchard and H. Harry Taylor, Department of Zoology, Uni-
versity of Canterbury, 58 Brodie Street, Upper Riccarton,
Christchurch, New Zealand.

The magnitude and rate of change of oxygen consumption in
water following short and long-term temperature changes were
quantified using closed box respirometry. Aerial oxygen consump-
tion was measured at different temperatures using a flow-through
system and Ametek Oxygen Analyser. On emersion, aerial oxygen
consumption decreased at first but progressively recovered during
extended emersion. We hypothesized that this increase in oxygen
consumption might be due to either: a) drying of the gills in air,
leading to improved diffusive conductance of the gills; or b) an
increase in the oxygen affinity of the hemocyanin after prolonged
emersion. Blood gas analysis (pre- and post-branchial Po, and
oxygen content) and acid-base analysis (pH and I-lactate concen-
tration of the hemoly mph) was carried out at 5 °C, 1 2 °C and 1 8 °C
in water and in air after various emersion times. Oxygen equilib-
rium curves were generated from hemolymph taken from lobsters
emersed at 1 2 °C for 4 or 24 h and from aquatic controls. These
data do not support an increase in gill conductance but suggest that
there was an increase in hemocyanin oxygen affinity following 24
h emersion, compared with the values measured at 4 h emersion
and the control values. L-lactate concentration increased during
emersion and may be partially responsible for the increase in oxy-
gen affinity following 24 h emersion.

680 Abstracts. September 19-22, 1999

International Symposium on Lobster Health Management

DETERMINATION OF TOTAL PROTEIN IN
HEMOLYMPH OF THE WESTERN ROCK LOBSTER
{PANULIRUS CYGNUS GEORGE) BY REFRACTOMETRY.
Brian D. Paterson, Centre for Food Technology. Queensland De-
partment of Primary Industries, Queensland ,19 Hercules street,
Hamilton, Queensland 4007; Glen W. Davidson and Patrick T.
Spanoghe, Centre for Food Technology, Queensland Department
of Primary Industries, C/- School of Biomedical Sciences, Curtin
University of Technology, GPO Box U1987, Perth, Western Aus-
tralia 6845.

Research on western rock lobsters {Paniiliriis cygniis) has
shown that refractometry is a simple non-destructive field tech-
nique for assessing the hemolymph protein concentration and
hence the tissue mass or "condition" of rock lobsters (Dall, W.
1975. J. Exp. Mar. Biol. Ecol. 18:1-18). However, a conversion
from refractive index (RI) to protein concentration has not been
published for this species. Hemolymph samples were obtained
from the pericardial sinus of lobsters via the arthrodial membrane
between the posterior margin of the carapace and the abdomen
using disposable hypodermic syringes. In order to convert RI val-
ues to protein concentrations, the refractive index of a number of
hemolymph samples were measured at ambient temperature using
a Shibuya S-1 salinometer calibrated at ambient temperature with
distilled water. Total protein concentrations were determined co-
lourimetrically by the biuret method. Comparing the data gave the
following regression equation:

Total protein (mg/mL) = (5402,398 x RI) - 7214.877, r- =
0.947, n = 28

This analytical method represents a simple, and useful way to
obtain important information about the condition of rock lobsters
entering commercial handling and transport. The measurement in-
volves a straightforward physical phenomenon and it is probably
not surprising that the conversion equation obtained in this study is
similar to that derived from a study of the American lobster (Leav-

itt, D.F. & Bayer, R.C. 1977. Aqiiacitltiire 12:169-171). These
conversions may be a satisfactory method of estimating blood
protein concentration in other large marine crustaceans. If large
numbers of measurements are to be made for other species using
this method, it is relatively easy to establish a calibration to ensure
the accuracy of the technique.

INVESTIGATIONS OF BACTEREMIA IN SPINY ROCK
LOBSTERS. Seema Fotedar, Anne Barnes, and Louis Evans,

Aquatic Science Research Unit, Curtin University of Technology,
GPO Box U1987, Perth, Western Australia 6845; Mike Geddes,
Department of Environmental Biology, University of Adelaide,
Adelaide. South Australia 5005; Ruth Renter, Veterinary Pathol-
ogy Services, PO Box 445, Glenside, South Australia 5065.

Bacteremia was studied as part of various laboratory based and
factory based stress trials in the spiny lobster, Paiiulirus cygnus
and in a dietary trial in southern rock lobster, Ja.nis edwardsii.
Bacteremia was assessed in three different ways: I ) based on per-
cent prevalence; 2) based on mean colony rank (ranging from 0 (0
CFU/ml) to 13 (>8.45 x 10' CFU/mU): and 3) based on mean
colony count (CFU/ml). Under unstressed conditions very low
bacteremia levels were observed (rank 0-1 ). Minor stressors such
as handling disturbance and repetitive sampling led to increased
prevalence of bacteremia in Paiudirus cygnus. In two simulated
transport and shipment trials significant levels of bacteremia were
observed. The frequency distribution of colony ranks showed
larger numbers within ranks 0-5 and rank 13 and few in between.

The dietary trial on Jasiis edwardsii involved on-site sampling
following collection from cages and repetition of sampling after air
freight (approx 1 h) and 16-24 h storage in a cold room at 4 °C.
Bacteremia results were consistent with those of Panulirus cygnus,
with increased prevalence of bacteremia in the 24 h samples. The
dietary treatment had no significant effect on the level of bacter-
emia.

Joiirmil of Shellfish Research. Vol. 19. No. 1. 681-690. 2000.

ABSTRACTS OF TECHNICAL PAPERS

Presented at the 53rd Annual Meeting

PACIFIC COAST OYSTER GROWERS ASSOCIATION

&
NATIONAL SHELLFISHERIES ASSOCIATION

(Pacific Coa.st Section)

Vancouver, Washington

September 29-October 1, 1999

681

PCOGA & NSA. Vancouver. Washington Abstracts. September 29-October 1. 1999 683

CONTENTS

Elizabeth M. Carr and Brett R. Dumbauld

Progress implementing a plan to monitor and control populations of the European green crab (Carciniis maenas) in

Washington coastal estuaries 685

Russell P. Herwig, Robyn M. Estes. Cindy L. Messey and Daniel P. Cheney

Distribution of Vibrio paraliaemolyticiis in Puget Sound oysters, water, and sediments — preliminary results using a

molecular method 685

Daniel P. Cheney, Ralph A. Elston, Brian F. MacDonald, Gary N. Cherr. Amro M. Hemdoim and Jacqueline L. Jacobsen

An update on the ongoing oyster summer mortality study: mortality of the Pacific oyster. Crassostrea gigas: health

screening, environmental links and management options 685

Micaela Schnitzler Parker

Population genetics of Protothaca smminea and Mcdcoma baltliica in Puget Sound, Washington 686

Brent A. Vadopalas, Patrick T. O'Reilly and Paul Bentzen

Isolation of novel microsatellites in geoduck clams (Paiiopea abrupta) by magnetic bead hybridization selection 686

Laura L. Hauck and Sylvia Behrens Yamada

Current status of the European green crab. Caninus maenus. and native crab species in Yaquina and Coos bays 686

Chris Hunt

The distribution of the European green crab, Carciniis maenas. in Yaqina Bay, Oregon 686

Brett R. Dumbauld, Daniel P. Cheney, and Brian F. MacDonald

Developing a pest monitoring plan for burrowing shrimp: a step towards integrated pest management 687

Patrick Baker and Nora B. Terwilliger

Reestablishment of a native oyster: implications for population distribution and structure 687

Alex Kalin and Sylvia Behrens Yamada

Growth of 1997/1998 year class of the green shore crab, Carciniis maenas. in Oregon 687

Anita E. Cook and Sandra Hanson

Progress implementing a plan to monitor for presence of the European green crab {Carciniis maenas) in Puget

Sound, Washington 687

Chris J. Langdon, Dave P. Jacobson, Ford Evans, Ebru Onal and Sean E. Matson

Development of a specific-pathogen-free (SPF) hatchery and nursery for production of Pacific oyster seed 688

Chris J. Langdon, Dave P. Jacobson, Ford Evans and Mike S. Blouin

Interim performance of progeny of selected broodstock from the molluscan broodstock program 688

Ralph Elston and Dan Cheney

Shellfish high health program 688

Amilee Caffey and Brady Blake

Fecundity study and preliminary field trial results of the butter clam (Sa.xidomiis giganteiis) 689

Jonathan P. Davis, Clea Barenburg and David Pederson

Burrowing response of juvenile geoducks iPanopea abnipra) to changes in temperature and salinity 689

Jonathan P. Davis and Clea Barenburg

Post settlement geoduck clam [Panopea abrupta) growth and survivorship in sand and screen-based

nursery systems 689

Alex Bradbury, Don P. Rothaus, Robert Sizemore and Michael Ulrich

A tag method for estimating the natural mortality rate of geoducks (Panopea abrupta) 690

PCOGA & NSA. Vancouver. Washinslon

Ahsinicls. September 29-October 1. 1999 685

PROGRESS IMPLEMENTING A PLAN TO MONITOR
AND CONTROL POPULATIONS OF THE EUROPEAN
GREEN CRAB {CARCIiVUS MAENAS) IN WASHINGTON
COASTAL ESTUARIES. Elizabeth M. Carr and Brett R.
Dumbauld, Washington State Department of Fish and WildHfe.
P.O. Box 190. Ocean Park. WA 98640.

The European green crab (Carciniis maenas) was first noted in
Washington Slate waters during the summer of 1998. having stead-
ily progressed northward from California where its presence in San
Francisco Bay was first recorded in 1989. The two distinct year
classes present appear to have resulted from strong larval recruit-
ment events and not from new introductions. The response to this
bio-invasion was rapid and resulted in a plan to monitor and con-
trol green crab populations in Willapa Bay and Grays Harbor.
Washington which was approved and implemented in 1999. Moni-
toring results suggest the same two year classes are still present.
Although mating couples and females with viable eggs have been
found, no new recruitment has been noted to date. Catch per unit
effort has declined as the population has apparently spread out in
both estuaries making control efforts potentially more difficult.
Trapping methods continue to be refined as the primary control
technique.

DISTRIBUTION OF VIBRIO PARAHAEMOLYTICUS IN
PUGET SOUND OYSTERS, WATER, AND SEDIMENTS-
PRELIMINARY RESULTS USING A MOLECULAR
METHOD. Russell P. Herwig and Robyn M. Estes, School of
Fisheries, University of Washington. 3707 Brooklyn Ave NE, Se-
attle. Washington 98105; Cindy L. Messey and Daniel P.
Cheney, Pacific Shellfish Institute. 120 State Ave NE. #142.
Olympia, Washington 98501.

Vibrio parahaemolydcus (Vp) is a bacterium that is naturally
found in estuarine and marine waters around the world. Selected
strains of Vp may cause food borne illness. This organism can
accumulate in filter-feeding organisms, such as oysters. Vp may be
a problem with oysters that are harvested in the warm summer
months. During the summers of 1997 and 1998. several outbreaks
of Vp gastroenteritis were caused by the consumption of raw oys-
ters harvested from the Pacific Northwest. Federal and state regu-
latory agencies have established a concentration of 10,000 cells per
gram of oyster meat as the level of concern.

During the summer of 1999. oyster, sediment, and water
samples were collected from four sites in Puget St)und. Washing-
ton. Levels of Vp in the various samples were determined using a
newly-developed molecular Most Probable Number (MPN)
method. Samples were blended, diluted, and inoculated into a se-
ries of tubes containing Alkaline Peptone Water (APWl. This me-
dium was incubated overnight at 37 "C. Small aliquots of liquid
were removed from each incubated APW tube and used in a poly-
merase chain reaction (PCR) procedure. This molecular method
was used to amplify a specific fragment of DNA that is found only

in Vp. The enumeration of V); in the molecular MPN method was
performed within 2 days, a much shorter time compared to the
FDA-approved method.

Our results indicated that levels of Vp in Puget Sound oysters,
sediment, and water were not detectable or very low in June 1999,
increased at selected sites in July, and remained elevated in Au-
gust. Concentrations of Vp were much higher in sediment com-
pared to water samples. Although the different Puget Sound sites
had comparable water temperatures and salinities during the sum-
mer, a site on Hood Canal had much higher concentrations of the
potential human pathogen in samples collected during July and
August. Interestingly, the site with the highest Vp levels had sedi-
ments that were very coarse compared to the other sites. The
reasons for the elevated levels of Vp at particular locations in Puget
Sound is not understood.

AN UPDATE ON THE ONGOING OYSTER SUMMER
MORTALITY STUDY: MORTALITY OF THE PACIFIC
OYSTER, CRASSOSTREA GIGAS: HEALTH SCREENING,
ENVIRONMENTAL LINKS AND MANAGEMENT OP-
TIONS. Daniel P. Cheney, Ralph A. Elston, and Brian F. Mac-
Donald, Pacific Shellfish Institute. 120 State Avenue N.E. #142.
Olympia. WA 98501; Gary N. Cherr, Amro M. Hamdoun, and
Jacqueline L. Jacobsen, Bodega Marine Laboratory, Bodega
Bay. CA 94923.

This project tests hypotheses relating to mortalities of Pacific
oysters on the West Coast. Four sites in Puget Sound experienced
limited mortality at all stations through the end of June 1999. By
the end of August increasing mortalities of up to 15% between
sample dates were seen in several treatment groups. These mor-
talities paralleled a similar pattern of very high seed oyster mor-
talities in Tomales Bay. Puget Sound ambient air and water tem-
peratures through late spring 1999 were similar to 1998; however
after June, peak air temperatures were below 1998 levels. Dis-
solved oxygen. pH and salinity levels at all stations remained
nominal throughout most of this period. There was a correlation
between densities of Gymnodiniiini sangidneum. Ceratiwn spp.,
Pseiido-nitzschia spp. and other dominant taxa with the onset of
summer mortality. In addition, moribund oysters had lesions con-
sistent with an acute toxic effect. A biotoxin. most likely of algal
origin, was considered as a probable cause. No infectious diseases
considered certifiable or reportable by regulatory agencies were
observed in these oysters. Investigations into the physiological
responses of oysters to stress are now underway to provide further
insights into the survival process. We are currently field testing
outplants to see if a correlation exists between the biochemical
response and the organismal response.

This research is supported by a grant from the National Sea
Grant College Oyster Disease Research Program and matching
contributions from West Coast shellfish growers.

686 Abstracts. September 29-October I, 1999

PCOGA & NSA. Vancouver. Washington

POPULATION GENETICS OF PROTOTHACA STAMINEA
AND MACOMA BALTHICA IN PUGET SOUND, WA. Mi-
caela Schnitzler Parker, School of Oceanography, University of
Washington. Campus Box 357940. Seattle. Washington 9819.').

Individuals from three populations of Protothaca staminea and
Macoma balthica were examined electrophoretically and scored at
5 allozyme loci. Each of the three populations is located in a
different hydrologically-defined basin of Puget Sound. WA.
Highly significant differences between the three Protothaca sta-
minea populations were found at all 5 loci. However, only at one
locus could the Macoma balthica populations be differentiated.
Genetic distances between the three Protothaca staminea popula-
tions were determined using both Cavalli-Sforza and Edwards
(1967) chord distance and Nei's ( 1972) genetic distance measures.
A genetic similarity dendrogram is presented and discussed in the
context of Puget Sound hydrology.

Between two and four of the allozyme loci demonstrated het-
erozygote deficiencies in Protothaca staminea, depending on
population. Only one locus was heterozygote deficient in each of
the three Macoma balthica populations. Implications of these de-
ficiencies, as well as the differences in population differentiation
between these two taxa, are presented.

ISOLATION OF NOVEL MICROSATELLITES IN GEO-
DUCK CLAMS (PANOPEA ABRUPTA) BY MAGNETIC
BEAD HYBRIDIZATION SELECTION. Brent A. Vadopalas,
Patrick T. O'Reilly, and Paul Bentzen, Marine Molecular Bio-
technology Laboratory, University of Washington. Seattle, Wash-
ington 98105.

Information regarding the genetic population structure of Puget
Sound geoduck clams should guide culture and fishery manage-
ment of this economically valuable species. If population substruc-
ture exists, both the avoidance of genetic intermixing cau.sed by
aquaculture practices and the re-designation of wild tracts avail-
able to fishers may be prudent for genetic conservation. Because of
their high variability and their ability to be assayed from extremely
small samples, microsatellite DNA markers are generally consid-
ered the best tools available for the detection of genetic variation.

Novel di- and tetranucleotide microsatellite loci were devel-
oped in geoduck clams by magnetic bead hybridization .selection
as markers for population genetic analyses. Polymerase chain re-
action (PCR) screening of a 155 clone library yielded 82 positive
clones. DNA sequencing revealed that 62 of the positive clones
contained microsatellite arrays, primarily (GATA),,. .some in com-
bination with other tetranucleotide array motifs. Three of the
clones contained short (AC),, arrays. PCR primer sets were de-
signed for 16 candidate loci. Mono- or di-allelic PCR profiles were
observed at 10 loci and all were polymorphic. These markers will
be used to examine population structure and variance in reproduc-
tive success.

Preliminary screening revealed a deficiency of heterozygotes at
many loci. If these heterozygote deficiencies are not due to labo-
ratory artifacts, larvae of type parents may be screened in an at-
tempt to ascertain the geoduck clam life history stage(s) associated
with these heterozygote deficient loci.

CURRENT STATUS OF THE EUROPEAN GREEN CRAB,
CARCINUS MAENUS. AND NATIVE CRAB SPECIES IN
YAQUINA AND COOS BAYS. Laura L. Hauck, Student. De
partment of Biology, Oregon State University, Corvallis, Oregon
97331; Sylvia Behrens Yamada, Department of Zoology, Oregon
State University, Corvallis, Oregon 97331.

Four different sampling methods were employed at four sites in
Yaquina Bay and five sites in Coos Bay to monitor the progress of
the invasive Carcinus maenas (CM) population. We also recorded
the status of native crab species: Cancer magister. Cancer pro-
diictiis. Hemigraphus oregonensis. Hemigrapsiis nudus. and
Pachxgrapsiis crassipes. in the intertidal zone at the current inva-
sion level.

The first method used at each site was a tethered snail predation
line. This method gives the best natural estimation of predation
rate at a given location, and allows us to compare predation rates
from site to site. Crab species hitting the line are identified by their
shell cracking technique when the lines are checked. Trapping
followed the first method to verify the identity and presence of
crab species. Rock turning was the third method used which
yielded information on shore crab species. The last method used
was to search for molts of a new year class at the high tide water
line.

No recruitment evidence of a CM new year class has yet been
found in either bay by an sampling method. Data recorded in 1997
on all crab species was compared to data collected this year.

THE DISTRIBUTION OF THE EUROPEAN GREEN CRAB,
CARCINUS MAENAS. IN YAQUINA BAY, OREGON. Chris
Hunt, Environmental Science Department. Oregon State Univer-
sity, Corvallis, Oregon 97331-2914.

Carcinus maenas. first discovered in Coos Bay. Oregon in
1997, was found in at least eight Oregon estuaries in 1998. With
the absence of a successful 1999 year class to date, their present
population exists of what is believed lo be two and three year old
crabs. These older, and much larger, crabs are present lower in the
estuary than last year, creating the potential for more interactions
with the larger nali\e Cancrids. An intensive trapping effort during
the summer of 1999 documented the distribulion of (his older year
class of C. maenas and other crab species in ■*> aquina Bay. It
appears that C. maenas coexists in areas with the native Dungeness
crab. Cancer magister. but appears to be absent, or limited, in
areas suitable lor another native crab, the Red Rock crab. Cancer
prmlucnrs. Although C. nuicnas appears to be abundant in areas

PCOGA & NSA, Vancouver. Washincton

Abstracts. September 29-October I, 1999 687

with fewer of these larger crabs, it appears dense populations of
adult C. productus may be a primary limiting factor in the lower
estuary for this new invasive species.

DEVELOPING A PEST MONITORING PLAN FOR BUR-
ROWING SHRIMP: A STEP TOWARDS INTEGRATED
PEST MANAGEMENT. Brett R. Dumbauld. Washington State
Department of Fish and Wildlife. P.O. Bo.x 190. Ocean Park. WA
98640; Daniel P. Cheney, and Brian F. MacDonald, Pacific
Shellfish Institute. 120 State Avenue N.E. #142. Olympia. WA
98501.

Several attempts have been made to develop an integrated pest
management (IPM) plan for burrowing shrimp in Washington state
coastal estuaries. Burrowing shrimp are an impoilant pest to the
oyster aquaculture industry and also cause habitat changes for
other benthic organisms in coastal estuaries across the Pacific
Northwest. Oyster farmers in Washington State have applied the
pesticide carbary! to control these shrimp on privately owned es-
tuarine tidelands since the early 1960's. A recent IPM feasibility
study identified several critical issues that needed to be addressed
before IPM could be successfully implemented.

A project to monitor shrimp populations in Grays Harbor and
Willapa Bay, Washington was initiated in July 1999 to address
some of these issues. Objectives are to; ( 1 ) examine and monitor
the overall efficacy of burrowing shrimp control measures; (2)
follow patterns and rates of shrimp recruitment and survival and
compare these with oyster survival and production through typical
grow-out cycles on treated and un-treated oyster beds; and (3) use
the data collected to develop a long term monitoring plan for
burrowing shrimp. Preliminary information on inherent variability
in burrow count data and efficacy of the 1999 treatment are pre-
sented.

REESTABLISHMENT OF A NATIVE OYSTER: IMPLICA-
TIONS FOR POPULATION DISTRIBUTION AND STRUC-
TURE. Patrick Baker, Department of Fisheries and Aquatic Sci-
ences. University of Florida. Gainesville. FL 32653; Nora B. Ter-
williger, Oregon Institute of Marine Biology, University of
Oregon, Charieston. OR 97420.

The reappearance of the Olympia oyster. Ostrea coiuluiphila
(= lurida) in Coos Bay, Oregon, raises questions about its popu-
lation structure and dispersal. On the outer coast from Washington
to central California, the Olympia oyster occurs only in certain
estuaries. This species went extinct in Coos Bay prior to European
settlement, and both deliberate and accidental inoculations prior to
1988 failed to reestablish it. Since 1988, the Olympia oyster has
become abundant in Coos Bay, with heavy and regular recruit-
ment. Why did the Olympia oyster become reestablished in 1988,
but not previously? From where did it reinvade Coos Bay, and
how?

Coos Bay's geographic isolation may prevent reestablishnient
of Olympia oyster from other populations by larval dispersal, but
does not explain the failure of human introductions. Our research
suggests, however, that the Olympia oyster has returned partly
because of human modifications to the estuary. Incremental deep-
ening of the bay for navigation has "restored" a prehistoric salinity
regime, making it similar to modem Olympia oyster habitat else-
where. Olympia oysters currently occur in the most modified por-
tion of Coos Bay, in which modal winter salinities have increased
strongly since at least 1950. If this scenario is correct, habitat
limitation, not dispersal, is probably the primary limiting factor for
the Olympia oyster in this region. Molecular genetic analysis is
underway to determine the source population of the Coos Bay
population, and the degree of isolation between Olympia oyster
populations. Knowing the source population will also allow us to
develop hypotheses for the vector of reestablishment.

GROWTH OF 1997/1998 YEAR CLASS OF THE GREEN
SHORE CRAB, CARCINUS MAENAS, IN OREGON. Alex
Kalin and Sylvia Behrens Yamada, Department of Zoology, Or-
egon State University, Corvallis, Oregon 97331.

During the spring of 1997, a strong new year class of Carciinis
maenas appeared in seven Oregon estuaries. The carapace width of
these crabs averaged 14 mm June. 27 mm in July and 45 mm in
September 1998. By the summer of 1999, the crabs had reached
between 44 and 80 mm in carapace width. The growth of tagged
crabs and a molt increment study of captive crabs support these
rapid size increases.

Carapace width data collected from Oregon bays suggests that
C, maenas is growing faster in Oregon than in the North Sea or
Maine. Our data supports the theory that C. maenas reaches sexual
maturity within one year in Oregon, while in the North Sea and
Maine sexual maturation may take two to three years. Molt incre-
ment data, however, suggests that growth per molt is constant in C.
maenas populations regardless of geographic location. Regression
equations of molt increment data of Oregon specimens showed no
statistically significant difference from similar regression equa-
tions describing North Sea and Maine populations. The molt in-
crement statistical analyses lead to the inference that C. maenas
molts more frequently in Oregon than in the North Sea or Maine.

PROGRESS IMPLEMENTING A PLAN TO MONITOR
FOR PRESENCE OF THE EUROPEAN GREEN CRAB
iCARCINUS MAENAS) IN PUGET SOUND, WASHINGTON.
Anita E. Cook and Sandra Hanson, Washington State Depart-
ment of Fish and Wildlife (WDFW). Point Whitney Shellfish Lab,
Brinnon, WA 98320.

A significant population of the European green crab, whose
first persistent presence on the U.S. west coast was recorded in

688 Abstracts. September 29-October I. 1999

PCOGA & NSA. Vancouver, Washington

1989 in San Francisco, was first noted in Washington State in 1998
in coastal Willapa Bay and Grays Harbor. The green crab likely
arrived in Washington via larval drift on ocean currents. To date no
European green crab have been confirmed in Puget Sound.

A large-scale Puget Sound green crab monitoring program was
established in 1999. with WDFW as the coordinating agency. The
primary aim of this initial phase was thorough geographical sam-
pling coverage of Puget Sound (including the Strait of Juan de
Fuca and the San Juan Islands) to maximize the potential of de-
tecting any green crab that might have spread to Puget Sound by
larval transport or other means. This was accomplished by enlist-
ing and training various volunteers to set crayfish traps at moni-
toring sites spread throughout the Puget Sound. Over 15 groups
sampled more than 50 monitoring stations in 1999. Participants
included non-profit volunteer organizations, shellfish growers.
tribes, marine science centers, government agencies, schools, and
the general public. In addition to providing information about the
potential presence of green crab in Puget Sound, the trapping
supplied some general baseline data about populations of small
native crab in the sampling areas. In the year 2000 WDFW will
focus on increasing the number of sample sites (for higher poten-
tial of discovering green crab presence), identifying sites with the
highest likelihood for introductions, and examining other green
crab detection techniques.

DEVELOPMENT OF A SPECIFIC-PATHOGEN-FREE
(SPF) HATCHERY AND NURSERY FOR PRODUCTION
OF PACIFIC OYSTER SEED. Chris J. Langdon, Dave P.
Jacobson, Ford Evans, Ebru Onal, and Sean E. Matson, Hat-
field Marine Science Center, Oregon State University, Newport.
Oregon 97365.

The Molluscan Broodstock Program (MBP) is sited on the
Yaquina Bay, Oregon, and produces Pacific oyster seed for testing
at commercial grow-out sites on the West Coast, U.S. in order to
identify families with superior yields. In 1998, an adult Pacific
oyster from Yaquina Bay was found to be infected with a hap-
losporidian — later shown to positively react with a DNA probe for
Seaside Organism (SSO). In order to prevent exposure of MBP
seed to potential haplosporidian infectious agents in Yaquina Bay,
seawater supplied to culture systems was filtered to I \xm micron
and subsequently either filtered to 0.22 (xm for larval culture or
UV-treated (>90,0()0 iaW sec/cnr ) for spat culture.

Growth of larvae and spat in treated seawater was significantly
poorer than in non-treated water. Experiments indicated that ad-
dition of antibiotics (2 mg/L chloramphenicol alternated with 2
mg/L erythromycin) improved larval growth in cultures supplied
with 0.2 |j.m-filtered water, while addition of 2 mg/L calcium
bcntonitc improved growth of both larvae and spat in treated water.
No haplosporidian infections were found in a sample of 168 spat
reared in MBP facilities, indicating that the adopted SPF proce-
dures were effective.

INTERIM PERFORMANCE OF PROGENY OF SELECTED
BROODSTOCK FROM THE MOLLUSCAN BROOD-
STOCK PROGRAM. Chris J. Langdon, Dave P. Jacobson,
and Ford Evans, Hatfield Marine Science Center, Oregon State
University, Newport, Oregon 97365; Mike S. Blouin, Zoology
Department. Oregon State University, Corvallis, Oregon 97331.

The Molluscan Broodstock Program (MBP) was established to
improve yields of Pacific oysters on the West Coast, U.S., by
genetic selection. Fifty full-sib families were planted in Tomales
Bay, California, in October 1996 and the top nine families with the
highest yields (meat weight per bag) were identified in July 1997.
In 1998, 45 fill-sib families were produced by crossing the top nine
families among and within themselves to produce out-crossed and
inbred families, respectively. The progeny were planted at Totten
Inlet, Puget Sound, Washington, in August 1998. In addition, prog-
eny of non-selected "wild" oysters together with samples of in-
dustry-produced seed were planted for comparison. An interim
weighing of oysters (before they reached market size) was carried
out in June 1999.

Results indicate that the mean yield (live weight per bag) of
progeny of selected MBP families was significantly greater (Fish-
er's PLSD test: P < 0.05) than those of both non-selected and
inbred families but not significantly different from that of industry-
seed. Heritability for yield was estimated to be 0.54, indicating that
family yields should improve through genetic selection. The poor
performance of inbred families underscores the importance of
avoiding inbreeding in commercial oyster production.

SHELLFISH HIGH HEALTH PROGRAM. Ralph Elston, Pa

cific Shellfish Institute, PO Box 687, Carlsborg, WA 98324. Dan
Cheney, Pacific Shellfish Institute, Olympia, WA.

An oyster high health program was designed and implementa-
tion begun. The purpose is to provide a health database for Pacific
oysters and to establish high health guidelines for live shellfish
producers that would facilitate entry into markets with regulatory
requirements for shellfish disease free status. Voluntary implemen-
tation of a shellfish high health program by seed producers or other
exporters of live shellfish destined for receiving waters should
expand markets and facilitate the process of obtaining import per-
mits.

Oyster brood stocks Irom Calilornia. Oregon, and Washington
sites were examined to provide 95Vr confidence of delecting con-
ditions occurring at a 2''/c prevalence or greater. Nocardiosis was
found in 3.2'* of oysters from one site while Mytilicola infesta-
tions were found in several samples at prevalences less than 2'}i.
Ruptured reproductive follicles were found in up to 27'^f of oys-
ters. Various other non-infectious conditions were found at preva-
lences of less than 5'/r. No examples of infectious diseases cur-
rently considered certifiable, including haplosporidia, Mikrocytos
imukini. Hfikiiisiis spp., Bonamiu spp., Marteilia spp.. unidenii-

PCOGA & NSA. Vancouver. Washinston

Abstracts. September 29-October 1. 1999 689

tied protistan parasites or histological evidence of viral infections
were found.

Components of the proposed voluntary program include a sys-
tem of health certifications, records and documentation, mainte-
nance of brood stock integrity, hatchery and nursery operations
protocols as needed to maintain infectious disease exclusion and a
response plan for infectious disease outbreaks.

FECUNDITY STUDY AND PRELIMINARY FIELD TRIAL
RESULTS OF THE BUTTER CLAM {SAXIDOMVS GIGAN-
TEUS). Amilee Caffey and Brady Blake. Point Whitney Lab..
Washington Department of Fish and Wildlife. Brinnon. WA
98320.

In 1996. the Point Whitney Shellfish Hatchery began a series of
spawning trials on the butter clam. Saxidomns gigaiiteiis. The main
goal of the study was to determine values for a population man-
agement model used for clam resources. The first value obtained
through this experiment was the smallest clam observed spawning:
29.21 mm male shell length/29.95 female shell length. The second
value included overall mean egg production at 2.07 million eggs
per female.

This study is a continuation of that work for 1997-1999.
Spawning trials included not onh' the smallest clam observed
spawning and overall egg production, but also looked at values
such as brood stock holding temperature, algal concentrations dur-
ing spaw ning trials, and potential egg production of repeat spavvn-
ers in one season. Overall mean production for the combined years
dropped to 1.07 million eggs per female with the smallest female
clam observed spawning remaining at 29.95 mm and the smallest
male clam observed spawning at 29.21 mm shell length. Results
show that butter clams are repeat spawners with an oxerall egg
production of 1.08 million and were observed spawning at this
level up to three times in one month. The highest egg production
occurred when the brood stock was conditioned at 10.5 °C. During
the spawning trial, the highest egg production also occurred at
algal concentrations of 950.000 cells/ml during spawning trials.

Seed from the study was then planted at the Point Whitney
beach and sampled survival and growth rates after a two year
period. Mean shell length of butter clams at time of plant was
19.62 mm and mean shell length at recovery after two years was
42.34 mm. The average growth of the butter clam w as 22.72 mm
over two years. Survival rate was 7.109c.

BURROWING RESPONSE OF JUVENILE GEODUCKS
iPANOPEA ABRUPTA) TO CHANGES IN TEMPERATURE
AND SALINITY. Jonathan P. Davis. Clea Barenburg. and
David Pederson, Taylor Resources Bivalve Hatchery. 701 Broad
Spit Road. Quilcene. WA 98376.

Geoduck clams. Panopea abnipta. are a newly cultured species
and the development of geoduck culture techniques, coupled with
out planting methods have not been perfected. Environmental pa-

rameters likely have a significant effect on the burrowing behavior
of clams which in turn may greatly influence the level of survi-
vorship of newly planted seed.

The burrowing behavior of three size classes of juvenile geo-
duck clams was measured in response to exposure to a suite of
temperature and salinity conditions. Seed were exposed to all com-
binations of six temperature (8, 1 1, 14. 17. 20, & 23 °C) and six
salinity (20. 22. 24. 26. 28. & 30 ppt) treatments. Three different
seed classes were tested; small (4.6 mm mean shell length), me-
dium (7.2 mm) and large (9.5 mm) geoducks. All clams were
maintained under common conditions prior to testing burrowing
response.

Results indicate that all seed size classes showed maximal bur-
rowing response at median temperatures (11. 14. and 17 °C) and
higher salinities (26. 28, and 30 ppt). The response for all size
classes indicated a proportionate increase in burrowing rate as
conditions neared ambient salinity (30-32 ppt). Size was also a
significant factor as large and medium seed demonstrated high
burrowing response only between 1 1 and 14 ^C and at higher
salinities, and reduced burrowing response at low (8 and 11 °C)
and high (23 "C) treatment temperatures. The burrowing response
of small seed in all treatments was uniformly higher compared to
medium and large seed across all temperature and salinity treat-
ments; however as also seen for large and medium sized cohorts,
burrowing behavior at salinities less than 26 ppt. was greatly re-
duced. Avoiding extremes in temperature and in particular salini-
ties less than 26 ppt. even for short periods of time, may signifi-
cantly increase overall planting success for culture operations.

POST SETTLEMENT GEODUCK CLAM (PANOPEA
ABRUPTA) GROWTH AND SURVIVORSHIP IN SAND
AND SCREEN-BASED NURSERY SYSTEMS. Jonathan P.
Davis and Clea Barenburg, Taylor Resources Bivalve Hatchery,
701 Broad Spit Road. Quilcene. WA 98376.

Post settlement growth and survivorship in sand and screen
based down-welling silos was examined for geoduck clams in
order to assess the viability of different nursery systems during this
critical life history stage. Geoduck pediveligers were introduced to
three sand-based substrates including fine (500-600 micron) and
course (700-800 micron) sands and screened (500-600 micron)
dolomite particles. Three screen-based treatments were assessed
using density as a dependent variable. Three initial stocking den-
sities tested were; 10. 40. and 80 geoduck plantigrades per cm",
respectively. All sand-based treatments were set at an initial stock-
ing density of 10 clams per cm~. All setting systems were rinsed
with filtered sea water once weekly over the 25 day experiment.
Sea water used for the downwelling setting system was 5 micron
filtered and UV treated and set on a recirculation mode with ap-
proximately 1 .5 L per minute replacement. A variety of cultured
algae was fed to the clams on a continuous basis for up to 1 8 h per
day.

690 Absrmcrs. September 29-October 1. 1999

PCOGA & NSA. Vancouver. Washington

Results indicated that all sand-based systems had significantly
higher post-set survivorship than any of the screen-based treat-
ments. The following was observed in order of survivorship after
25 days: small-grained sands (6.07 clams per cm") < large-grained
sands (4.44) < dolomite sands (3. .^6) < screen-based at 80 clams
per cm~ (0.60) < 10 clams per cm" (0.40) < 40 clams per cm"
(0.33). Growth rate of surviving clams in all of the treatments was
similar, although the onset of mortality in sand and screen-based
systems showed a tendency for clams to survive to a larger size in
the sand-based systems.

A TAG METHOD FOR ESTIMATING THE NATU-
RAL MORTALITY RATE OF GEODUCKS {PANOPEA
ABRUPTA). Alex Bradbury, Don P. Rothaus. Robert
Sizemore, and Michael Ulrich, Washington Department of Fish
and Wildlife. Point Whitney Shellfish Laboratory, Brinnon, Wash-
ington 98320.

We tested a tagging method for estimating the instantaneous
rate of natural mortality (M) of subtidal geoduck clams {Panopea
abmpta) at a previously unfished site in Hood Canal. Washington.
Divers "tagged" 1 128 adult geoducks (>3-4 yrs) in May 1998 by
placing thin plastic stakes next to geoduck siphons at a distance of
7.6 cm. Geoducks were tagged within I m of three lines running
offshore and anchored in depths of -6 m to -23 m MLLW. One
year later, divers found 875 of the original 1 128 tags remaining in
the substrate. Over a 6-day period, siphons were visible next to 856
of the tags. Divers used a venturi dredge to excavate the 19 tags
with no visible siphons; 4 of these geoducks were alive, 14 were
dead, and one tag had no sign of a living or dead geoduck. The
annual survival rate (S) was estimated as N|/N|, = 861/875 =
0.984 yr~' and the coiresponding estimate of M was 0.016 yr~'
(95% CI = 0.025 - 0.007). This point estimate is lower than the
value currently used in Washington's equilibrium yield model (M
= 0.0226), but is not significantly different. The direct estimate of
M makes fewer assumptions than catch curve estimates, and is less
expensive.

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fish Research. P.O. Box 465, Hanover, PA 17331.

Daniel P. Cheney, Brian F. MacDonald, and Ralph A. Elston

Summer mortality of Pacific oysters. Cnissostrea gigas (Thunberg): initial findings on multiple environmental stressors in Puget

Sound. Washington, 1998 353

Richard Dame, David Biishek, Dennis Allen, Don Edwards, Leah Gregory, Alan Lewitus, Sarah Crawford, Eric Koepfler, Chris Corbett,
Bjbrn Kjerfre, and Theo Prins

The experimental analysis of tidal creeks dominated by oyster reefs: the premanipulation year 36!

Denise L. Breitburg, Loren D. Coen, Mark W. Luckenbach, Roger Mann, Martin Posey, and James A. Wesson

Oyster reef restoration: convergence of harvest and conservation strategies 371

Brett R. Dumbauld, Eileen P. Visser, David A. Armstrong, Lauren Cole-Warner, Kristine L. Feldman, and Bruce E. Kauffman

Use of oyster shell to create habitat for juvenile dungeness crab in Wa.shington coastal estuaries: status and prospects 379

Francis X. O 'Beirn, Mark W. Luckenbach, Janet A. Nestlerode, and Gregory M. Coates

Toward design criteria in constructed oyster reefs: oyster recruitment as a function of substrate type and tidal height 387

John Supan

The Gulf Coast oy,ster industry program: an initiative to address industry's research needs 397

Michael A. Rice, April Valliere, and Angela Caporelli

A review of shellfish restoration and management projects in Rhode Island 401

Anita E. Cook, J. Anne Shaffer, Brett R. Dumbauld, and Bruce E. Kauffman

A plan for rebuilding stocks of Olympia oysters [Ostreola conchaphila. Carpenter 1857) in Washington State 409

Joel Haamer and Johan Rodhe

Mussel Myiilus edidis (L.) filtering of the Baltic Sea outflow through the Oresund — an example of a natural, large-scale

ecosystem restoration 413

S. de Luca-Abbott, G. D. Lewis, and R. G. Creese

Temporal and spatial distribution of enterococcus in sediment, shellfish tissue, and water in a New Zealand harbour 423

David Lapola, Gunther Rosen, Jaelyn Chock, and Connie H. Liu

Red and green ahalone seed growout for reseeding activities off Point Loma. California 43 1

Walter R. Keithly, Jr., Assane Diagne, and Ronald J. Dugas

The demand for oyster relaying activities in Louisiana: 1976-1995 439

Odele da Silva Pinho

Community involvement in projects to reduce nonpoint source pollution 445

William F. Dewey

Maintaining the shellfish niche in the new millennium: the Pacific Coast shellfish growers approach 449

S. L. MacFarlane, J. Early, T. Henson, T. Balog, and A. McClennen

A resource-based methodology lo assess dock and pier impacts on Pleasant Bay. Massachusetts 455

Richard C. Karney

Poor water quality? Not in my backyard! The effectiveness of neighborhood pond associations in the protection and improvement of

shellfish growing waters on Martha's Vineyard 465

Robert D. Brumbaugh, Laurie A. Sorabella, Carene Oliveras Garcia, William J. Goldsborough, and James A. Wesson

Making a case for community-based oyster restoration: an example from Hampton Roads. Virginia. U.S. A 467

Nancy M. White, Daniel E. Line, J. D. Potts, William Kirby-Smith, Barbara Doll, and W. F. Hunt

Jump Run Creek Shellfish Restoration Project 473

Suzanne Colson and Leslie N. Stunner

One shining moment known as Clamelot: the Cedar Key story 477

S. W. Abadie and M. A. Poirrier

Increased density of large Rangia clams in Lake Pontchartrain after the cessation of shell dredging 481

M. J. Stewart and R. G. Creese

Evaluation of a new tagging technique for monitoring restoration success 487

Abstracts of papers presented at the 4th International Abalone Symposium. February. 2000 493

Abstracts of papers presented at the NAFO-ICES-PICES Symposium on Pandalid Shrimp Fisheries "Science and Management at the

Millenium." Halifax. Nova Scotia. September 8-10. 1999 543

Abstracts of technical papers presented at the 20th annual meeting. Milford Aquaculture Seminar. Milford. Connecticut. February. 2000... 563

Abstracts of technical papers presented at the 92nd Annual Meeting. NSA. Seattle. Washington. March 19-23. 2000 585

Abstracts of technical papers presented at the International Symposium on Lobster Health Management. Adelaide. Australia. September

19-22. 1999 669

Abstracts of technical papers presented at the 53rd Annual Meeting of the PCOGA & NSA. Vancouver. Washington. September

29^ctQber. .1... 1999 681

COVER PHOTO: Eight daj-old larvae of the Pacific oyster {Cnissostrea gigiis) viewed under epinuoresccnl light (excitation
355—425 nm. emission 525 nm) at x400 magnification. Larvae were fed on ribonavin-containing lipid spray heads (50 beads/jjil) for
one hour, followed by a two hour period of I'eeding on Isnrhrysis gcilhaim (T-ISO) alone. Free rihollavin is evident as a diffuse
greenish fluorescence in ihc guts of larvae while rihollavin crystals present in intact or partially digested SB are evident as bright
yellow points. The digestive systems of some larvae also fluoresce red due to the presence of chlorophyll from ingested algae.
Average larval shell length = 122 ixm. (C. Langdon)

The .liuiriial of .Shellfish Research is indexcil in ihc lollovving: Science Cilalion Index*. Sei Search*. Research Alert*. Current
Conlcnts"7Agnciillurc. Biology and Knvironnicnial Sciences. Biological ,'\bslracts. Chemical Absiracls. Nulrilion Abstracts. Current
Advances in Ecological Sciences. Deep Sea Research and Oceaiiographic Literature Review. Hnviionnienlal Periodicals Bibliography,
Aquatic Sciences and Pisheries Abstracts, and Oceanic Abstracts.

175

203

David Mills

Combined effects of temperature and algal concentration on survival, growth and feeding physiology of Pimiada maxima

(Jameson) spat '^'

Bodil Henirolli. Annhild Larsson and Lars Edebo

Influence on uptake, distribution and elimination of Salmonella typhimuriwn in the blue mussel, Mvlilu.s edulis. by the cell surface

properties of the bacteria '°'

Bodil Hernrolh, Annhild Larsson. Magne Alpslen and Lars Edebo

On the possibility of u.sing radioactive labeling and gamma camera technique to study Salnumclla lyphiiniirimn In the blue mussel,

Mylilus Citiilis

Guillaume Mitta, Philippe Roch and Jean-Paul Cadoret

Isolation and characterisation of a cDNA encoding an actin protein from the mussel, Mylilus lialkipidYincialis 183

J. M. F. Babarro, M. J. Ferndndez-Reiriz and U. Labaria

Growth of seed mussel (Mxtiliis gallninovincialis Lmk): effect.s of environmental parameters and seed origin 187

J. M. F. Babarro, M. J. Ferndndez-Reiriz and U. Labaria

Feeding behavior of seed mussel Mxiiliis fialUipriiYiucialis: en\ ironmental parameters and seed origin 195

David W. Hicks. Doyle L. Hawkins and Robert F. McMahon

Salinity tolerance of brown mussel Penia pcrna (L.l from the Gulf of Mexico: an extension of life table analysis to estimate median

survival time in the presence of regressor variables

Karl B. Andree, Carolyn S. Friedman, James D. Moore and Ronald P. Hedrick

A polymerase chain reaction assay for the detection of genomic DNA of a rickettsiales-like prokaryote associated with withering

syndrome in California abalone -'■'

ScoresbyA. Shepherd, Douglas Woodby, Janet M. Rumble and Miguel Avalos-Borja

Microstructure, chronology and growth of the pinto abalone, Haliolis kumtschatkana. in Ala.ska 219

P. E. Penchaszadeh. G. Darrigran, C. Angulo. A. Averbuj, M. Briigger, A. Dogliotti and N. Pirez

Predation of the invasive freshwater mussel Limiwpema fonunei (Dunker, 1857) (Mytilidae) by the fish Leporimts obtusidens

Valenciennes. 1 846 ( Anostomidae) in the Rio de la Plata, Argentina 229

Michelle R. Barlsch. Diane L. Waller. W. Gregory Cope and Sieve Gutreuter

Emersion and thermal tolerances of three species of unionid mussels: survival and behavioral effects 233

Junemie Hazel L. Lebata

Elemental sulfur in the gills of the mangrove mud clam Aniidoiuia edeimda (Family Lucinidae) 241

David P. Lemarie. David R. Smith. Rita F. Villella and David A. Weller

Evaluation of tag types and adhesives for marking freshwater mussels (Mollusca: Unionidae) 247

Clifford E. Starliper and Patricia Morrison

Bacterial pathogen contagion studies among freshwater bivalves and salmonid fishes -51

Laura Chanes-Miranda and Maria Teresa Viana

Development of artificial lobster baits using fish silage from tuna by-products 259

Jean Lavallee, K. Larry Hammelli, Elizabeth S. Spangler, Richard J. Cawthorn and Ian R. Dohoo

Descriptive .statistics of fishing practices, postharvest health status, and transport conditions in the Prince Edward Island lobster

(Hnmanis americantis) industry -"-*

Jean Lavallee, Elizabeth S. Spangler, K. Larry Hammelli, Ian R. Dohoo and Richard J. Cawthorn

Analytical assessment of handling, fishing practices, and transportation risk factors on lobster {Homarus americumis) health in

Prince Edward Island, Canada -''

M. Mascara and R. Seed

Foraging behavior of Carcinus maenas (L.): comparisons of size-selective predation on four species of bivalve prey 283

M. Mascara and R. Seed

Foraging behavior of Carcinus macmis (L.): species-selective predation among four bivalve prey 293

Christina M. Mikulski, Louis E. Burnett and Karen G. Burnett

The effects of hypercapnic hypoxia on the sur\ ival of shrimp challenged with Vibrio parahaemolyuciis 301

Kelly M. Curtis, Vera L. Trainer and Sandra E. Shumway

Paralytic shellfish toxins in geoduck clams iPanope ahnipta): variability, anatomical distribution, and comparison of two toxin

detection methods - ' ^

Andrew G. Bander and Allan D. Cembella

Viability of the toxic dinofiagellale Prorocemriiiu lima following ingestion and gut pas.sage in the bay xaWop Argopecten irradians 321

Alain Paugam, Marcel Le Pennec, and Andre-Fontaine Genevieve

Immunological recognition of marine bivalve larvae from plankton samples 325

Selected papers from the 1998 International Conference on Shellfish Restoration: "Forging Partnerships to Improve the Health of Coastal

Ecosystems Through Shellfish Restoration" 333

Roger Mann

Restoring the oyster reef communities in the Chesapeake Bay: A commentary 335

Aswani K. Volety, Frank O. Perkins. Roger Mann, and Paul R. Hershberg

Prouression of diseases caused by the oyster parasites, Perkinsiis mariiui.s and Haplosporidium iielsoni, in Crussoslreu virginica on

constructed intertidal reefs -'41

Shawn M. McLaughlin and Mohamed Faisal

Prevalence of Perkinsiis spp. in Chesapeake Bay soft-shell clams, Mya arenaria Linnaeus, 1758 during 1990-1998 349

CONTENTS CONTINUED

JOURNAL OF SHELLFISH RESEARCH
Vol. 19, No. 1 June 2000

CONTENTS

JN MEMORIAM

John Carl Mcdcof 1

IN MEMORIAM

Riilh Dixon Turner 7

Neil Bourne

Honored Life Member: Kenneth Kendall Chew 13

Joseph A. Mihursky

Honored Life Member: Victor Samuel Stuart Kennedy 15

William Fisher

Honored Life Member: Sammy M. Ray 17

Liberato V. iMurela and Evelyn T. Marasigan

Habitat and reproductive biology of angelwings, Pluihis orieiitulis (Gmelin) 19

Teresa J. Naimo, W. Gregory Cope. Emy M. Monroe, Jerry L. Farris and Cristin D. Milam

Inlluencc of diet on survival, growth, and phy.siological condition of fingernail clams Musculium tnmsver.sum 23

Kurt Jnrdaens, Hans de Wolf, Tania Willems. Stefan van Dongen, Carlos Brito, Antonio M. Frias Martins and Thierry Backetjau

Loss of genetic variation in a strongly isolated A/orean population of the edible clam, Tapcx deciissanis 29

Clyde L. Mackenzie, Jr. and Shawn M. MclMughlin

Life history and habitat observations of softshell clams Mvu urciiariti in northeastern New Jersey 35

Gregory S. MacCallum and Sharon E. McGladdery

Quahog parasite unknown (QPX) in the northern quahog Mercenaria mercenarki (Linnaeus, 1758) and

M. mercenaria var. natatu from Atlantic Canada, survey results from three maritime provinces 43

Arnold G. Eversole. Nathalie Devillers and William D. Anderson

Age and size of Mercenaria mercenaria in Two Sisters Creek, South Carolina 51

A. Hoffman, A. Bradbury and C. L. Goodwin

Modeling geoduck, Panopea ahnipla (Conrad. 1 849) population dynamics. L Growth 57

A. Bradbury and J. V. Tagart

Modeling geoduck, Panapea ahnipla (Conrad. 1849) population dynamics. II. Natural mortality and equilibrium yield 63

Karin B. Lohrmann, Stephen W. Feist, and Andrew K. Brand

Microsporidiosis in queen scallops [Aeqiiipeclen operciilaris L. ) from U.K. waters 71

Luis Freites V, Anibal Velez and Cesar Lodeiros

Evaluation of three methods of bottom culture of the tropical scallop Eiivola iPecten) ziczac (L. 1758) 77

J. E. Perez, O. Nusetti, N. Ramirez and C. Alfonsi

Allo/yme and biochemical variation at the octopine dehydrogenase locus in the scallop Etivola ziczac 85

Pedro Cruz, Carmen Rodriguez-Jaramillo and Ana M. Ibarra

Environment and population origin effects on first sexual maturity of catarina scallop, Art^iipeclen ventricosiis (Sowerby II, 1842)., 89

Bertha Patricia Ceballos-VazQuez. Marcial Arellano-Martinez. Federico Garci'a-Dominguez and Marcial Villalejo-Fuerte

Reproductive cycle of the rugose pen shell. Pinna rui;(/.\a Sowerby, 1835 (Mollusca; bivalvia) from Bahia Conccpcicin. Gull of

California and its relation to temperature and pholopcriod 95

Huiping Yang. Huayong Que. Yichao He and Fusui Zhang

Chromosome segregation in fertilized eggs from Zhikong scallop Clilanixs farreri (Jones & Preston) following polar body I

inhibition with cytochalasin B 101

Antonio Luna-Gonzdlez, Carlos Cdceres-Martinez, Claudia Ziiniga-Pacheco, Silverio iMpez-l-dpez and Bertha Patricia Cehallos-Vdzquez

Reproductive cycle o'i Argapecten ventricosiis (Sowerby 1842) (Bivalvia: Pectinidae) in the Rada del Puerto de Pichilingue, B.C.S,.

Mexico and its relation to temperature, salinity, and food 107

R. W. Penney and T. J. Mills

Biocconomic analysis of a sea scallop, I'lacopcclcn inaKcllaniciis. aquacullure production system in Newfoundland. Canada 113

John E. Supan, Charles E. Wilson and Standish K. Allen, Jr.

The effect of cytochalasin B dosage on the survival and ploidy of Crassosirea virf^inica (Gniclin) larvae 125

C. J. iMugdon, C. Seguineau. B. Ponce, J. Moal and J. /•', Samain

Delivery of ribollavin to larval and adult Pacific oy.stcrs. Crassosirea .i;/,i;i/.v Thunberg by lipid spray beads 129

I). J. Brousseau and J. A, Baglivo

McHJelling seasonal proliferation of (he parasite, I'erkinsiis niariniis (l)enno) in field populations of (he oyster. Crassosirea viixinicn . . . . 133

Caroline E. O'Farrell. Jerome /■', Im Peyre, Kennedy T. Paynter and Eugene M. Burreson

Osmotic loleranee and volume regulation in in vilro cultures of the o\s(er pathogen Perkinsiis nuinniis 139

Maoxiau lie. Yueguang I. in. Qi Shen. Jianxin llu and Weiguo Jiang

Produclion of (clraploid pearl oyster {I'inclaila nuulensii Dunker) by mhibiling the first polar body in eggs from Iriploids 147

Katsuyuki Numaguchi

Evaluation of five microalgal species for (he growth of early spat of (he Japanese pearl oyster Pinckula fucaia mariensii 153

CONTENTS CONTINUED ON INSIDE BACK COVER

JOURNAL OF SHELLFISH RESEARCH

VOLUME 19, NUMBER 2

DECEMBER 2000

The Journal of Shellfish Research (formerly Proceedings of the

National Shellfisheries Association) is the official publication

of the National Shellfisheries Association

Editor

Dr. Sandra E. Shumway

Natural Science Division

Southampton College, Long Island University

Southampton, NY 11968

Dr. Standish K. Allen, Jr. (2000)
School of Marine Science
Virginia Institute of Marine Science
Gloucester Point. VA 23062-1 1346

Dr. Peter Beninger (2001)

Laboratoire de Biologic Marine

Faculte des Sciences

Universite de Nantes

BP 92208

44322 Nantes Cedex 3

France

Dr. Andrew Boghen (2001)
Department of Biology
University of Moncton
Moncton. New Brunswick
Canada El A 3E9

Dr. Neil Bourne (2001)
Fisheries and Oceans
Pacific Biological Station
Nanaimo, British Columbia
Canada V9R 5K6

Dr. Andrew Brand (2001)
University of Liverpool
Marine Biological Station
Port Erin. Isle of Man

Dr. Eugene Burreson (2001)
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062

Dr. Peter Cook (2000)
Department of Zoology
University of Cape Town
Rondebosch 7700
Cape Town, South Africa

EDITORIAL BOARD

Dr. Simon Cragg (2000)
Institute of Marine Sciences
University of Portsmouth
Ferry Road
Portsmouth P04 9LY
United Kingdom

Dr. Leroy Creswell (2001)
Harbor Branch Oceanographic

Institute
US Highway 1 North
Fort Pierce, Florida 34946

Dr. Lou D'Abramo (2000)
Mississippi State University
Dept of Wildlife and Fisheries
Box 9690
Mississippi State, Mississippi 39762

Dr. Ralph Elston (2001)
Battelle Northwest
Marine Sciences Laboratory
439 West Sequim Bay Road
Sequim, Washington 98382

Dr. Susan Ford (2000)

Rutgers University

Haskin Laboratory for Shellfish

Research
P.O. Box 687
Port Norris, New Jersey 08349

Dr. Raymond Grizzle (2001)
Randall Environmental Studies Center
Taylor University
Upland, Indiana 469S9

Dr. Mark Luckenbach (2001)
Virginia Institute of Marine Science
Wachapreague, Virginia 23480

Dr. Bruce MacDonald (2000)
Department of Biology
University of New Brunswick
P.O. Box 5050
Saint John, New Brunswick
Canada E2L 4L5

Dr. Roger Mann (2000)

Virginia Institute of Marine Science

Gloucester Point, Virginia 23062

Dr. Islay D. Mursden (2000)
Department of Zoology
Canterbury University
Christchurch, New Zealand

Dr. Tom Soniat (2000)
Biology Department
Nicholls State University
Thibodaux, Louisiana 70310

Dr. J. Evan Ward (2001)
Dept. of Marine Sciences
LIniversity of Connecticut
Groton, CT ()634()-6()97

Dr. Gary Wikfors (2000)

NOAA/NMFS

Rogers Avenue

Milford. Connecticut 06460

Journal of Shellfish Research

Volume 19, Number 2
ISSN: 00775711
December 2000

Jimrihil of Shellfish Research. Vol. 19. No. 2. 6yi-7U(). 20()().

PREVALENCE AND PROGRESSION OF SHELL DISEASE IN AMERICAN LOBSTER,
HOMARUS AMERICANUS, FROM RHODE ISLAND WATERS AND THE OFFSHORE CANYONS

JAN ^ 2 2001

KATHLEEN M. CASTRO' AND THOMAS E. ANGELL^

'University of Rhode Island

Department of Fisheries Animal and Veterinary Science
Kingston. Rhode Island 02881

'Department of Environmental Management
Division of Fish and Wildlife Coastal Fisheries Laboratory
1231 Succotash Road
Wakefield. Rhode Island 02879

ABSTRACT From 1995 to 1999. shell disea.se in lobsters. Homanis amerkwms. was monitored in research trawl and trap surveys
conducted in Rhode Island waters including Narragansetl Bay. Rhode Island and Block Island Sound, and the offshore areas of Block
and Hudson Canyons. In the inshore population a significant increase in frequency and severity of the disease was documented
beginning in 1996. reaching 20% infected by 1999 with over 50% of ovigerous females affected. Shell di.sease was noted in the offshore
lobster population beginning in 1998. A tag-recapture study conducted in Narraganselt Bay between 1997 and 1999 followed the
change in severity over a .Vyear period in the field for 86 individuals. Observed disease pathways point to potential affects for
reproductive behavior and mortality. The proportion infected with disease appeared to diminish over the molting period, but sub.se-
quently increased in infection rate and shell coverage during the months of September and October.

KEY WORDS: Lobster, shell disease, field observations, tag-recapture

INTRODUCTION

Parasites and disease can be significant factors in population
dynamics. There is evidence that disease presents a major force
that shapes populations that is as profound as the forces of preda-
tion or resource utilization (Anderson and May 1979. Hart 1990).
Disease outbreaks have been known to produce significant losses
in all life history stages of cultured and wild American lobsters
(Bayer elal. 1993).

Shell disease has been studied extensively in relation to high
winter mortality rates in commercial lobster pounds where ani-
mals are confined in high densities for up to 6 mo in duration. Shell
disease is caused by an external infection that occurs when
chitinoclastic organisms, including several bacteria and fungal
species, attack the exoskeleton. Vibrio spp are most commonly
found and several other gram-negative bacteria are usually
present in necrotic pits. Researchers believe that it is the com-
bined action of these chitinoclastic organisms that interact to
cause the general appearance of shell disease. Very little infor-
mation is available about shell disease in wild lobsters. In the
natural environment chitinoclastic bacteria are believed to cause
little harm, living in the substrate and on the animal it.self (Johnson
1983).

Shell disease lesions typically begin as small dark brown or
black pits in the exoskeleton, indicating that the calcified layers
have been eroded (Getchell 1989), The blackening is due to the
melanization of the necrotic area. In minor cases the non-calcified
layers reinain intact; however, in more severe cases, inner tissues
become damaged. Damage to the exoskeleton is not a prerequisite
to shell damage, although it can be a precursor (Stewart 1984).
Malloy (1978) was able to experimentally transmit the disease in
H. americaniis after abrasion, however, this has only been shown
to occur under degraded or crowded conditions (Getchell 1989).

Bullis el al. (1988) described two general forms of shell disease
in red crabs, one associated with injuries that appeared as random

localized areas and one that was manifested as bilateral lesions in
areas of microscopic sensory organelles on the carapace. Later
stages of this latter type were characterized by extensive areas of
darkened melanization leading to loss of large areas of the exo-
skeleton. Sindermann (1991) hypothesized that shell disease was
an external indication of some metabolic disturbance that results in
the failure of the animal to keep up with chiton deposition. Me-
tabolism can be affected by a number of environmental stressors
such as pollutants, anoxia, or physiological changes .such as poor
nutrition.

Estrella (1991) found that shell disease prevalence in Massa-
chusetts in 1989 was significantly higher in larger lobsters, sug-
gesting an inverse relationship with molting frequency in hard
shelled lobsters. Ovigerous females displayed a significantly
higher percentage of disease and mature non-ovigerous females
displayed higher percentages than males. If the appearance and
worsening of shell disease are related to the frequency of molting,
then we would expect smaller, immature animals to be less in-
fected than larger, mature animals. We would also expect mature
females to be more infected than tnales or immature females since
they carry eggs for up to 9 to 1 1 mo and may not molt for two
years (Waddy et al. 1995). However, if shell disease is a conse-
quence of internal metabolic disturbance, then the new shell may
only provide a short respite before the disease reappears.

The recent increase in extensive shell damage in lobsters in
Massachusetts, Rhode Island, and Long Island Sound waters has
not been previously recorded for wild stocks. The appearance
of the disease also coincides with the discovery of a "limp lobster
syndrome" in the Gulf of Maine that is causing weakness and
mortality of lobsters in pounds (Bayer pers. commun., Maine
Lobster Institute) and a large mortality in Western Long Island
possibly caused by a paramoeba infection. The contribution ot
shell disea.se to natural mortality of lobsters may be significant
(Taylor 19481, as are the effects on other life history characteris-
tics such as growth or reproduction. This study describes the

691

692

Castro and Angell

Mass3chusells

Hhodc ^ .J

fslaml .

/ ^

New
Jersey

G^^

TABLE 1.

Rating system used to score shell disease index (SDI) score. Three
areas of the lobster were visually examined and assigned a score

0 to 100 corresponding to the percentage of each body section
affected. These were added up and assigned a rating from 0 to 4.

SDI

Percentage of coverage of
abdomen, claws, and carapace

None

1-100

101-200

201-250

251-300

P^

Figure 1. Rhode Island offshore sea sampling areas (above). Location
of URI trawl survey sites (a). Wickford (4r34.4S'N/7r24.34'W to
4r33.45"N/7r24.34' W) (b). Whale Rock (4r26.55"N/7r25.14W to
41°2S.10W/71°25.S4"W), and (c) Trap survey Dutch Harbor. Rhode
Island Inshore Sea sampling sites (below).

prevalence and progression of shell disease in the lobster
population in Rhode Island waters and offshore canyons from
1995 to 1999.

MATERIALS AND METHODS

returned to the water. Bottom water temperature was recorded on
the URI trawl survey using a Nanson bottle and a thermometer.

The occurrence of shell disease in the URI surveys was noted
by drawing a symbol indicating the location and size of the occa-
sional lesion. However, this system was later modified into a shell
disease index (SDI) which describes the percentage of covered
area in three body areas: carapace, abdomen, and claws. Each body
section has a maximum of 100 points for a maximum score of 300
(Table 1 ). The DEM survey recorded presence and absence of shell
disease only.

Each survey was evaluated as proportion infected by sex cat-
egory and weighted by number captured to obtain proportion in-
fected by year. Differences between years were statistically ana-
lyzed using an approximate z test based on normal approximations
to the binomial (Ostle and Malone 1988). The URI trawl and trap
data were examined for independence of variables using the Spear-
man Rank Correlation procedure followed by partial correlation
analysis (SPSS, Base 10). The Mann-Whitney two-sample test or
the chi-square test of independence was applied to examine the
effects of variables on disease incidence (Sokal and Rohlf 1981,
SPSS, Base 10). A regression analysis was used to test relation-
ships between infection rate and both temperature and proportion
.soft. Differences in size frequency distributions were evaluated
using large sample Kolmogorov-Smirnov tests and a student's

Shell condition of (he American lobster, Homanis ciiiwricainis
has been routinely monitored during several state and research
surveys in Rhode Island and offshore waters (Fig. I ). The Univer-
sity of Rhode Island (URI) conducted a weekly research trawl
survey at two sites (Wickford and Whale Rockl in Narragansett
Bay between 1995 and 1999. Sea sampling surveys were con-
ducted monthly from 1996 to 1999 by Rhode Island Department of
Environmental Management (DEM) biologists aboard fishing ves-
sels Ihrinighout Narragansett Buy. Rhode Island and Block Island
Sound, and offshore canyons. Twenty-four inshore trips per year
and four otfshore trips per year were sampled. During spring,
summer, and fall of 1997 to 1999, a weekly trap survey for tag-
recapture studies was conducted at three sites (associated with an
artificial reef project) in Dutch Harbor in the West Passage of
Narragansett Bay. using six commercial traps covered with small
mesh at each site.

Lobsters from all surveys were sexed. measured (for carapace
length |CL|). examined for molt stage, egg-bearing sialus. cull
status, and shell disease, and those from the URI studies were
tagged with a numbered t-bar anchor tag (Floy lag) before being

Survey Results
Proportion of Lobsters with Shell Disease

-^ DEM Inshore '
-•-URI Trawl
-i- URI Trap
-»- DEM Offshore

. ■ ^"""^ Z 1 '

1997
Year

Figure 2. Proportion of lobsters with shell disea.se weighted by sex and
number for each survey (Rl DEM inshore sea sampling survey, Rl
DEM offshore sea sampling survey, URI trawl survey, and Dutch
Harbor trap survey).

Shell Disease in American Lobsters

693

TABLE 2.

Proportion of lobsters with shell disease and total number sampled (in parentheses) from inshore and offshore areas (RI DEM sea sampling

trap data). Significant values are indicated with an asterisk.

Lobster type

Males

Non-ovigerous
female

Ovigerous
females

Weighted means

(z scores)

Inshore
1996
1997
1998
1999

Offshore
1996
1997
1998
1999

0.0043 (8,754)
0.0304(6.216)
0.0956(5.325)
0.1023(6.236)

0(12.087)

0(11.864)

0.0032(10.160)

0,0056(11.176)

0.00063(14.163)

0.0125 (10..M3)
0.104(10.182)
0.092 (9,806)

0(16.704)

.00005(18.881)

.0031 (14.437)

.0066(13.622)

0.0142(3.022)

0.16663(3.151)

0.4984 (4.438)

0.53 (5.235)

0(1.019)

0(1,182)

0.0074(1,631)

0.0522(1.053)

0.00345

0.0427 (26.43*)

0.1895(46.94*)

0.203 (3.39*)

0
2.96E-05 (0.99)
.002415 (8.09*)
.00803 (8.93)*

r test was used for evaluating mean sizes. All differences were
tested for significance at a = 0.05.

For the tag/recapture study, lobsters received a shell disease
difference score for each recapture event. Shell disease difference
(SDD) was obtained by subtracting the recapture SDI from the
previous capture SDI. Results were statistically analyzed using a
chi-square test of independence (SPSS, Base 10) using Pearson's
chi-square statistic at a = 0.05 for effect of size. sex. and season
on shell disease outcome score (Sokal and Rohlf 1981).

RESULTS

The presentation of shell disease typically began with small
lesions occurring in the epicuticle of the carapace and abdomen
that were characterized by erosion and melanization. Many of
these lesions affected deeper layers of the cuticle. The site of
deepest penetration was an area directly behind the rostrum. In
later stages the entire shell became flexible. The last area affected
was the claws. Many newly molted lobsters had areas of mela-
nization already present in the new epicuticle.

The DEM inshore survey examined a total of 86,871 lobsters.
Total numbers of lobsters from all combined sites showed a sig-
nificant increa.se for every year in the proportion of shell diseased
lobsters in the inshore areas beginning in 1996. with the highest
infection rate of 20% in 1999 (Fig. 2; Table 2). The highest in-
fection rates were for ovigerous females, with over 50% displaying
signs of shell disease in 1999. A total of 87.865 lobsters were
examined in the offshore canyon survey. This area is beginning to

show low infection rates (Fig. 2; Table 2). In 1999 there was an
infection rate of 0.8%, with highest rates in ovigerous females
(5.22%).

The URI trawl survey examined a total of 18,841 lobsters. Shell
disease was first noted in the Wickford upper bay site in October
1995. The first occurrence of shell diseased lobsters in Whale
Rock occurred in May 1996. Both URI sites showed a significant
increase from 1995. with the highest infection rate in ovigerous
females in 1998 (Fig. 2; Table .3). Overall, proportions infected
from the trawl survey were much lower than the DEM survey, only
I'eaching 4.35% in 1998. However, infection rates for ovigerous
females were high: 52% in Whale Rock and 33% in Wickford.

Shell disease prevalence from the URI trawl survey was posi-
tively correlated with site, sex, size, and molt and was negatively
correlated with temperature. No correlation was found with cull
status (Table 4). Third order partial correlation controlling for
temperature, site, and molt status resulted in significant positive
correlations between shell disease and sex (P < 0.001 ) and size (P
= 0.028). There was a very weak relationship between proportion
infected and bottom temperature (R- = 0.1087, P = 0.835).

Mean size of diseased lobsters from the URI trawl survey
(66.64 mm CL) was significantly larger than the mean size of
non-diseased lobsters (64.3 mm CL: r test, df = 18827, P =
0.001 ) and size frequency distributions were significantly different
(Kolmogorov-Smirnov, D = 0.1197; Fig. 3).

There was no relationship found between proportion soft and
proportion infected (R" = 0.0078, P = 0.32). However, a pattern
of decreasing proportion infected occurred over the molt season.

TABLE 3.

Proportion of lobsters with shell disease and total number sampled (in parentheses) in Wickford in Narragansett Bay and Whale Rock in
Rhode Island Sound (URI trawl data). Significant values from the previous year are indicated with an asterisk.

Whale Rock

Wickford

Non-ovigerous

Ovigerous

Non-ovigerous

Ovigerous

Weighted mean

Males

females

females

Males

females

females

(z scores)

1995

0(1,011)

0(750)

0(17)

0.0011 (918)

0.00195(513)

0(4)

5.82 E-06

1996

0.006(1.725)

0.0033(1.504)

0.0145 (69)

0.0043(1.629)

0.0096(935)

0(2)

0.00551 (5.69)*

1997

0.02(1,166)

0.014(947)

0(0)

0.0087(1.031)

0.0022 (453)

0(6)

0.01292(3.5)*

1998

0.062 (598)

0.088 (506)

0.52(41)

0.018(1.555)

0.0235 (809)

0.33 (9)

0.0435 (7.8)*

1999

0.054(78)

0.024(84)

0.063(17)

0.0198(303)

0.019(155)

0.11 (9)

0.0267 (2.32)*

694

Castro and Angell

TABLE 4.

Spearman's correlation matrix for variables tested in association with shell disease prevalence in the URI Trawl Survey. Variables include

site, bottom temperature, legal or sublegal size, molt status, and cull status.

Spearman's
rho

Disease

Site

Temperature

Sex

Size

Molt

Cull

Disease

1.00

Site

0.037**
0.000

1.00

Temperature

-0.062**
0.000

0.032**
0.000

1.00

Sex

0.026**
0.001

0.120**
0.000

-0.034**
0.000

1.00

Size

0.025**

-0.056**

-0.103**

-0,076**

i.on

0.001

0.000

0.000

0.000

Molt

0.039**

0.016*

-0.024**

-0.016*

0.143**

1. 00

0.000

0.027

0.001

0.027

0.000

Cull

0.001

0.054**

-0.001

-0.006

-0.061**

0.069**

1.00

0.859

0.000

0.845

0.457

0.000

0.000

** Correlation is significant at the .01 level (two-tailed). * Correlation is significant at the .05 level (two-tailed). Listwise N. 18,291.

with an increasing trend in disease after the molt season that was
consistent between sites. (Fig, 4),

Trap sampling in Dutch Harbor sites examined 2,909 lobsters.
Shell disea.se showed a significant increase for each year between
1997 and 1999, reaching an infection rate of 19.1 1% (Fig. 2). The
highest rates were seen in ovigerous females at 56.4% infected
(Table 5),

Correlation analysis indicated positive relationships between
disease and date, size and recapture status, and a negative rela-
tionship with molt and cull status and no correlation with sex or
site (Table 6), A fourth order partial correlation controlling for
date, molt status, cull, and recapture status showed a significant
relationship between shell disease and size (P < 0,001 ). but not sex
{P = 0,071). However, an evaluation of effect of sex on proportion
infected using the Mann-Whitney test showed significant differences
between males and non-ovigerous females (z = -2.51. P = 0,012),
males and ovigerous females (z = -7,72. P < 0,001). and non-
ovigerous females and ovigerous females (z = -9,002, P < 0,001),

Lobsters captured from the trap survey were significantly larger
than those from the trawl survey (Kolmogorov-Smirnov. D =
0,236). Mean size of diseased lobsters (75,3 mm CL) was signifi-
cantly larger than mean size of non-diseased lobsters (70,4 mm
CL; I test, df = 344S, P < 0.001 ) and size frequency distributions
were significantly different (Kolmogorov-Smirnov, D = 0,2476),
There was a small peak in prt)portion infected at 50 mm CL.
followed by an increasing trend from 70 to 100 mm CL (Fig, 3),

Low proportions of diseased lobsters were observed from April
to July 1997. but increased over the summer and fall; 199S had
decreasing proportions from April to July when spawning and
molting, increasing again in September and October (note: the
large proportion of ovigerous females infected seen in 1998 may
be due to small sample size for this sex category in that year; Fig,
5), The degree of disease coverage (SDI) was not indcpendenl of
year sampled (chi-st|uare = 146,8. P< 0.001 ); 1999 had increased
proportions of SDI I and 2 (Table 7),

In the tag-recapture study 295 males were followed over the
study period (Fig. 6), Of these, 19,79!^ became diseased. Of the
total non-diseased lobsters, 25.3% molted and 74,7% did not.
Thirty-five of them that were disease free at first capture were
recaptured multiple limes. Thirty-lour of these developed the dis-

ease during the study; one molted and the disease did not reappear
after 83 days. Twenty lobsters were diseased at first capture and
followed with multiple recaptures. Once diseased. 81,8% of them
did not molt. All but one of them worsened over time. Two mor-
talities (3.4%) were recorded for males with minor shell disease
(SDI-1 ) and two mortalities were observed for non-diseased males
(0.84%; Table 8).

One hundred thirty-eight non-ovigerous females were followed
of which 10,9% of them became diseased (Fig, 7), Of those that
were not observed to contract the disease. 42,6% molted, while
57,4% did not. One mortality was observed (0,72%). Seventeen
lobsters were recaptured multiple times and all developed the dis-
ea.se. Before acquiring the disease. 73% of them did not molt and
27% did molt. After acquiring the disease, 36.4% of them molted
and did not show signs of the disease after a mean of 88,3 days.
Forty-four females became ovigerous during the study period and
25% of them became infected. Of those that were not diseased.
9.1% molted and lost eggs. Of the ovigerous diseased females.
27,3% of them molted and/or lost eggs (Table 9),

Shell disease outcome was independent of size (chi-square. df
= 2. P < 0,058), season (chi-square = 2,24. df = 3, P = 0,524),

Size Frequency and Percent with Shell Disease

URI Trawl and Tnp Survoys

A

03S

— ■ — Proporlioft Trawl

• Proponion Traps

<-

\

- * - %Stioii Disoasod T<awl

/

\

0 30

- « - % Shell DlseBJBd Traps

h

\

i°"

/

r

\^

\

0

/

\

\

= 0 20

/

1

\

\

to.

/

/

/

\

A

010

/

/

/

\^

->,

oos

/

'/

/,

- -X '

pt '

■\

V\_

- .

^_

=^

:_c_

-*

r -* •

-* .

- -pt'^^

so w

e

5

Figure 3. Si/.e lre(|uenc> of shell diseased and non-diseased lobster
from 11 RI trawl sur\ev and trap sur\e> friini Dutch Harbor.

Shell Disease in American Lobsters

695

0.2

0.18

0.16

0.12

c
o
■E

O 0.1

a.

o

0.06

0.04

0.02

Proportion Shell Diseased By Month and Year
URI Trawl Survey

D Whale Rock
■ Wlckfor(d

■

n

,n ,n ,

fl

1

1

n

1

-> S E

Month/Year

Figure 4. Proportion of lobsters witli stiell disease by month and year from the URI trawl survey. Molt usually occurs from April to June and
August to October of each year. Sampling occurs year round.

and sex (chi-square = 5.095, df = 4. P = 0.278) (Fig. 8). No
relationship was found between days between recaptures and shell
disease difference; in some cases the greatest increase in infection
occurred in short recapture periods. By examining short recapture
intervals (less than 36 days) the first appearance of shell disease
was estimated (from 0-1 SDI). These were confined to September
through November in 1997 {n =11); June, September and Octo-
ber in 1998 ill = 6); and August through October in 1999 in =
10). Worsening of shell disease condition was rapid between Sep-
tember and October.

DISCUSSION

The routine surveys described here opportunistically recorded a
shell disease episode affecting the inshore Rhode Island lobster

population. Beginning in 1995, there were low proportions of mi-
nor shell diseased lobsters observed in the URI trawl survey; how-
ever, by 1999. a greater percentage of lobsters were showing evi-
dence of infection in all surveys, with ovigerous females being the
most affected. Each survey revealed slightly different trends and/or
proportions observed. The prevalence of shell disease observed in
1997 and 1999 from the DEM survey coincides with that observed
in Dutch Harbor. The URI trawl survey reported much lower over-
all proportions than other surveys.

The differing sampling designs and gear types probably ac-
count for the majority of the variation observed, although no in-
formation is available regarding changes in lobster behavior (i.e.
catchability) as a result of disease. The large sample size examined
from the DEM survey may increase observation error and may not
represent the population as a whole because catchability in the

TABLE S.

Proportion of lobsters with shell disease and total numbers sampled (in parentheses) in Dutch Harbor (URI trap survey). Significant

differences from the previous year are indicated with an asterislc.

Dutch Harbor

Males

Non-ovigerous
females

Ovigerous

Weighted mean

(z scores)

1997
1998
1999

0.0599(901)

0.099(383)

0.1625(449)

0.0298 (436)
0.053(225)
0.186(339)

0.1 96 (.56)
0.625 (8)
0.564(39)

0.056

0.08903 (2.54*)

0.1911 (5.71*)

696

Castro and Angell

TABLE 6.

Spearman's correlation matrix for variables tested in association with shell disease infection in Dutch Harbor. Variables include site, sex,

size, molt status, cull status, date, and recapture status.

I

i

Spearman's rho

Disease

Site

Sex

Size

Molt

Cull

Date

Recap

Disease

1.00

Site

0.010
0.547

1.00

Sex

0.019
0,277

0.116**
0.000

1.00

Size

0.164**
0.000

-0.055**
0.001

-0.085**
0.000

1.00

Molt

-0.067**
0.000

0.011
0.518

-0.022
0.194

-0.114**
0.000

1.00

Cull

-0.060**

-0.050**

0.006

-0.061**

-0.001

1.00

0.000

0.004

0.742

0.000

0.954

Date

0.206**

-0.067**

0.073**

0.164**

-0.230**

-0.115**

1.00

0.000

0.000

0.000

0.000

0.000

0.000

Recap

0.069**

-0.068**

-0.006

0.214**

-0.084**

-0.004

0.100*

1.00

0.000

0.000

0.726

0.000

0.000

0.811

0.000

Listwise N, 3.388. ** Correlation significant at the 0.01 level (two-tailed).

traps increases shortly after the molt. If the propoilions infected are
at their lowest value at the time of highest catchability, the infected
estimate may be low. The size frequency may also be different
than those captured in the other two surveys because of escape

vents. However, the DEM survey covers a larger area of the in-
shore waters and may be more representative of the whole. Many
differences between the URI trawl survey and other surveys have
been noted (ASMFC 2000), especially in reference to trends and

Proportion with Shell Disease and Total Captured
Dutch Harbor -Traps

0.4

0.35

0.3

0.25

c
o

t

O 0.2

a
o

0.15

0.1

0.05

0 i

,n,n,n

JllL

D Proportion Infected
■ -Total Captured

350

300

250

200 S

3
Q.

n
O

«
150 -g

100

50

a

cr>
a

S

Month/Year

Figure 5. Proportion of lobsters with shell disease by month/year from Dutch Harbor trap survey. Molting usually occurs from April to June
and August to October each year. Note: no sampling occurs from December 1997 to March 1998 and November 1998 to March 1999.

Shell Disease in American Lobsters

697

NON DISEASED
MALE (35)

Range61-93minCL

V''

\ •

MOLT (1)

NO MOLT (34)

a

r

DISEASE (2)

DISEASED (20)

MOLT (5)

_ik

i.

NO MOLT (27)

JZ

i.

JL

JL

la.t

1_

~±

JZ

JZ

J.

J.

Figure 6. Pathways followed by shell diseased males followed during tag-recapture study. Numbers inside boxes corresponds to numbers of
individuals observed: numbers in bold indicate mean days between recaptures, unless only one individual is represented. STS, stays the same;
None, no disease observed.

numbers. This may be a consequence of the fixed stations, the
weekly sampling, and/or the habitat type. The URI trawl may be
capturing migrating lobsters since it occurs over soft featureless
bottom consisting of mu(d/sand substrate. The trawl captures a
larger range of size classes. The incomplete year of sampling in the
URI trawl survey in 1999 most likely is responsible for the de-
crease seen that year, especially since there was an early molt.
Dutch Harbor represents only one area and may have unusually

TABLE 7.

Proportion of lobsters (sexes combined) with shell disease index
(SDI) 0 to 4 in years 1997 through 1999 from Dutch Harbor.

high or low infection rates. The lobster reef site is in a voluntary
no-fishing zone and proportionally larger lobsters are captured
there than at other sites. These have a longer time to develop shell
disease than those removed by the fishery and may present a more
accurate size-related effect of shell disease. The higher rates could
also be related to habitat type as well. Since many of these lobsters
are handled through multiple recaptures, stress may influence the
progression of shell disease. Tagging and handling may affect
molting and reproduction as well. Oocyte resorption has been as-

TABLE 8.
Proportion of male lobsters following designated pathways.

1997

1998

1999

SDI-0

0.94.^

0.911

0.808

SDI-1

0.032

0.047

0.138

SDI-2

0.007

0.013

0.041

SDI-3

0.009

0.008

0.002

SDI-4

0.009

0.021

0.011

Lobster types

Molt

No molt

Non-diseased males
Diseased males'
Diseased males"

0.253

0.03

0.28

0.747

0.97

0.818

Males that entered pathway non-diseased.
• Males that entered pathway diseased.

Die

0.0084

0

0.034

698

Castro and Angell

Non-Diseased Females (17)

{15) Non- Ovigcrous

Range 72-82 mm CL

(2) Ovigerous

1

(l)Bemed

(4) Molt

1 , 1

1.

(1 1) No Molt

-|716

484

(1) Disease

(3) Disease

(3) Bemcd

(2) No Molt

125.5

nor

(l)None

17

(3) Disease

(2) Disease

(3) None

in

(5) Disease

(1) Berried

IL

(2) Disease

J_

(1) Disease

(8) Diseased

Non-
Ovigcrous

(4) Diseased
Ovigerous

(4) Molts

IL

(7) No Molls

(1) Molt
Looses eggs

7

1

1

I

(l)None

(10) No Molt

6.3

I 95J

y_c

523 r

( 1 ) Looses

(4) None

(7) STS

(3) STS

(1) Molts

( 1 ) Looses
eggs

Figure 7. Pathways followed by shell diseased females followed during tag-recapture study. Numbers inside boxes corresponds to numbers of
individuals observed; numbers in bold indicate mean days between recaptures, unless only one individual is represented. STS, stays the same;
None, no disease observed; Berried, egg-bearing.

sociated with stressful environmental conditions (Waddy et al.
1995). Cooper (1970) reported a delayed molt or reduced molt
increment under certain circumstances as a result of tagging.

Sampling time had a large impact on observed proportion in-
fected, with pre-molting peaks seen in both URI sites in the spring.
The peak after the second molt period in Whale Rock corre-
sponded to the time period in which infection rates also rose in
Dutch Harbor. Malloy (1978) observed that more disease was
established in lobsters in post-ecdysis than in pre-ecdysis, which
could explain the higher proportions observed in the fall. This may
be the more serious indication of problems in the population since

those lobsters must overwinter with the degraded shell condition.
This may result in a weakened state that makes these lobsters more
vulnerable to other pathogens, predators, or adverse environmental
conditions (Smolowitz et al. 1992). There were verbal accounts of
lobsters being consumed by starfish in traps over the winter fish-
ery. It is unknown if healthy lobsters would be susceptible to
attacks by starfish, even when they are lethargic because of low
water temperatures.

There was evidence from this study that the proportion infected
increased with size, especially in the no-fishing area where larger
lobsters were present. This supports the hypothesis that molting

TABLE 9.
Proportion of female lobsters following designated pathways.

Lobster types

Molt

No molt

Die

Non-diseased/nnn-ovigerous females
Discased/non-ovigcrous females'
Di.seased/non-ovigemus females'
Non-tliseasedAivigerous Icmales
Diseascd/ovigcrmis females

,42fi

27

.164

,091

27.1

0..'i74

0.7,1

0.6.^6

0.909

0.727

().(K)72

0

0

0

0

' Females that entered pathway non-diseased.
" Females that entered pathway diseased.

Shell Disease in American Lobsters

699

Shell Disease Difference

Tag-Recapture

Remains same

Shell Disease Difference

Figure

8. Proportion of shell diseased lobsters by sex that improve,
the same, or worsen during tag-recapture events.

frequency affects proportion diseased. However, there were con-
siderable numbers of smaller lobsters infected as well. The molt
may physically remove signs of shell disease, however, the degree
of damage to the new shell may be related to when the diseases
occurs in the molt cycle and how quickly it progresses through the
shell layers. If the disease is the result of an internal metabolic
disturbance, molting might not provide extended respite from in-
fection. Many of the lobsters followed in the tag-recapture study
redeveloped shell disease after molting. Field observations of
newly molted lobsters revealed many with darkened areas.

There was no relationship found in this study between bottom
temperature and proportion of shell diseased lobsters. This was not
surprising since shell disease occurs worldwide in crustaceans
from all climatic conditions ranging from ice-covered lakes to
semi-tropical estuaries and deep water (Rosen 1970). Malloy
(1978) reported more disease occurred in lobsters held in water
temperatures of 2 °C to 5 " rather than at higher temperatures,
which would imply that the disease does not recess during the
winter months, but continues to progress. Hood and Meyers (1974)
found the highest populations of chitinoclastic bacteria in the en-
vironment occurred during spring and early summer when median
temperatures were above 16.9 C. but shell disease in blue crabs
was more prevalent during late fall and winter.

Lobsters fed an insulTicient diet may also be more vulnerable to
shell disease (Malloy 1978). Dietary deficiencies were a factor in
the development of shell disease in juvenile American lobsters (4th
through 12th stage) because of problems in epicuticular repair
(Fisher et al. 1976). Prince et al. (1995) reduced shell disease
incidence in pounds by feeding pellets containing a higher protein
and crude fat content.

Cook and Lofton (1973) found that in blue crabs, only me-
chanically damaged areas were susceptible to shell disease. Me-
chanical injuries caused by handling, ecdysis, aggressiveness, and
high stocking densities have been blamed for shell disease in
prawns (Delves-Broughton and Poupard 1976). Johnson (1983)
reported that chitinoclastic bacteria did not cause harm in natural
unstressed environments. Getchell (1989) reported that only in
degraded or crowded conditions does shell disease appear to be
highly contagious. Other degraded conditions may include expo-
sure to sewage, sludge, heavy metals, or dredge spoils. Healthy
lobsters held in aerated seawater with sewage for up to 6 wk
developed shell disease, whereas control lobsters held in clean

water did not (reported in Stewart 1980). However, no field study
to date has been able to effectively demonstrate a direct cause and
effect relationship (Young and Pearce 1975. Estrella 1984.
Ziskowski et al. 1996).

Several of these factors may play a role in the observed in-
creased lobster infection rates. It appears to be occurring on a large
geographic scale, which would point to a common environmen-
tally induced cause. A similar hypothesis has been proposed for the
recent increases in abundance reported for recruit sized lobsters in
South of Cape Cod Long Island Sound Stock (SCCLIS) asses.s-
ment area (ASMFC 2000). These very high abundance estimates
may increase densities, creating more opportunities for aggressive
encounters because of food or space limitations or stressed condi-
tions leading to metabolic dysfunction or contagious conditions.
However, few of the observed shell-diseased lobsters displayed
obvious physical damage; juvenile and adult lobsters are not
thought to be habitat limited (Wahle and Steneck 1992) and since
lobsters are opportunistic feeders, it is unlikely that food supply is
limiting. The observed shell disease etiology fits the description
- provided by Bullis et al. (1988) for the endogenous origin of shell
disease which may be a consequence of immunosuppression or of
failure of metabolic processes associated with shell repair and
maintenance. The etiology and mechanism for this are not obvious.

Possible consequences for the population are numerous. Taylor
(1948) found a 71% mortality associated with shell-diseased lob-
sters in a laboratory setting compared with 6% in control lobsters.
Large lesions may cause mortality during ecdysis if there is adhe-
sion between the exoskeleton and underlying tissues (Martin and
Hose 1995). Winter mortalities due to shell disease have histori-
cally been recorded in Nova Scotian pounds (Hess 1937, Taylor
1948, Malloy 1978, Getchell 1989). Mortality rates attributed to
shell disease during pounding were reported to be 6.5% (Prince et
al. 1995). Secondary mortality effects due to predation or parasites
are unknown.

It was surprising to find that three diseased and one non-
diseased ovigerous female lobsters who molted late in the season
shed their eggs. Several area fishermen have also reported finding
cast shells with eggs attached in their traps. However, the eggs
have not been shown to be fertilized or even viable. It is known
that some females will reabsorb the yolk from mature oocytes
(Waddy et al. 1995). which may be related to warm winter tem-
peratures. However, it is rare for lobsters to molt while carrying
eggs (Waddy and Aiken 1991 ). Usually the molting and spawning
cycles are synchronized by temperature and are under endocrine
control, and molting is believed to take precedence over reproduc-
tion (Waddy et al. 1995). The consequences for egg production
may be severe, regardless of the ultimate pathway followed. Since
the ovigerous females are displaying the highest rate of infection
and if they experience the extreme mortality rates as indicated by
Taylor (1948) then our reproducing lobsters may suffer higher
mortality rates if they continue to brood their eggs. If they molt,
then the potential egg production is lost and they may recontract
the disease and legal-sized females then become vulnerable to the
fishery.

This is the first study that has been conducted that examines the
effects of shell disease on lobsters in the field. There is a great need
for future work, especially concerning site differences, mortality
rates, and biological and economic consequences to the population
and the fishery. With the example of the recent devastating mor-
tality event in Western Long Island Sound lobsters, we need to

700

Castro and Angell

gain an understanding of the mechanisms and causes of disease
outbreak in our valuable marine crustaceans.

ACKNOWLEDGMENTS

There are many people who have contributed to this collabo-
rative effort. Many thanks to the RI DEM personnel who have
collected the sea sampling information and provided the data for
this analysis. This state sea sampling program is entirely voluntary
and the participating fishermen should be acknowledged for their
interest and willingness to assist in the assessment and manage-

i

ment of the resource. Many thanks to Drs. Jeremy Collie and Perry
Jeffries from the University of Rhode Island, Graduate School of
Oceanography for allowing us to collect information on the weekly
trawl survey on board the R/V Cap'n Bert. Collectively, I thank the
many undergraduate and graduate students, staff, and volunteers
who have helped over the 5-year study. 1 would also like to express
my deep-felt gratitude to the late Dr. Ann Durbin for her valuable
advice. Many thanks to Rhode Island Sea Grant for their continu-
ing support for our outreach program that has made this all pos-
sible.

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Bayer, R.. H. Hodkins, M. Loughlin & D. Prince. 1993. Lobster Health

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Bullis, R., L. Leibovitz, L. Swanson & R. Young. 1988. Bacteriologic

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Cooper, R. A. 1970. Retention of marks and their effects on growth,

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Shellfi.'ih Res. 8(1): I -6.
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strategies. Neurosci. Biohehavior. Rev. 14:273-294.
Hess, E. 1937. A shell disease in lobsters {Homanis americamis) caused by

chitinivorous bacteria. J. Biol. 3:358-362.
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in natural estuarine waters and aquarial systems, pp. 1 1.5-121. In: R. S.

Amborski, M. A. Hood and R. R. Miller (eds.). Proceedings of Gulf

Coast Symposium on Diseases of Aquatic Animals. Louisiana Stale

University, Baton Rouge.
Johnson, P.T. 1983. Diseases caused by viruses, rickellsiae. bacteria, and

fungi, pp. 1-78. In: J. P. Provenzano (ed.). The Biology of Crustacea.

Palhobiology. Academic Press. New York.
Malloy, S. C. 1978. Bacteria induced shell disease on lobsters, Homarus

americamis. J. Wildlife Dis. 14:2-10.
Martin, G.G. & J.E. Hose. 1995. Circulation, the blood and iliscasc. pp.

465^96. In: J. R. Factor (ed.l. The Biology of llic Lobster. Homarus

americamis. Academic Press, New York.

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Techniques for Research Workers. 4th ed. Iowa Sate University Press.
Ames, 664 pp.

Prince. D. L.. R. C. Bayer. M. L. Gallagher & M. Subramanyam. Reduction
of shell disease with an experimental diet in a Nova Scotian lobster
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F. Snieszko (ed.). A Symposium on Diseases of Fishes and Shellfishes.
American Fisheries Society. Washington, DC, Special Publication
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Sindermann, C. J. 1991. Shell disease in marine crustaceans: a conceptual
approach. / Shellfish Res. 10(2):491-f94.

Sokal, R. R. & F. J. Rohlf 1981. Biometry. 2nd ed. W.H. Freeman and
Company, New York, 843 pp.

Smolowitz, R. M., R. A. Bullis & D. A. Abt. 1992. Pathologic cuticular
changes of winter impoundment shell disease preceding and during
intermolt in the American lobster, Homarus americamis. Biol. Bull.
183:99-112.

SPSS. 1999. SPSS BaselO.O Applications Guide. SPSS, 426 pp.

Stewart. J.E. 1980. Disease, pp. 301-344. In: J. S. Cobb, and B. F. Phillips
(eds.). The Biology and Management of Lobsters, vol. 1: Physiology
and Behavior. Academic Press. New York.

Stewart, J.E. 1984. Lobster diseases. Helgolander Meeresiinlers 37:243-
254.

Taylor, C.C. 1948. Shell disease as a mortality factor in the lobster,
Homarus americamis. Fisheries Circular no. 4. State of Maine, Depart-
ment of Sea and Shore Fisheries, Fisheries Laboratory. Boothbay Har-
bor. Maine. 8 pp.

Waddy. S. L & D. E. Aiken. 1991. Egg production in the American lob.sler.
pp. 267-299. In: F. R. Schram (ed.). Crustacean Issues, vol. 7: Crus-
tacean Egg Production. Balkema, Rotterdam, The Netheriands.

Waddy, S. L.. D. E. Aiken & D. P. V de Kleijn. 1995. Control of growth
and reproduction, pp. 217-266. In: J. R. Factor (ed.). The Biology of
the Lobster, Homarus americamis. Academic Press, New York.

Wahle, R. A. & R. S. Steneck. 1992. Habitat restrictions in early benthic
life: experiments on habitat .selection and in situ predation with Ameri-
can lobster. / E.xp. Mar. Biol. Ecol. 157:91-1 14.

Young. J. S. & J. B. Pearce. 1975. Shell disease in crabs and lobsters from
the New York Bight. Mar. Poltui. Bull. 6:101-105.

Ziskowski, J., R. Spallone, D. Kapareiko, R. Robohm, A. Calabrese & J.
Pereira. 1996. Shell disease in American lobster in the offshore North-
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Mar. Environ. Enf>incer. 3(2-4):247-271.

Jourmil of Shellfish Research. Vol. 19. No. 2. 701-709. 2000.

BRACHYURAN COMMUNITY IN UBATUBA BAY, NORTHERN COAST OF SAO PAULO

STATE, BRAZIL

FERNANDO LUIS MEDINA MANTELATTO' AND
ADILSON FRANSOZO-

' Departamento de Biologia, FFCLRP

Unhersidade de Sao Paulo. Cep. 14040-901

Ribeiido Preto. SP, Brasil
'Departamento de Zoologia

Institiito de Biociencias. UNESP

Botiicatu. Cep. 18618-000

Botucatii. SP. Brasil

ABSTRACT A brachyuran crab assemblage from eight transects at a non-consolidated sublittoral site in Ubatuba Bay was studied
on a monthly basis from September 1995 to August 1996. Data about number of individuals of ."iO species found and other information
such as distribution of the dominant crabs are reported. The family Majidae was represented by 13 species, followed by Xanthidae (13).
Portunidae (10). Leucosiidae (5). Calappidae (2), Dromiidae (2). Parthenopidae (2). Goneplacidae (1). Pinnotheridae (1). and Ocy-
podidae (1). The brachyuran taxocoenosis was dominated by Callinecres onmtiis (60.4%). Callinecres liiiiuie (18.8%). and Hepatus
pudibundus (1.1%). representing together 86.9% of the total number of collected brachyurans. The Shannon-Weaver diversity index
ranged from 1.10 to 2.06 between transects, and from 1.34 to 2.22 between months, depending more on equitability than on richness.

KEY WORDS: Biodiversity. Brachyura. crabs. Crustacea. Ubatuba. Brazil

INTRODUCTION

With approximately 5.000 species described worldwide, the
Brachyura is a highly significant group of marine crustaceans play-
ing an important role in the marine trophic chain (Melo 1996).
Recently our group listed 315 brachyuran species living along the
Brazilian coast (Pohle et al. 1999) and this number continues to be
modified both by new species descriptions and new records of
exotic brachyurans.

The South Atlantic coastal zone can be functionally divided
into two regions: the subtropical Brazilian coast (from 22°S to
33°S) of about 3.000 km. and the Patagonian region consisting of
the temperate coasts of Southern Brazil. Uruguay and Northern
Argentina (from 33°S to 42°S) extending over 2,900 km. Located
along the northern coastline of the State of Sao Paulo, the Ubatuba
region is an important area for crustacean investigations, particu-
larly on Brachyura. This region comprises the coastal portion of
the Biogeographic Province of the State of Sao Paulo, a zone of
faunal transition (Palacio 1982). The area as a whole possesses a
mixture of faunas of both tropical and Patagonian origin (Sumida
and Pires-Vanin 1997). In addition, Ubatuba Buy is its fairly pris-
tine and is used as a standard for comparison with other marine
habitats strongly influenced by man (Mantelatto and Fransozo
1999a). For this reason in recent years there has been an impres-
sive number of studies of the intertidal zone and continental shelf
centered on the crab faunal composition of a variety of habitats in
the Ubatuba area. Fomeris ( 1969) performed the pioneering study
which provided a brachyuran check-list in Flamengo Bay; Abreu
(1980) described ecological aspects in an estuarine area of
Ubatuba; Pires (1992) reported the structure and dynamics on the
continental shelf offshore of Ubatuba; Fransozo et al. ( 1992) es-
tablished the composition and the distribution at the non-
consolidated sublittoral from Fortaleza Bay; Hebling et al. ( 1994)
reported the crabs sampled in the Anchieta Island region; Man-
telatto and Correa ( 1996) described the composition and seasonal
variations of the species living on the algae Sargassum cymo.sum

C. Agarth, 1820 from three different Ubatuba beaches; Pinheiro et
al. (1997) studied the composition and the relative abundance of
crabs associated with sand reefs created by Phragmatopoma lapi-
dosa Kimberg, 1867. and Mantelatto and Souza-Carey (1998) re-
ported the species inhabiting the bryozoan colonies of Schyzoporella
unicornis (Johnston 1847). The purpose of the present paper is to
report the species composition of brachyuran crabs from a sublittoral
location with non-consolidated sediments in Ubatuba Bay as a con-
tribution to the study of the biodiversity of Brachyura from the Sao
Paulo coast.

Figure I. Map of Ubatuba Bay (Sao Paulo State) showing the position
of the sampling tran.sects.

701

TABLE 1.

Total species composition and number of Individuals In each transect in Ubatuba Bay calculated for whole year. (CN, constancy; Co,

constant: Ac, accessory, and Ad, accidental).

Transects

Family/Species

III

IV

VI

VII

VIII

TOTAL CN

DROMIIDAE

Cryptodromiopsis antillensis (Stimpson 1X58)

Hypoconcha arcuata (Stimpson 1858)
CALAPPIDAE

Hepaliis piulihwulus (Herbst 1785)

Calappa nalliis ( Herbst 1 803 )
LEUCOSllDAE

Litluulia hra.siliensis (von Martens 1872)

Persephona criniui (Rathbun 1931 )

Persephona lichlensleinii (Leach 1817)

Persephona mediterranea (Herbst 1794)

Persephona punctata (Linnaeus 1758)
MAJIDAE

Apiomithrax violaceus (A. Milne Edwards 1868)

CoHodes inermis (A. Milne Edwards 1878)

CoUoiles rostratus (A. Milne Edwards 1878)

Lihinia ferreirae (Brito Capello 1871)

Lihinia spinosa (H. Milne-Edwards 1834)

Microphrys biconnitiis (Latreille 1825)

Nololopas brasiliensis (Miers 1886)

Pelia rotunda (A. Milne Edwards 1875)

Pitho Iherminieri (Schramm 1867)

Podochela gracilipes (Stimpson 1871)

Pndochlae riisei (Stimpson 1860)

Pyromaia lubercuhita (Lockington 1876)

Stenorhyiichiis seticornis (Herbst 1788)
PARTHENOPIDAE

Partheniipe (Parthenope) agona (Stimpson 1X71)

Purtheniipe (Platytanihrus) gueriiii (B. Capello 1871 )
PORTUNIDAE

Arenaeus cribrarius (Lamarck 1818)

Callinectes danae (Smith 1869)

Callinectes ornatus (Ordway 1863)

Callinectes supidiis (Rathbun 1896)

Charybdis hellerii (A. Milne Edwards 1867)

Croniiis ruber (Lamarck 1818)

Portunus ordwayi (Stimpson I860)

Portunus spinkarpus (Stimpson 1871)

Portunus spinimanus (Latreille 1819)

Portunus ventralis (A. Milne Edwards 1879)
XANTHIDAE

Eurypaimpeus ahbrcviutus (Stimpson 1860)

Hexiipunopeus sp.

Hexupanapeus puulensis (Rathbun 1930)

Hexapanopcus schniitti (Rathbim 1930)

Menippe nadifrnns (Stimpson 1859)

Mkropanope nuttingi (Rathbun 1898)

Panopeus americanus (Saussure 1857)

Panopeus bermudensis (Benedict and Rathbun 1981)

Pilumnoides hussleri (A. Milne Edwards 1X80)

I'iluinnus diomeileae (Rathbun 1894)

Pilumnus reticulatus (Stimpson I860)

Piluinims spiniisissimus (Kalhbun IS9S)

Xanthidae sp.
C.ONEPI.ACIDAR

liiiiialopxis crassinianu.'. I Dana 1X52)
PINNOTHERIDAE

Pinnixa sp.
OCYPODIDAE

Ucides cordutus (Linnaeus 1763)
TOTAL
Number of species

-

-

-

3

8

-

-

-

11

Co

-

-

-

-

2

-

-

-

2

Ad

266

50

86

96

35

368

70

15

986

Co

-

-

-

5

2

-

-

-

7

Ac

-

-

1

-

1

2

2

-

1
5

Ad
Ad

-

-

4

-

-

14

6

1

25

Co

105

-)

1

12

2

3

1

-

126

Co

20

5

5

70

68

181

11

-

360

Co

_

_

_

_

9

1

_

_

10

Ac

-

-

-

-

4

-

-

-

4

Ad

-

-

-

1

-

-

-

-

1

Ad

5

7

5

12

5

9

7

-

50

Co

7

1

3

1

-

3

1

-

16

Ac

1

-

1

-

-

-

-

-

2

Ad

-

-

-

-

6

-

-

-

6

Ac

-

3

2

1

-

-

-

-

6

Ac

~

~

~

1

1

1

-

~

~

1
1

1

Ad
Ad
Ad

Ad
Ac

Ad

;

-

-

1

1
11

1

"

~

~

1

1
11

1

213

98

2

1

1

85

47

1

446

Ad
Co

4

16

86

9

-

393

272

1.626

2.406

Co

436

422

489

l.lll

834

2,910

657

X66

7,725

Co

-

-

-

-

-

2

1

1

4

Ac

-

1

-

4

131

3

-

-

139

Co

-

-

-

1

1

-

-

-

2

Ad

-

-

-

-

7

-

-

-

7

Ad

-

4

-

-

10

-

-

-

14

Ad

:

1

:

65

143
1

5

1

:

215
1

Co
Ad

-

-

-

-

-

1

1

-

-

1
1

Ad
Ad

-

4

1

9

21

15

-

-

50

Co

-

1

_

24

35

19

-

-

79

Ac

-

-

-

-

1

1

1

-

3

Ad

-

-

-

1

-

-

-

-

1

Ad

_

_

_

T

1

-

-

-

3

Ad

-

-

1

-

-

-

-

-

1

Ad

-

-

-

4

2

-

1

-

7

Ad

-

-

-

-

1

-

-

-

1

Ad

-

-

-

1

20

2

-

-

11
1

Co
Ad
Ad

-

-

-

I
2

-

-

-

-

1
->

-

-

-

13

7

1

-

-

21

Ac

-

-

1

-

-

-

1

-

2

Ad

1 ,057

615

688

1.4.50

1.372

3,9.M

I.II7

1

2,557

1
12,790

Ad

9

14

15

25

31

20

15

7

Brachyuran Crabs of Ubatuba Bay

703

TABLE 2.
Total number of individuals per month for all eight subareas combined, collected from September 1995 (S) to August 1996 (A).

Months

Family/Species

S

O

N

D

J

F

M

A

M

J

J

A

Total

DROMIIDAE

Cryplodroiniopsis antillensis

-

1

1

-

1

3

1

1

-

-

-

3

11

Hypocomha aniiala

2

-

-

-

-

-

-

-

-

-

-

-

2

CALAPPIDAE

HepciUis ptulihimdiis

133

101

117

40

88

84

55

39

29

53

130

117

986

Calappa callus

1

-

1

1

-

-

-

-

-

1

2

-

7

LEUCOSIIDAE

Lilhadia brasiliensis

1

-

-

-

-

-

-

-

-

-

-

-

1

Persephona crinita

-

-

-

1

-

-

-

-

-

-

2

T

5

Persephona lichtensleinii

6

-

-

3

-

1

1

1

-

6

-)

25

Persephona mediterranea

21

11

18

3

15

3

9

-

1

3

29

13

126

Persephona piinctara

70

17

43

6

9

11

6

12

10

8

54

114

360

MAJIDAE

Apiomithrax violaceiis

1

1

-

-

-

4

3

-

-

-

-

10

Collodes inermis

-

-

-

-

-

-

-

1

-

-

2

4

CoUodes robusuis

-

-

-

-

-

-

-

-

-

-

-

1

Libinia ferreirae

2

11

2

-

2

-

2

1

1

14

11

50

Lihinia spinosa

-

4

-

1

-

-

-

6

-

4

1

16

Microphiys bicornutus

-

-

1

-

-

-

-

-

-

-

-

2

Nololopas brasiliensis

-

-

-

1

-

2

-

-

-

1

1

6

Pelia rotunda

-

1

-

-

-

-

1

-

-

1

2

6

Phito Iherminieri

-

-

-

-

-

-

1

-

-

-

_

-

1

Podochela gracilipes

-

-

-

-

-

-

1

-

-

-

-

-

1

Podochela riisei

-

-

-

-

-

-

-

-

-

-

-

1

1

Pyromaia luherciilara

1

-

-

-

-

-

-

-

-

-

-

-

1

Stenorhynchiis sericomis

-

-

4

-

-

-

1

1

-

-

2

3

11

PARTHENOPIDAE

Parthenope af^ona

-

-

-

-

-

-

1

-

-

-

-

-

1

Parthenope iPlarylanibrus) giierini

-

-

-

-

-

-

-

-

1

-

-

-

1

PORTUNIDAE

Arenaeus cribrarius

33

33

28

20

34

66

35

27

39

45

61

25

446

Callinectes danae

104

47

82

28

114

515

542

234

162

269

189

120

2406

Culliiiecles ornalus

824

426

592

466

696

1031

692

662

430

466

648

792

7725

Cullinecres supidiis

1

1

-

-

1

1

-

-

-

-

-

-

4

Char\hdis hellerii

7

1

7

-

-

-

16

40

32

2

15

19

139

Cronins rtilyer

-

-

-

-

-

-

-

1

-

-

-

-

2

Portuniis ordwayi

-

-

-

-

-

-

-

7

-

-

-

-

7

Portunus spinicarpus

-

-

-

5

-

9

-

-

-

-

-

-

14

Portunus spinimanus

61

15

35

6

15

5

2

23

21

11

8

13

215

Portunus ventralis

-

-

-

-

-

-

-

-

1

-

-

-

I

XANTHIDAE

Eurypanopeus abhrevialus

-

-

-

1

-

-

-

-

-

-

-

-

1

Hexapanopeus sp.

-

-

-

-

1

-

-

-

-

-

-

-

1

Hexapanopeus paulensis

1

-

-

3

7

3

4

18

5

2

2

5

50

Hexapanopeus schmitti

1

-

-

3

9

s

47

14

-

-

-

-

79

Menippe nodifrons

-

-

-

-

-

1

1

1

-

-

-

-

3

Micropanope nuttingi

-

-

-

-

-

-

-

1

-

-

-

-

1

Panopeus americanus

1

-

-

-

-

2

-

-

-

-

-

-

3

Panopeus bennudensis

-

-

-

-

-

-

-

1

-

-

-

-

1

Pihimnoides hassleri

1

1

-

-

5

-

-

-

-

-

-

-

7

Pihnnnus diomedeae

-

-

-

-

1

-

-

-

_

_

_

-

1

Pihimnus reticulatus

-

1

4

1

-

-

12

2

-

-

1

1

22

Pihnnnus spinosissimus

-

-

-

-

-

-

-

-

-

-

1

-

1

Xanthidae sp.

-

-

-

-

-

-

-

-

-

-

2

-

2

GONEPLACIDAE

Eucratopsis crassimanus

2

-

-

-

4

9

")

3

-

-

1

-

21

PINNOTHERIDAE

Pinnixa sp.

-

-

-

1

1

-

-

-

-

-

-

-

2

OCYPODIDAE

Ucides cordatus

-

-

-

-

-

-

-

-

-

-

1

-

1

Number of species

21

20

16

17

19

16

21

■>-)

15

11

-1-)

20

704

Mantelatto and Fransozo

MATERIALS AND METHODS

Ubatuba Bay (23''26'S and 45°02'W) is adjacent to the town of
Ubatuba situated on tfie northern coast of Sao Paulo, Brazil. The
area of the bay is about 8 km" with a width of approximately 4.3
km at the entrance.

The study site was divided into eight subareas selected for their
relation to the bay mouth, the presence of a rocky wall or a beach
along the boundaries, the inflow of fresh water, the proximity of
offshore water, depth, and granulometric composition. Each
transect was assigned to a subarea for sampling of crabs and mea-
surement of environmental factors (Fig. 1). During the study the
environmental data was sampled throughout full transect and was
checked at the beginning, middle, and end of each transect sample.
There was no change in this data throughout each transect. Depth
ranged from 2.5 (subarea 2) to 18.5 m (subarea 1), temperature
ranged from 19.2 °C to 20.1 °C, salinity ranged from 33.5%p to
34.8%o, and dissolved oxygen ranged from 5.21 to 5.87 mg/L. The
overall organic matter content in bottom sediments ranged from
2.0% (subarea 2) to 30.27f (subarea 5| and fine sediments (<0.250
mm) prevailed in most subareas. Water samples were collected
from the bottom using a Nansen bottle. Temperature was measured
with a thermometer attached to the bottle, salinity was measured
using an optical refractometer (Atago S/IOOO). and dissolved oxy-
gen was measured by the Winckler method modified by the addi-
tion of azide. Depth was measured in each sampling station using
a graduated rope that was attached to the Van-Veen grab sampler

(1/40 m') used for sampling sediment. Sediment (= 200 g) was
dried at 70 °C for 72 h before organic matter and grain size analy-
ses. The Wenthworth (1922) scale was used for the grain size
analyses. The phi (<t> = mean diameter) value was u.sed according
to Suguio (1973) to calculate the central sediment tendency. Or-
ganic matter was obtained by ash-weighting, three aliquots of 10 g
each per subarea per month were heated in porcelain crucibles for
3 h at 500 °C and then reweighed. Detailed descriptions of physical
and chemical features characterizing this area and statistical simi-
larity of environmental factors among transects can be found in
Mantelatto and Fransozo (1999a).

Sampling of crabs occurred monthly from September 1995 to
August 1996. The sample was performed at a diurnal 1-km-long
trawl transect at each of the eight sampling subareas during a three
consecutive days per month. The catches of crabs were done by
trawler equipped with double rigged nets (3.5-m wide mouth, 10
mm of mesh size cod end). Each trawl was performed with veloc-
ity and time adequate to prevent significant scape from each net in
function of the differences on bottom substrate surface and tidal
currents. Immediately after capture all crabs were placed on ice
and frozen until being examined in the laboratory.

The Constancy Index (C) for each species was calculated ac-
cording to Dajoz (1983): C = Px 100/P, where "P" is the number
of samples in which a given species was recorded, and "P" is the
total number of samples analyzed. Species were then classified
into three different constancy categories; i.e. constant (C > 50 %),
accessory (25% < C < 50%), and accidental (C s 25%). Diversity

25

15

25

20

15

10

-•- - - Organic Matter
-4 — N of individuals

4500

4000

3500

3000

2500

2000

1500

1000

500

0

B

• Organic Matter
-4 — N of species

IV V

Transects

VIII

Figure 2. Total nunihcr or individuals (A) and species (B) as a function
of organic matter in the sediment (|H'rcentage of dry weighll in each
transect sample in Ubatuba B;i> for whole year.

45

4
35

3
25

2
1 5

1
05

0

45

35

4
35

M

3

25

25

iii

[iZ

o

2

20

s

tf>

1 5

1b

?!

F

05

10

3
Z

0

5

0

l.-io...-.

IV V

Transects
Figure i. Numher of individuals (A) and species (B) as a function of
the central sediment tendency (11 = <J)I in each transect sample of
llbatuhu Hay for whole year. The <j) was calculated according to Su-
guio (l'>7.1) from the formula <t> = - log,d, where d = grain diameter
(mm I. The distribution curve was obtained by the fornuila <|) 16 + <|) 50
+ ct> S4/,V

Brachyuran Crabs oh Ubatuba Bay

705

was calculated using the Shannon-Weaver index (Shannon and
Weaver 1963): H' = S,', i Pi- log^ P,. where ".v" is the number of
species and "P" is the proportion of /''' species. The equitability
index (J') was calculated as indicated by Garcia Rase and Fernan-
dez Munoz (1987): J' = H'/log2 s. Pearson coefficient was used
to check relationships between the absolute values of each envi-
ronmental factor studied and the number and frequency of species
for all subareas combined.

RESULTS

A total of 12,790 brachyurans belonging to 50 species and 10
families was collected (Tables 1 and 2). The brachyuran taxo-
coenosis was dominated by Callinectes onuitiis (60.4%), Calli-
necies danae (18.8%), and Hepatus pmlihtindus {1.1%). together
representing 86.9% of the total collection. These three dominant
species are differentially distributed in Ubatuba Bay (Table I ).

The greatest number of species was recorded for transects VI
(30.8%) and VIII (20.0%). Species richness was significantly cor-
related with coarse grains sediments {P = 0.00017; r = -0.96)
and with high organic content (Figs. 2 and 3). No correlation was
observed between the above factors and number of individuals (P
= 0.00265; r = 0.90). Pearson's analysis revealed significant
coefficients between some species and group correlation (Table 3).

Continuous and heterogeneous occurrence throughout the sam-
pling period was recorded for H. piidihundits. P. punclata. A.
cribrarius. C. danae. C. oiiatus. and P. spinimamis. The number

of species and their respective frequencies did not show significant
seasonal variation, although both parameters increased slightly
during the warmest months (Fig. 4) when the abundance of om-
nipresent species was greatest. In terms of their temporal pattern of
occurrence, 26% of species were classified as omnipresent or con-
stant, 18% as accessory, and 56% as accidental. The data of the
most abundant species from monthly sampling taken on eight dif-
ferent subareas are shown in Table 4.

The diversity index ranged from 1. 10 to 2.06 within transects
and from 1.34 to 2.22 within months, depending more on equita-
bility than on richness (Table 2 and Fig. 5). The lowest richness
values were detected in transect VIII (7.0) which was influenced
by fresh water inflow and significantly contrasted with those ob-
served in transects IV (31.0), which had a high percentage of
medium sand and organic content. The highest diversity and eq-
uitability were recorded in transects V (high percentage of medium
sand and organic content) and I (highest depth and high percentage
of fine sand), while the lowest values were obtained in transect
VIII. During the study period both indexes showed wide variation
along.

DISCUSSION

Examination of the species collected in Ubatuba Bay provided
new information on the Brachyura fauna of the area and confirmed
the biological potential of this region. Of all the brachyuran species
recorded from Brazilian waters, 15.9% were found in Ubatuba

TABLE 3.

Coefficients of Pearson's Linear Correlation carried out between the abundance of total individuals (TO), the most abundant species, and

the sampled environmental factors for all subareas combined.

Coefficients

Variables

TO

Hp

Pm

Pp

Lf

Ac

Cd

Co

Ch

Ps

Hx

Hs

Depth

0.135

-0.200

-0.174

0. 1 7 1

-0. 1 89

-0.173

0.094

0.178

0.225

-0.417

0.350

0.295

Dissolved oxygen

0.047

0.097

0.218

0.304

0.114

-0.398

-0.037

0.009

0.130

0.071

-0.234

0.412

Temperature

0.417

-0.436

-0.452

-0.678**

-0.670**

0.190

0.664*

0.339

-0.008

-0.174

0.363

0.607*

Salinity

0.123

-0.216

-0.056

-0.065

-0.147

0.106

0.252

0.010

0.360

-0.421

0.568*

0.362

Organic matter

-0.282

0.364

0.173

-0.008

0.056

-0.091

-0.391

-0.200

-0.327

0.230

-0.320

-0.297

Sediment

Gravel

-0.275

0.087

0.005

-0.244

0.055

0.231

-0.118

-0.375

0.038

0.122

-0.086

-0.264

Very coarse sand

0.048

0.243

0.083

-0.088*

-0.065

0.503

0.106

-0.024

-0.360

0.096

-0.301

-0.313

Coarse sand

0.156

0.622*

0.501

0.160

0.133

0.205

-0.053

0.160

-0.567**

0.555

-0.628

-0.205

Medium sand

0.279

0.280

0.054

0.564*

0.422

-0.161

-0.019

0.387

-0.140

0.077

-0.159

-0.181

Fine sand

0.250

-0.477

-0.574

-0.268

-0.294

-0.112

0.269

0.359

0.253

-0.151

0.536

0.191

Very fine sand

-0.401

-0.371

-0.096

-0.404

-0.219

-0.378

-0.251

-0.321

0.028

-0.240

0.184

0.236

Silt and clay

0.141

-0.078

-0.023

0.453

0.228

0.005

0.159

-0.007

0.622*

-0.200

0.291

0.131

Species
TO

0.3 1 1

0.164

0.246

0.054

0.524

0.749*

0.927*

0.031

-0.035

0.084

0.390

HP

0.879*

0.723*

0.669*

0.178

-0.265

0.414

-0.346

0.412

-0.481

-0.338

Pm

0.581*

0.663*

0.164

-0.288

0.197

-0.234

0.337

-0.436

-0.168

Pp

0.692*

-0.139

-0.316

0.357

0.105

0.377

-0.183

-0.336

Lf

0.115

-0.305

0.068

0.069

0.030

-0.262

-0.390

Ac

0.560*

0.388

-0.165

-0.289

-0.231

-0.089

Cd

0.504

0.095

-0.423

0.135

0.667*

Co

-0.079

0.110

0.105

0.168

Ch

0.087

0.705*

0.204

Ps

-0.077

-0.314

Hx

0.277

The pairs of variables show a tendency tu increase (*) or to decrease (**) correlation together [P < 0.05). Hp, Hepatus pudibundus: Pm. Persephona
medilerranea: Pp. Persepluina punclata: Lf, Libinia feneirae: Ac. Arenaviis cribrarius: Cd, Callinectes danae: Co, Callinectes ornatus: Ch, Charvhdis
hellerii. Ps. Portunus spinitminus: Hx, He.xapunopeus paulensis: and Hs, He.xapanopeiis sclumilli.

706

Mantelatto and Fransozo

2000

Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug

B

30

25

<J 20

« 15

10

-•- - - Temperature
-■ Nof species

25

20

15 S

10 s

e

0

Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug

Months

Figure. 4. Number of individuals (A) and species (B) as a function of

temperature throughout the study period (September 1995 to August

1996) in Ubatuba Bay for transects and subareas combined.

Bay. As discussed by Fransozo et al. ( 1998) in a study of anonui-
rans, the above percentage may be regarded as relatively high,
considering the small area of this bay compared to the extent of the
Brazilian coast. The present study revealed a diversity at least two
times higher than that obtained in similar studies in the Ubatuba
region carried out by Fransozo et al. ( 1992) in Fortaleza Bay. and
by Negreiros-Fransozo and Nakagaki (1998) in Ubatuba Bay. It is
likely that this difference was partly due to the sampling method-
ology (higher capture effort associated with the higher number of
sampled subareas).

In all three study areas the dominant families in terms of num-
bers of individuals were the Portunidae and Calappidae. The larg-
est temporal variation in species composition and density was
strongly influenced by two species. C. umatiis and C. danac. The
relatively large abundance t)f both species is probably due to their
high fecundity as they have more than one reproductive cycle a
year in this bay (Costa and Negreiros-Fransozo 1998, Mantelatto
and Fransozo 1999b). Even though C. daime was common in the
three aforementioned transects it only dominated the transect VIII
collections. Ctillincites onuiiiis was the most common brachyiiran
species in every other transect in addition to being common in
transect VIII. This pattern was also found by Negreiros-Fransozo
and Fransozo ( \995) in Fortaleza Bay. adjacent to Ubatuba. Calli-
iH'clcs ilaiuic was most frequent in subareas 6. 7. and 8 influenced
by freshwater. Furyhalinc species such as C. ornalus and C. danac
are found al both low and high salinity environments, as function
of their growlh. de\elopmenl. spawn, and lar\ac ilispcrsion phases.

Among the environmental factors that influence the occurrence
of brachyuran crabs in Ubatuba Bay. the sediment texture and
organic content may be the most important agents. Both param-
eters accounted for the spatial distribution of//, pudibiindus and C.
ornalus in the Ubatuba region, studied by Mantelatto et al. ( 1995)
and Mantelatto (2000), respectively.

Although environmental factors can delimit the distribution of
benthic species (Pinheiro et al. 1996), their relative importance
may differ among species, for the same species in different re-
gions, or in the same region in different years. According to Fran-
sozo et al. (1998), organic matter was deposited among sediment
particles or laid over the substratum as a covering layer in Ubatuba
Bay, with biogenic fragments mainly consisting of remains of
polychaetes, mollusks, crustaceans, and echinoderms. In this study
the organic matter content of the substrate was much higher than
data reported previously for three other areas of Ubatuba region
(see Mantelatto and Fransozo 1999a). Since organic matter has
been known to play an es.sential role in benthic crustacean distri-
bution, it along with abiotic conditions such as salinity, sediment
size, and temperature, may determine the development and estab-
lishment of benthic invertebrates in Ubatuba.

One purpose of this study was to delimit both spatially and
chronologically the brachyuran distribution in Ubatuba Bay so as
to identify important parameters for experimental investigations to
determine their relative influence on specific brachyuran species.

The number of individuals collected and the species diversity

■ 0.15

IV V VI

Transects

B

Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug
Months
Figure. 5. .Spatial and monthly oscillation of diversity (H'» and equi-
tability (.I'l for both each transect area (Al and month (B) during the
study period (.September 1995 to August 1996) in Ubatuba Bay for
transects and subareas combined.

Brachyuran Crabs of Ubatuba Bay

707

TABLE 4.
Number of individuals or the most abundant species from monthly sampling on eight subareas.

)areas

Months

Sut

S/95

O

N

D

J/96

F

M

A

M

J

,1

A

Total

Hepams pii

lihunjus

I

32

20

17

6

16

60

30

-

1

18

55

11

266

II

9

4

5

-)

11

4

5

T

1

0

-

5

50

III

20

5

8

4

21

1

1

1

1

7

12

5

86

IV

2

5

1

-

-

-

-

13

14

2

33

26

96

V

22

-

4

1

-

-

-

5

2

-

-

1

35

VI

4?

47

71

18

20

16

17

18

8

20

25

63

368

VII

3

18

7

9

19

3

1

-

2

2

T

4

70

VIII

-

2

4

-

1

-

1

-

-

2

3

2

15

Persephona

meJiterranea

1

19

8

17

3

14

3

8

-

-

1

24

8

105

11
III
IV

-

-

-

-

-

-

-

-

-

-

2
1

-

02
01

12

_

3

1

_

_

_

_

_

1

1

1

2

4

V

2

-

-

-

-

-

_

_

-

-

-

-

02

VI

-

-

-

-

-

-

1

-

-

1

-

1

03

VII
VIII
Persephona

-

-

-

-

1

-

-

-

-

-

-

-

01

punctata

I

I

-

4

-

-

2

1

-

-

-

3

9

30

II

I

-

-

-

3

-

-

-

-

1

-

-

05

III

2

-

-

2

-

-

-

-

-

-

-

1

05

IV

I

2

-

-

-

1

_

5

4

-

25

32

70

V

52

-

12

1

1

-

-

1

1

-

-

-

68

VI

13

15

27

">

f

8

4

6

4

6

25

69

181

VII
VIII
Libinia ferr

-

-

-

I

3

-

1

-

I

1

1

3

11

eirae

I

-

2

1

-

-

I

-

-

-

-

1

-

05

II

-

-

1

-

-

-

-

-

1

-

1

4

07

III

_

-

2

1

-

-

-

-

-

1

~

1

05

IV

-

-

3

1

-

-

-

-

-

-

4

4

12

V

2

2

-

-

-

-

-

-

-

-

-

1

05

VI

-

-

4

-

-

-

-

2

-

-

3

-

09

VII
VIII
Arenaeus c

-

-

-

-

-

I

-

-

-

-

5

1

07

■ihrariiis

I

16

17

19

8

8

49

28

1

12

23

22

10

213

II

15

8

5

-)

11

14

7

3

13

9

7

4

98

III
IV
V
VI
VII

-

-

1

~

1

:

-

:

I

-

:

-

02
01

2

6

-

10

8

1

-

11

11

8

17

11

85

VIII

-

2

3

-

6

2

-

12

2

5

15

-

47

Callinecles

T

danae

1

2

1

1

04
16

i
II

1

1

1

_

1

1

4

1

1

5

1

_

III

5

2

3

-

-

5

23

4

15

16

11

7

86

IV

V
VI

1

-

-

-

-

-

5

1

I

-

1

-

09

20

20

38

7

5

34

33

35

30

72

56

43

393

VII

10

5

20

7

9

40

86

18

39

16

10

12

272

VIII

66

19

21

13

99

433

391

175

76

160

110

63

1626

Catlinectes

omatiis

I

41

19

23

21

16

154

114

5

7

27

8

1

436

II

47

7

11

39

81

108

32

22

17

33

9

16

422

III

41

12

13

71

60

51

103

23

17

36

21

41

489

708

Mantelatto and Fransozo

TABLE 4.
continued

Months

Subareas

S/95

O

N

D

J/96

F

M

A

M

J

J

A

Total

IV

42

52

47

58

5

-)

5

277

155

43

164

261

nil

V

400

15

89

5

8

107

13

117

45

6

21

8

834

VI

215

236

359

173

296

230

246

157

134

198

309

357

2910

VII

25

48

28

72

126

110

65

12

38

38

42

.■13

657

VIII

13

37

22

27

104

269

114

49

17

85

74

55

866

Chaiyhtlis IwUerii
I

II
III
IV

-

-

-

-

-

-

-

1

-

-

-

-

01
04

-

-

-

-

-

-

1

1

1

-

I

I

-

V

07

01

06

-

-

-

15

36

32

1

14

19

131

VI
VII

-

:

01

-

:

:

:

2

:

:

-

-

03

VIII

Poinmus spininiiinn.s
I
11

ni

IV

_

_

_

_

_

I

_

_

_

_

_

_

01

65

14

-

5

6

8

-

9

16

3

2

2

V

47

14

29

-

5

3

2

14

5

8

6

10

143

VI

-

1

1

-

1

1

-

-

-

-

-

1

05

VII
VIII

Hexapaniipcus ptnilensis
I

II
III
IV

-

-

-

-

1

-

-

-

-

-

-

-

01

-

-

-

-

2

-

-

I

-

I

I

-

04
01
09

_

_

_

_

3

1

_

4

_

_

1

1

V

I

-

-

3

2

1

4

4

-

1

-

4

21

VI

VII

-

-

-

-

-

-

-

9

5

-

1

-

15

Vlll
Hi'-xapunopcti.s srlmiilti
1

U
ffl
IV

_

_

_

_

_

_

_

1
12

_

_

_

_

01

24

1

-

-

2

-

4

5

-

-

-

-

V

-

-

-

1

9

1

23

1

-

-

-

-

35

VI

VII

-

-

-

-

-

-

19

-

-

-

-

-

19

VIII

-

-

-

-

-

-

-

-

-

-

-

-

-

increased durinj; the Miniincr inonllis in Ihe Ubatuba region. Tliis
can be explained by the interaction of two water masses, i.e.
Coa.stal Water (CW) and South Atlantic Central Water (SACW),
with temporal and spatial effects dependent on the penetration
intensity of SACW. The SACW is rich in nutrients and when it
reaches shallow areas in summer primary production increases
causing pronounced eutrophicalion. Consequently, more food
should be channeled to the benthos in summer, which would ex-
plain the seasonal variation in its biomass (Pires 1992).

We infer that both brachyuran community composition and
diversity are controlled, at least in part, by seasonal abundance k'i^
dominant species associated with monthly changes in environmen-
tal conditions (Table .3). In this respect, intra- or interspecific fac-
tors (segregation of the sexes, competition, prey-predator relations,
reproductive, and molt cycles, among others) coidd act to partition
the resources of living space during a specific period when more

food is available to adults or larvae (Mantelatto 2000). Alterna-
tively, the presence of rare species such as Collodes iiwrmis, Potlo-
rheki riisci. and Porliiiuis ventralis reported by Goes et al. (1998)
might be the result of accidental introduction by offshore fisher-
men who sort their catch in I'batuha Hay before taking it to com-
mercial wholesalers.

Because of the large number of species and individuals docu-
mented in this study, it is difficult to explain in full the brachyuran
distribution in this bay, but we may infer, as reported for Ctilli-
lU'ch's onuiliis in a previous study, that the presence or absence in
an area results from interdependence between phases of ontoge-
netic development and the conditions ol ihc physical environment
(Mantelatto 2()(K)). Field observations on the extent of wave in-
tensity, buoy movements, fishing sites, commercial trawling ac-
tivity, scuba diving, and deposition of particles in the subareas
implied the existence of a strong circulation, with a predominant

Brachyuran Crabs of Ubatuba Bay

709

inflow reaching successively the following subareas; 1— >2— >7-^8.
Mantelatto and Fransozo (1999a). found subareas 4 and 5 to be
reproductive sites judging by the numerous ovigerous females of
Brachyura and Anomura collected there, suggesting that these sub-
areas are favorable to brooding and larval dispersion. The greater
number of species (31) in subareas 4 and 5 (southern portion of
bay) and the presence of 22 species found nowhere else in the bay
probably is related to the higher density of biogenic fragments and
the proximity of a steep, protective coastal shoreline. This idea is
supported by the presence of predominant species such as A. vio-
laceus. N. hrasiliensis, S. selicomis. C. hellerii. and P. reticulatus,
which live in consolidated habitats (reefs) or in association with
algae, and not in the non-consolidated area. In this way the abiotic
conditions and intra- or interspecific relationships may be cross-
correlated and lead to the distribution observed.

The present study documents the distribution of some 50 spe-
cies of Brachyura found in Ubatuba Bay and points out the need

for more detailed studies on the environmental parameters, biodi-
versity, larval dispersion, and larval settlement in different
biotopes to improve knowledge of the underlying factors deter-
mining population structure and dynamics of the brachyuran com-
munity of this important faunal transition zone on the northern
coast of Sao Paulo State.

ACKNOWLEDGMENTS

The authors are grateful to FAPESP (grant no. 95/2833-0) for
financial support. Special thanks are due to the NEBECC co-
workers for their help in field and laboratory work. A special
thanks is due to Dr. Gustavo A. S. de Melo (Zoology Museum of
University of Sao Paulo. Brazil) for assistance with species iden-
tification. Thanks are also due to Dr. Jack O'Brien (University of
South Alabama) and anonymous reviewer for helpful criticism and
English correction.

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Dajoz. R. 1983. Ecologia Geral. Editora Vozes. EDUSP. Sao Paulo. 472 pp.

Fransozo. A., M. L. Negreiros-Fransozo. F. L. M. Mantelatto. M. A. A.
Pinheiro & S. Santos. 1992. Coniposii^ao e distribuii;ao dos Brachyura
(Crustacea. Decapoda) do sublitoral nao consolidado na Enseada da
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Fransozo. A.. F. L. M. Mantelatto. G. Bertini. L. Fernandes-Goes & J. M.
Martinelli. 1998. Distribution and assemblages of anomuran crusta-
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Fomeris. L. 1969. Fauna bentonica da Baia do Flamengo. Ubatuba. SP:
aspectos ecologicos. PhD Thesis. Bioscience Institute. University of
Sao Paulo. Brazil. 215 pp.

Garcia Raso. J. E. & R. Fernandez Munoz. 1987. Estudio de una comu-
nidad de Crustaceos Decapodos de fondos "corali'genos" del alga cal-
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l):30l-322.

Goes. J. M., V. J. Cobo. A. Fransozo & F. L. M. Mantelatto. 1998. Novas
ocorrencias de caranguejos marinhos (Crustacea. Decapoda.
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sistema.s Brasileiros. PMiiai;ao .ACIESP M 1041:426-430.

Hebling. N. J.. F. L. M. Manlelatto. M. L. Negreiros-Fransozo & A. Fran-
.sozo. 1994. Levantamento e distribuii^'ao de braquiiiros e anomuros
(Crustacea. Decapoda) dos sedimentos sublitorais da regiao da llha
Anchieta, Ubatuba (SP). B. Inst. Pesca 21(iinico):l-9.

Mantelatto, F, L. M. 1000. Allocation of the portunid crab Callinectes
onuitii.s (Decapoda: Brachyura) in Ubatuba Bay. northern coast of Sao
Paulo State. Brazil. Cnisl. Issues 12:431-443.

Mantelatto, F. L. M.. A. Fransozo & M. L. Negreiros-Fran.sozo. 1995. Dis-
tribui(;ao do caranguejo Hepanis piidihiindus (Herbst. 1785) (Cru.sta-
cea. Decapoda. Brachyura) na Enseada da Fortale/a. Ubatuba (SP).
Brasil. Bolm. Inst. Oceanogr. 43(1):51-61.

Mantelatto. F. L. M. & E. K. Correa. 1996. Composition and seasonal
variations of the brachyuran crabs (Crustacea, Decapoda) living on
Sarga.t.sum cymosum in Ubatuba region. Sao Paulo. Brazil. Bioikos
9-10(l/2):22-31.

Mantelatto, F. L. M. & M. M. Souza-Carey. 1998. Brachyura (Cru.stacea.
Decapoda) associated to Schizoporella unicornis (Bryozoa. Gymnolae-

mata) in Ubatuba Bay (SP). Brazil. Bra.-. Arch. Biol. Tecnol. 41(2):
212-217.

Mantelatto. F. L. M. & A. Fransozo. 1999a. Characterization of the physi-
cal and chemical parameters of Ubatuba Bay. Northern Coast of Siio
Paulo State. Brazil. Rev. Brasil. Biol. 59(1):23-31.

Mantelatto. F. L. M. & A. Fransozo. 1999b. Reproductive biology and
moulting cycle of the crab Callinectes onuitus (Decapoda. Portunidae)
from the Ubatuba region. Sao Paulo. Brazil. Cnistaceona 72( I ):63-76.

Melo. G. A. S. 1996. Manual de identifica(,-ao dos Brachyura (Ciiranguejos e siris)
do litoral brasileiro. Editora Pleiade/FAPESP, Sao Paulo. Brasil. 604 pp.

Negreiros-Fransozo, M. L. & A. Fransozo. 1995. On the distribution of
Callinectes ornatus Ordway. 1863 and Callinectes danae Smith, 1869
(Brachyura. Portunidae) in the Fortaleza Bay. Ubatuba. Brazil. Iherin-
gia. Ser. Zool. 79:13-25.

Negreiros-Fransozo. M. L. & J. M. Nakagaki. 1998. Differential benthic
occupation by crabs in the Ubatuba Bay. Sao Paulo, Brazil. / Shellfish
Res. 17(1):29.V297.

Paliicio. F. J. 1982. Revision zoogeografica marina del sur del Brasil. Bolm.
hut. Oceanogr. 3l(l):69-92.

Pinheiro. M. A. A., A. Fransozo & M. L. Negreiros-Fransozo. 1996. Dis-
tributional patterns of Arenaens cribrarius (Lamarck, 1818) (Crusta-
cea, Portunidae) in Fortaleza Bay. Ubatuba (SP). Brazil. Rev. Brasil.
Biol. 56(4):7n5-716.

Pinheiro. M. A. A., G. Bertini. L. C. Fernandes-Goes & A. Fransozo. 1997.
Decapod crustaceans associated to sand reefs of Phragnmlopoma lapi-
dosa Kinberg. 1867 (Polychaeta. Sabellariidae) at Praia Grande.
Ubatuba. SP. Brazil. Naiipliiis 5(2):77-83.

Pires. A. M. S. 1992. Structure and dynamics of benthic megafauna on the
continental shelf offshore of Ubatuba, southeastern Brazil. Mar. Ecol.
Progr. Ser. 86:63-76.

Pohle. G.. F. L. M. Mantelatto. M. L. Negreiro,s-Fransozo & A. Fransozo.
1999. Larval Decapoda (Brachyura). /;;.■ D. Boltovskoy (ed.). South
Atlantic Zooplankton. Backhuys Publishers. Leiden, pp. 1281-1351.

Suguio. K. 1973. lntrodu(;ao a Sedimentologia. //;: E. Blucher (ed.).
EDUSP. Sao Paulo. 317 pp.

Sumlda. P. Y. G. & A. M. S. Pires- Vanin. 1997. Benthic associations of the
shelfbreak and upper slope off Ubatuba-SP. south-eastern Brazil. Es-
tuar. Coast. Shelf Sci. 44:779-784.

Shannon, C. E. & W. Weaver. 1963. The Mathematical Theory of Com-
munication. University of Illinois Press. Urbana, 177 pp.

Wenthworth. C. H. 1922. A scale of grade and class terms for clastic
sediments. J. Geol. 30:377-392.

Joimuil of Shellfish Research. Vol. 19, No. 2, 711-716. 2000.

LABORATORY OBSERVATIONS ON THE REPRODUCTIVE AND MOLT CYCLES OF THE
ROBINSON CRUSOE ISLAND LOBSTER J ASUS FRONTALIS (MILNE-EDWARDS, 1836)

ENRIQUE M. DUPRE

Department of Marine Biology
Universidad Catolica del Norte
Coquimho. Chile.
Casilla 1 17 Coquimho. Chile

.ABSTRACT Observations on molting, mating, embryo- carrying, hatching, and early larval development were made on the Juan
Fernandez lobster Jasus frontalis maintained in seawater aquaria at the Coastal Aquaculture Center of the Universidad Catolica del
Norte. Coquimbo, Chile. Males underwent their annual prereproduction molt from February through March at temperatures around 1 7
°C and females molted from mid-April to mid-June at temperatures of 13 to 15 °C. Growth at each molt was about 1.3 to 3.1 mm in
both males and females, and hardening of the carapace occurred over 3 days. Embryo- carrying was initiated between the months of
June and August, with hatching of larvae between October and December. Embryonic development lasted 1 15 days at 13 "C and 76
days at 18 =C. Hatching released a naupliosome larvae normally occurring between sunset and before midnight. The naupliosoma swam
actively for 10-20 min. then molted to produce the first phyllosome larvae. Five days after hatching. 2-S % of the egg mass remained
attached to the pleopods of the females, as did numerous empty capsules. These remaining eggs were in the first stage of development.
Six stages of larval development were obtained. The six larval stage were observed after 56 to 92 days, depending upon incubation
temperature. At 14-15 °C the fifth stage was obtained at 77 days; at 17-18 °C the sixth stage was obtained at 79 days, and after 56
days at 20-21 °C.

KEY WORDS: Jasus fiontalis lobster, reproductive cycle, molt

INTRODUCTION

Jasus fromalisAhs spiny lobster endemic to the Juan Fernandez
Archipelago, is a commercially valuable decapod crustacean in
Chile's oceanic waters. It has been exploited since about 1893,
thus constituting one of Chile's oldest fisheries. However, the
annual catch of this resource has decreased from 140 tons in 1964
to 20 tons in 1996 (Arana and Toro 1985, SERNAP 1996) despite
management measures implemented in the 1960s (Arana and Toro
1985. Yafiez et al. 1985). This decline emphasized the need for
new research on mating, spawning, and larval development that
would permit the design of more adequate regulations for the
protection of this species.

Early research on this lobster was oriented priiuarily toward
such fisheries-related parameters as growth (Arana and Marti'nez
1985), monality and yield (Diaz and Arana 1985), capture (Arana
and Melo 1973. Larrai'n and Yafiez 1985), and population structure
and dynamics (Gaete and Arana 1985, Yafiez et al. 1985). Little is
known about its reproduction and development. Arana et al. (1985)
determined size at first sexual maturity and fecundity, and de-
scribed 10 stages of embryonic development on the basis of mor-
phological characteristics and degree of coloration of the embryos.
The description of some of the larval stages ( VII. XI. XIII A and
B) was made from plankton samples obtained near the Robinson
Crusoe island (Baez 1973).

More recent research has begun to elucidate the reproductive
biology of this species. Each of the stages of embryonic develop-
ment has now been described using light (Dupre 1988) and scan-
ning electron microscopy (SEM) (Tavonatti 1998). The structural
organization of the ovary was described by Elorza (1998). The
duration and characteristics of different phases of the molt cycle as
revealed by analysis of pleopods. were described by Elorza and
Dupre (1996).

*Corresponding address: Department of Marine Biology. Universidad
Catolica del Norte. Coquimbo. Chile. Casilla 117 Coquimbo. Chile.
E-mail: edupre@uch.cl

Information on larval biology of the species is scarce and re-
cent. Dupre (1996) described the first stage phyllosoma larva and
later observed the first five phyllosoma stages in experimental
cultures over a 3-month period ( Dupre and Guisado 1996). Larval
cultures were subject to high mortalities because of infection of
larvae by Vibrio spp. and filamentous fungi (Dupre unpublished
data).

The present study evaluates the key events in the complete
reproductive cycle of this species, establishing the chronological
sequence of these events using specimens in captivity. The molting
period in males and females, mating period, time between mating
and appearance of embryo masses (berrying), periods of hatching
of larvae, and also the periods of embryonic and larval develop-
ment at different temperatures were observed. Our laboratory ob-
servations were made with the intention of duplicating the repro-
ductive cycle as it occurs in nature.

MATERIALS AND METHODS

Lobsters were obtained from Robinson Crusoe island (33 ° 40'
S; 78 ° 40' W) between 100 and 150 m depth and transported to the
Coastal Aquaculture Center of the Universidad Catolica del Norte
at Coquimbo (29 ° 58' S: 71 ° 22' W).

Females and males over the first sexual maturity size (cepha-
lothoracic length, CL = 75 mm for females and 82 mm for males;
Arana et al. 1985) were studied. Specimens included 31 females
(CL = 85.5 to 99.2 mm) and 16 males (CL = 92.6 to 1 16.7 mm).
Specimens were obtained in 1994 and 1995. and experimentation
was carried out in 1995 and 1996. Almost no mortality occurred
during the experimental period.

Males (M) and females (F) were distributed into four circular
500-L seawater tanks with constant flow of 50 ixm filtered sea-
water at ambient temperature, which ranged from a minimum of
13.1 X (6/95) to a maximum of 17.8 °C (12/96). Continuous
aeration was provided to each tank. The distribution of the speci-
mens was as follows: Tank 1-7 F. 7 M; Tank 2-9 F. 5 M: Tank
3-9 F, 3 M; and Tank 4-6 F and I M with a cephalotoracic lenght

711

712

DUPRE

(LC) of 1 16.7 mm. This male was able to fertilize three females
previous to the experiments.

The tanks were inside a laboratory greenhouse with ambient
light regime. The tanks were covered with shade netting that re-
duced total light entry by about 50 %. The lobsters were fed with
frozen fish ad VihUum every 2 days for 2 years. Unconsumed
residues and feces were removed from the tanks on each day
following feeding.

Tanks 1-3 were observed daily for evidence of molting; molts
were removed, and molted individuals were measured and trans-
ferred to individual 50-L tanks until their carapaces had hardened.
Later, each one of the molted female {/; = 8) were transferred to
Tank 4. where they were observed every 4 hours each day to detect
mating or presence of eggs on pleopods. The size increment of
recently molted individuals was measured, always using the length
from the postorbital margin to the dorsal termination of the cepha-
lothorax.

Embryonic development was determined at four different tem-
perature ranges: 12-14 °C; 15-17 °C. and 17-19 °C. Each embry-
onic development stage was determined according to Dupre
(1988). by light microscopy, of 10-15 embryos obtained from the
medial region of the abdomen of six ovigerous females carrying
225,350 to 287,200 embryos

To determine larval hatching time, females with embryos in
final developmental stages (Dupre 1988) were placed in 50-L
tanks to capture larvae upon their release. Seawater flowing out of
these tanks was passed through semisubmerged 300-|a,m screens
on which recently hatched larvae were captured. Larvae recovered
from these screens were washed in 10-(xm filtered seawater and
placed in 1-L glass beakers. Temperatures in the hatching obser-
vation varied from 15 ± 1 "C to 19 ± 1 °C.

Larval cultures were also carried out at four different tempera-
ture ranges: 14-15 °C. 17-18 °C, 19-20 °C, and 20-2 1°C main-
tained by a Jager thermostat. The water was changed daily in each
beaker, and larvae were fed ad libitum with Anemia naitpUi. Lar-
vae were observed daily in the microscope to follow developmen-
tal stages. Dead larvae lost the transparency and were observed
opaque-white. They were removed by a glass tube. To determine
the development time of the different stages, 325 larvae distributed
in eight 1-L glass beakers in groups of 30-50 larvae per L. were
used.

RESULTS

Molting and Reproductive Cycle

The annual reproductive and molting cycle is diagrammed in
Figure 1 . Molting in males occurred between February and March
each year, when the temperature reached an average of 1 7 ± 0.5 °C
(1995) and 16.9 ± 0.4 ° C (1996). Females molted from the middle
of April to the middle of June, when temperatures reached an
average of 15.0 ± 0.5 °C (1995) and 13.1 ± 0.2 "C (1996). Growth
(in CL) per molt varied between 1.4 and 3.6 mm in males and 2.2
and 3.1 mm in females; average increase by males was 2.3 and
females 2.7 Vr. Hardening of the carapace occurred slowly over 3
-5 days, during which the individual ceased feeding and showed
little activity.

Mating

Because no mating was observed during the day. it was con-
cluded that copulation occurred at night. In most cases, mating
occurred between postmolt females and males of a larger size.
When males with CL - ')2.6 and l()2.(i mm were presented to CL

19
18

.- 17

U

'Z i«

I 15

~ 13
12
11

/._

WaterTemp.

• B«m«<irenialc

i i ^ '■

JuD Jul Ako Sep Oct No\ Die Elle Feb Mar Abr Ma> Juo Jul Aro Sep Oct Nat Die

Figure I. Reproductive and molting cycle of the spiny lobster of Juan
Fernandez Jasus frontalis, reared in the laboratory. Key : male molt
(T) , female molt (i), embryos carrying females (#1, hatching of larvae
(A), temperature of the tank water (Temp.).

= 95 and 99.2 mm. respectively, recently molted females, none
were observed to be carrying embryos on their pleopods. In two
cases, using a CL < 95 mm males, eggs were found on the bottoms
of the aquaria, with minor amounts of eggs (100-300) adhered to
the pleopods of the females. The male in Tank 4 (CL = 116.7
mm) was the only one able to fertilize the oocytes of the post-
molted females; it fertilized all six moiled females introduced into
the tank between June and August. All females presented viable
embryos on the pleopods; these developed normally. Although no
mating was observed, this was estimated to occur sometime be-
tween 3 and 20 days postmolt, because egg-bearing females were
observed at 21 days postmolt.

Spawning

Recently spawned eggs were spherical, with diameters of 590-
610 p.m. They were covered with an adhesive coating (chorion or
vitelline coat), separate from the plasma membrane (Fig. 2a),
which allowed them to adhere to the pleopods and later formed the
funiculus. At the beginning of adhesion, the funiculus was short
(120- 250 |xm) and wide (450-500 |j.m) (Figs. 2b, c); within 30 to
40 min. eggs were observed adhering to the setae of the pleopods
(Fig. 2d) with the funiculus thinner (30-40 |jim) and three to four
times the initial length.

Embryonic Development

The total embryonic development, from spawning to the VII
stage, occurred between 1 15 and 76 days at temperatures average
of 13 ° C (ranging 12-14 C) and 18 C (ranging 17-19 °C).
respectively (Fig. 3). Average times of development were 76
(range ± 6) days at 18 °C (range 17-19 °C); 85 ± 4 days at 16 ±

1 C and 115 ± 3 days at 13 ± I °C. The major variation in
development time with temperature was observed with the first
stage (29 days at 13 °C. 22 days at 16 C, and 19 at 18 °C).
Between developmental stages IV and VII, variations caused by
temperature were not significant when incubated at 16 °C (average

= 10.5 days; SD = 1.4) and 18 C (average = 1 0.0 days; SD

= 1). but was significant between stages II and IV at 13 °C (av-
erage = 12 days; SD = 0.2) and 18 °C (average = 8.0; SD

= 0.41.

Hatching

The first larval stage to hatch trom the egg is the naupliosome
(Dupre 199fi). After 20 min of active swimming using the ex-

Reproductive Cycle of Jasus frontalis in Captivity

713

Figure 2. Recently spawned eggs adhered to pleopods setae. (A) Shows
the highly adhesive chorion at the beginning of the adhesion; initially
it is separated from the surface of the egg (arrows). The outer side of
the chorion has adhered to the setae of the pleopod and begun to
stretch (54X) . (Bl Eggs adhered to the setae of the pleopod 5 min
postspawn. Note chorion attached to the seta (arrowhead) without
formation of a funiculus (54X). (Cl Scanning electron microscopy
(SEM) view of the chorion adhered to a setae (s) (I.IOOX). (D) Eggs
adhered to a seta (s) of a pleopod forming a funiculus (f and arrows)
30 min after spawning (SOX).

opodites of the antennulae. these molted to produce the first phyl-
losome larvae. Hatching of larvae occurred between October and
November in each year of study, and in all cases occurred after
sunset and before midnight. Female lobsters actively initiated the
hatching process by extending and raising the abdomen to about
20° above horizontal and rhythmically agitating the pleopods to
produce a water current, which expelled larvae posteriorly.

The larvae were strongly phototactic. swimming toward the
surface, or toward any light source entering their environment.
Pleopodal beating of the females was active three to five times at
intervals of 30 min during the night. Activity suspended during
daylight hours. This induction of hatching was carried out over 3
to 5 consecutive days, with most larvae expelled during the first 3
days. Although natural hatching normally occurred during the
early evening, naupliosomes could be collected at any time by
removing the female lobster from the aquarium and directing a
gentle stream of seawater over the pleopods for 5 to 10 sec into a

7 -

y y

y ^

V

''/

c/'

o ^

E

/ ^

/

4. 17.19'C

1^

B. 15-irc
c. 12-irc

/^^

0 \^'^-^--

1 1

: I'll

1 11 21 31 41 51 61 71 81 91 101 111
Culture days
Figure 3. Embryonic development at different temperatures.

suitable receptacle (bucket). This method could be employed two
or three times a day without adversely affecting the females. At the
beginning of the hatching, embryos at different stages of develop-
ment were observed (Fig. 4a). Five days after initiation of hatch-
ing, about 2-8 % of the egg mass was still adherent to the pleopods
together to all the empty capsules left by enclosed larvae. The
notable of those remaining eggs had not progressed beyond the
first developmental stage (Fig. 4b) and they looked normal.

Figure 4. Embryos adhered to setae of a pleopod at the beginning of
hatching. (A) Shows early developed embryos (EE) and final developed
embryos at the distal end of the setae (8.3X1. (B) Embryos adhered to
a seta of a pleopod 2 days after the initial hatching. Observe empty
capsules (C), early developed embryos (EE). and final developed em-
bryos adhered to the same setae.

714

DUPRE

Larval Development

Six ditferent stages of larval development were observed be-
tween 56 to 92 days in culture, depending upon temperature (Fig.
5). Cultures carried out at 20-2 1 °C and 14-15 "C presented sig-
nificant differences between the time required to reached stage VI
and the time required in each development stage (average = 9.5
days per stage; SD = 1.04 and 15.3 days per stage; SD = 12.2,
respectively). However, at 17-18 °C and 19-20 °C the difference
between the averages time on each development stage was not
significant (13.0 days, SD = 4.1 and 11.5 days, SD = 4.4, re-
spectively). At all temperature ranges, the first two stages usually
required longer time periods for development than the intermediate
stages (III to IV), but less time than stages V and VI. Duration of
the first phyllosome stage was similar, between 17 and 20 °C (12
to 19 days). In contrast, at 14-15 °C their duration was 38 days.
After the second developmental stage, duration of each stage was
directly related to culture temperature (Fig. 5).

Filtration of culture water to 10 (xm was insufficient to prevent
infestation of larval cultures with filamentous fungi, which ad-
hered to larval pereiopods, antennae, antennulae, and buccal struc-
tures, and especially to the setae on swimmerets exopods of the
pereiopods. The best larval survival and most regular periods be-
tween development stages (9.5 days in average) were obtained at
20-21 °C (Fig. 5)

DISCUSSION

Molting

Molting of male lobsters occurring in the summer months of
February to March coincides with the beginning of seasonal tem-
perature decline typical of their natural habitat (17.8-16.8 °C)
(Neshiba and Silva 1985). Females began molting at the end of
April and beginning of May, typical of when habitat temperature
had declined to about 14 °C and onward through June as the
temperature droped to about 1 2.9 °C, after which habitat tempera-
ture began to rise in July.

Arana and Martfnez (1985), based on commercial captures in
the Robinson Crusoe Island, reported the highest percentages of
soft (recently molted) lobsters of both sexes were observed in
January and February, and in September, with a notable presence
of postmolt males in December to January. In our observations,
moiling commenced about one month later (Feb.-March).

This difference of molting periods between the captive speci-

(I 1(1 20 30 40 50 (ill 711 »(l IH

Culture days
I'ijjure 5. Karly larval development at different temperatures.

mens in aquaria and specimens in the field could be explained by
the gradual softening of the branchiostegal plates that start from 35
days before molt (Elorza and Dupre 1996), which can produce
errors in the estimation of the molt stage when it is determined by
palpation of the branchiostergal plates of the females in the field;
that is, field reports are based on different observations, and they
include the premolt stage within the postmolt stage. Observations
by Arana et al. (1985) that postmolt females are found in the
environment in May and June do not coincide with our observa-
tions in the laboratory.

Our values for cephalothoracic increases in length after molting
(1.5-3.6 mm) were lower than those made indirectly through the
analysis of the distribution of cephalothoracic length frequency, as
estimated by Arana and Martinez (1985) for the same species (8.9
mm). Our results represent the first time growth data have been
obtained by direct measurement. However, they estimated that
length increasing per molt is constant to all the molt stages, which
represents a decreasing rate per consecutive period of molt. Further
support for this position was that the growth values estimated in
our study were similar to those obtained for Jasiis lalandii (3.0 mm
for males and 1.0 mm for females with 6.0 to 9.9 cm cephalotho-
rax) (Beyers 1979) and Panulinis interntptus (Mitchell et al.
1969), where the annual cephalothoracic length increment was 3.7
mm for male and 4.4 for females, using individuals from 5.1 to 9. 1
cm of cephalothoracic length.

Following ecdysis, the carapace of Jasus frontalis has a soft
texture, which begins to disappear on the first day postecdysis to
the third to fifth day, when it has again become rigid. This result
is considerably different from observations made on Jasus lalandii
by Matthews (1962) and Heydorn (1969); the former author ob-
served the soft-textured condition lasted for 14 days; whereas, the
latter author estimated its duration at 15 to 32 days. The difference
between the cited results and our present results, other than inter-
specific variability, may be attributed to the abundant diet (rich in
Ca**) given to lobsters in our laboratory or to an error in the
determination of the molt stage by the compression of the bran-
chiostegal plates of lobsters, as mentioned above.

Mating and Spawning

Although we were unable to witness copulation in relation to
the precise time of molting, eggs appeared on the females about 2 1
days after males were placed with molted females. Mating oc-
curred between individuals of similar size or with males larger
than females. This observation may be of value in further attempts
to culture this species, especially because we noted that the sexu-
ally mature male of over 95 mm cephalothoracic length (Methods:
Tank 4) was able to fertilize at least six females in a period of 2
months.

tierried Phase

Our observations thai Icmales carried eggs Ironi June through
October coincided partially with observations of Arana et al.
( 1985) at Robinson Crusoe Island, who observed berried females
from July to February of the following year with a period of
maximum incidence between October and December. Our results
showing precocious embryos-carrying females of laboratory speci-
mens may be attributable to early molting, because maximum
ovarian development is obtained in the period immediately fol-
lowing ecdysis and is promoted by the hormone vitellogenine,
which is intimalelv rehilcd to the molting cycle (Meusy and Payen

Reproductive Cycle of Jasus frontalis in Captivity

715

1988). The advancement of molting, compared to that observed in
the natural habitat (Arana et al. 1985), may be attributable to the
abnormal rise in temperature experienced by specimens in the
laboratory. Lobsters in their natural habitat (100-200 m) experi-
ence less \ ariation in. and slower rates of change of temperature
than those kept in laboratory tanks.

Halcliiiig

Activity of the female lobsters, which produces liberation of
larvae (agitation of pleopods) suggests presence of an adaptive
mechanism to aid larvae unable to release themselves from the
chorion that envelops them. Simple mechanical agitation of the
pleopods presumably permits the releasing of the larvae.

Correct estimation of the fecundity of a species allows estima-
tion of the number of larvae available for recruitment into the
plankton. Previous estimates of this datum may be in error given
that our observations show that 2-8 % of the eggs carried by
females of J. frontalis may remain undeveloped on the pleopods
(they remain in the first stage) after most larvae have been re-
leased. Thus, the real contribution to the larval population made by
a female of 90-94 mm in cephalothoracic length that carries
131,000 to 301,000 eggs (Arana et al. 1985) would be diminished
by 2,620-6,020 (2 %) to 10.480-24,080 (8 %) larvae, plus an
unknown number of eggs lost during the normal incubation period
and the normal estimation error of the fecundity. In Palaemon
pandaliformis and Macrobrachium acanlhurus the loss of eggs
may reach 23 % (Anger and Moreira 1998).

As mentioned above, the presence of undeveloped eggs at-
tached to the pleopods after 76-1 15 days of incubation presumably
without deterioration of the yolk mass must be investigated. Be-
cause these oocytes were attached to the proximal end of the
pleopods setae, it suggests they were not fertilized.

Larval Development

In the temperature ranges of our study, development of em-
bryos and larvae was inversely related to culture temperature. Mor-
tality was not the same at each temperature range observed, with
larvae showing highest survival at 19-20 °C. This may be attrib-
utable to the higher frequency of molting in the higher temperature
ranges. Molting avoids formation of filamentous fungi on setae of
the exopodites of the pereiopods and the rest of the body, allowing
more freedom of motion and better feeding efficiency.

We conclude from our results that the reproductive cycle of this
lobster in captivity may not differ greatly from that observed in its
natural habitat. Further research is recommended to complete the
details missing for this valuable lobster species.

ACKNOWLEDGMENTS

This paper is dedicated to Dr. Claudio Barros R. Professor of
the P. Universidad Catolica de Chile. I thank Dr. Louis DiSalvo for
critical reading of the manuscript and I am also grateful to the
Robinson Crusoe Island fishermen, Albis Gonzalez and Deni
Gonzalez and to meteorologist Alex Meneses. This study was
supported by a research grant from the Chilean National Research
Fund (FONDECYT) 194-1121 to E.D.

LITERATURE CITED

Anger, K. & G. Moreira. 1998. Morphometric and reproductive traits of
tropical caridean shrimps. / Crust. Biol. 18:823-838.

Arana. P.. E. Dupre & V. Gaele. 1985. Cicio reproductivo, talla de primera
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Arana. P. & G. Martinez. 1985. Crecimiento por muda de langosta de Juan
Fernandez {Jasus frontalis), pp. 225-236. In: P. Arana (ed.). Investi-
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Arana. P. & T. Melo. 1973. La langosta de Juan Fernandez. II. Pesca
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Arana. P. & T. Toro. 1985. Distribution del esfuenzo, rendimento por
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Baez. P. 1973 . Larvas phyllosomas del Pacifico sur oriental (Crustacea,
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Beyers, C. J. 1979. Stock assessment and some morphometric and biologi-
cal characteristics of the rock lobster Jasus lalandii on Marshall Rocks,
its main commercial fishing area off South West Africa, 1971-1974.
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Di'az, P. & P. Arana. 1985. Estimaciones de mortalidades y de la edad
cri'tica en la langosta de Juan Fernandez {Jasus frontalis) de las islas
Robinson Crusoe y Santa Clara, pp. 237-250. In: P. Arana (ed.). In-
vestigaciones Marinas en el Archipielago de Juan Fernandez. Ediciones
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Dupre. E. 1988. Desarrollo embrionario de la langosta de Juan Fernandez
Jasus frontalis (Decapoda, Macrura, Palinuridae). Investigaciones Ma-
rinas 16:49-62.

Dupre. E. 1996. Primer estado de phyllosonia de la langosta de Juan
Fernandez. Jasus frontalis Revista Chilena de Historia Natural 69:
231-242.

Dupre. E. & Ch. Guisado. 1996. Identificacion de los pimeros estados de
Phyllosoma de la langosta de Juan Fernandez, Jasus frontalis. Investi-
gaciones Marinas 24:39-50.

Elorza, A. 1998. Efecto de la 17-alfa -dihidroxi-progesterona sobre la
maduracion ovarica de la langosta de Juan Fernandez, Jasus frontalis
(Milne Edwards, 1837), (Crustacea. Decapoda; Palinuridae). Tesis para
optar al Ti'tulo de Biologo Marino, Facultad de Ciencias del Mar.
Universidad Catolica del Norte. 175 pp.

Elorza, A. & E. Dupre. 1996. Determinacion de los estados del cicIo de
muda en la langosta de Juan Fernandez. Jasus frontalis (Milne Ed-
wards, 1837). Investigaciones Marinas 24:67-76.

Gaete. V. & P. Arana. 1985. Analisis de la proporcidn sexual en la langosta
de Juan Fernandez (Jasus frontalis), pp. 213-223. In: P. Arana (ed.).
Investigaciones Marinas en el Arachipielago de Juan Fernandez. Edi-
ciones Universitarias. Valparaiso, Chile.

Heydom. A. 1969. The rock lobster of the south African west coast. Jasus
lalandii (H. Milne Edwards). Notes on the reproductive biology and the
determination of minimum size limits for commercial catches. Dept.
Commerce and Industry Division Sea Fisheries South Africa. Investi-
gational Report 53:1-32.

Larrai'n, F. & E. Yanez. 1985. Diseno de un sistema de recolecciiion y
procesamiento de datos de captura y esfuerzo para la pesqueri'a de
langosta de Juan Fernandez, Jasus frontalis (H. Milne Edwards. 1837).
pp. 273-278. In: P. Arana (ed.). Investigaciones Marinas en el
Archipielago de Juan Fernandez. Ediciones Universitarias. Valparaiso.
Chile.

Matthews. J. P. 1962. The rock lobster of South West Africa {Jasus la-
landii) (Milne Edwards). Size frequency, reproduction, distribution.

716 DuPRE

and availability. Administratiim of Smiih West Africa Marine Research SERNAP. 1996. Anuario Estadi'stico de Pesca. Ser\icio Nacional de Pesca,

Laboratory, lnveslif;ational Repf. 7:1-61. Minislerio de Econonfa. Fomento. y Reconstruccion. Chile. 65-68.

Meusy, J. & G. Payen. 1988. Female reproduction in Malacostracan cms- Tavonatti, S. 1998. Analisis del desarrollo embrionario de la langosta de

tacea. Zool. Sci. 5:217-265. Juan Fernandez. Jasns frontalis, niediante microscopia electronica. Te-

Mitchell, C. T., C. Turner & A. R. Strachan. 1969. Observation on the sis para optar al ti'tulo de Biologo Marino. Facultad de Ciencias del

biology and behavior of the California spiny lobster. Paniilirns inter- Mar, Universidad Catolica del Norte. Chile. 143 pp.

ruiniis (Randall). Calif. Fish Game 55:121-131. Yaiiez, E., L. Pizarro, M. Barbieri & O. Barra. 1985. Dinamica del stock

Neshiba. S. & U. Silva. 1985. Algunos aspectos climatologicos de la isla de langosta Jasu.i frontalis explotado en el Archipi^lago de Juan

Robinson Crusoe y Santa Clara (Pacifico Seed Oriental), pp. 43-54. /».■ Fernandez), pp. 251-271. In: P. Arana (ed.). Investigaciones Marinas

P. Arana (ed.). Investigaciones Marinas en el Arachipielago de Juan en el Archipielago de Juan Fernandez. Ediciones Universitarias. Val-

Fernandez. Ediciones Universitarias, Valparaiso. Chile. parai'so. Chile.

7(>»;7i<;/ of Shellfish Research. Vol. 19, No. 2. 717-722, 2000.

COMPENSATORY GROWTH RESPONSE FOLLOWING PERIODS OF STARVATION IN
CHINESE SHRIMP, PENAEUS CHINENSIS OSBECK

LIXIN WU, SHUANGLIN DONG,* FANG WANG, AND
XIANGLI TIAN

Aquae II It lire Research Laboratory

Fisheries College

Ocean University of Qingdao

Qingdao, 266003. People's Republic of China

ABSTRACT The effects of previous periods of starvation on the subsequent changes in body mass, food consumption, food
utiM/ation. and body composition in Chinese shrimp. Penueiis chineiisis were investigated. Upon refeeding. shrimp responded to
various periods (4. 8. and 12 days) of food deprivation by exhibiting hyperphagia. There were the characteristic patterns that the
intensity of compensatory appetite increased in proportion to the length of the starvation periods and that the hyperphagic responses
of the starved .shrimp were not sustained, and within 8 days declined to levels not significantly different from those of the controls fed
continuously at satiation feeding. During subsequent refeeding. the previously 4-day-starved shrimp were only slightly higher, and the
8- and 12-day-starved shrimp were significantly higher in specific growth rates in terms of dry matter, protein, and energy content
(SGRj. SGRp. and SGR^.) than those of the controls. However, there were no significant differences in SGR„ (SGR in terms of wet
weight) among all the experimental groups. During the course of refeeding there were no significant differences in food conversion
efficiencies (FCE„. FCEj, FCE^, and FCE^.) among all groups, and only the shrimp previously starved for 8 days showed slightly higher
FCEs (FCEj. FCEp, and FCE^.) than the controls. There was a trend that, within the first 8 days of refeeding, FCE„ decreased with the
length of starvation periods, which may be attributable to changes in body water content. With food deprivation, lipid, protein, and
energy content decreased and water content increased. At the end of starvation the shrimp starved for more than 8 days .showed
significantly lower lipid, protein, and energy content and higher water content than the controls. After 32 days of refeeding no
significant differences in water, protein, and energy content were found between the starvation-satiation shrimp and the controls, except
that lipid content of the shrimp starved for 4 or 12 days was still lower than that of the controls. The results of this study suggest that
the shrimp regulate their appetite and growth rate in relation to their previous nutritional history.

KEY WORDS: Penaeus chinensis. compensatory growth, starvation, food utilization

INTRODUCTION

Aquatic animals, either in natural environments or under cul-
ture conditions, occasionally experience starvation or undernutri-
tion. The capacity to withstand and recover from nutritional stress
is an important adaptation for survival, growth, development, and
reproduction of any organism that must sporadically endure peri-
ods of limited food supply. Thus to investigate the recovery growth
in aquatic animals following a period of starvation or malnutrition
is not only of theoretical value in ecophysiology and evolution
(Russell and Wootton 1992. Nicieza and Metcalfe 1997). but also
of important applications in aquaculture (Quinton and Blake 1990.
Hay ward et al. 1997). In crustaceans there have been a consider-
able number of reports concentrating on survival, development,
and changes in metabolic activity and energy reserves during the
period of starvation in attempts to characterize the physiological
and biochemical effects of starvation (Anger et al. 1981. Barclay et
al. 1983. Dall and Smith 1986, Dawirs 1987). By contrast, few
studies have concerned on changes in biochemical composition
(Whyte et al. 1986. Stuck et al. 1996). weight gain (Bostworth and
Wolters 199.'i). and food consumption (Paul et al. 1994) during
recovery growth following transfer to plentiful rations.

As noted by Cui (1989). many animals subjected to variable
environment have the ability to actively regulate their growth.
When refed following a period of undernutrition aniinals will often
display a rapid growth spurt known as compensatory growth or
catch-up growth. Compensatory growth is known to occur in a
wide range of domestic mammals and birds (Wilson and Osbourn

*Corresponding author. E-mail dongsKa'mail. ouqd.edu.cn

1960. Mersmann et al. 1987. Yu et al. 1990). Amongst aquatic
animals compensatory growth has mainly been reported for fish
(Bilton and Robins 1973. Weatherley and Gill 1981. Dobson and
Holmers 1984. Quinton and Blake 1990. Russell and Wootton
1992,Joblingetal. 1993, Jobling et al. 1994. Hay ward et al. 1997).
However, little information on compensatory growth in crusta-
ceans is available (Bostworth and Wolters 1995). Moreover, there
is no further evidence on physiological mechanisms of compen-
satory growth in crustaceans, although in general, compensatory
growth in fish and other animals may be a result of hyperphagia,
improving food conversion efficiency, or both on subsequent re-
alimentation.

This study described the changes in weight gain, feed intake,
food conversion efficiency, body composition, and energy content
in Chinese shrimp, Penaeus chinensis Osbeck subjected to various
periods of starvation following by satiation feeding with the aims
to investigate the capacity for intrinsic growth regulation —
compensatory growth response displayed by the shrimp and to
determine the extent to which the relative severity of starvation
influenced the subsequent growth and the restoration of body com-
position.

MATERIALS AND METHODS

Rearing Condition

Chinese shrimp. P. chinensis were kept in glass aquaria (45 x
30 X 30 cm, water volume of 35 L). Each rearing unit was stocked
with 4 shrimp. The room temperature was controlled using an air
conditioner. Aeration was provided continuously and one-half to
two-thirds of volume water was exchanged every other day to

717

718

WU ET AL.

ensure high water quality. Seawater used in the experiment was
tlhered by composite sand filters. During the course of the experi-
ment, dissolved oxygen was maintained above 6.0 mg/L. the pH
was around 7.8, water temperature was 25.0 ± 0.5 °C. the salinity
of seawater was within 30%r to 33%o, and a .simulated natural
photoperiod (14 h of light: 10 h of darkness) was used.

Source and Acclimation of Shrimp

The experiment was carried out between August 26 and Octo-
ber 9. 1999 at the Laboratory of Aquacultural Ecology. Ocean
University of Qingdao, Qingdao, People's Republic of China. The
shrimp used in the experiment were collected from the Fengcheng
Shrimp Farm. Qingdao. Prior to the experiment the shrimp were
transferred into aquaria and underwent a 10-day acclimation pe-
riod during which they were fed polychaete worms. Neathes
japonicits (Izuka) at satiation level twice a day (at about 8:00 and
20:00).

Experimental Design

The experiment lasted for 44 days and was divided into two
periods: the food deprivation period and the refeeding period. Four
feeding groups were established: ( I ) Group C (control) was fed to
satiation twice a day (at 8:00 and 20:00) for 32 days; (2) Group S4
was starved for 4 days followed by 32 days of satiation feeding; (3)
Group S8 was starved for 8 days followed by 32 days of satiation
feeding: and (4) Group S 1 2 was starved for 1 2 days followed by 32
days of satiation feeding.

After 24 h of food deprivation, 1 14 shrimp with an initial wet
body weight of 2.164 ± 0.055 g (mean ± SE) were selected of
which 84 shrimp were randomly assigned to 21 aquaria using
randomized complete block design with the four treatments (3
aquaria for the control group and 6 aquaria for each of the three
starved groups). Each aquarium was stocked with 4 shrimp.

During the period of starvation, shrimp in Group S4. S8, and
S12 were placed individually in sections of aquaria divided into
quadrants by opaque plastic plates with holes ( 1 cm in diameter).
The plates were removed at the end of starvation. During the
refeeding period shrimp in all the four treatments were individu-
ally weighed every 8 days.

Samples Collection and Analysis

Three groups (10 shrimp each) were sampled from the origi-
nally selected batch for measurement of initial body composition.
At the end of starvation, three aquaria of Group S4, S8, and SI 2.
respectively, were randomly sampled and were weighed individu-
ally. Thus there were three aquaria that remained within each of
the three groups previously subjected to starvation during the

course of refeeding. After 32 days of refeeding the shriinp of all
groups were starved for 24 h and then sampled. The shrimp from
the same aquarium were pooled as a sample.

During the course of the experiment the daily food (polychaete
worms) supplied was recorded and uneaten food was collected
before the next ration was provided. To remove excess moisture,
shrimp and food were carefully blotted with paper towel and
weighed to the nearest 0.001 g using an electronic balance.

After the weight was obtained all the samples of shrimp and
food were dried timely in an oven at 70 °C to constant weight,
homogenized with a glass mortar, and stored at -20 °C. Before
chemical composition analysis the samples were re-dried at 70 °C
to constant weight.

Nitrogen content was measured using a PE-240C elemental
analyzer and crude protein content was calculated from nitrogen
content by multiplying nitrogen content by 6.25. Crude lipid was
determined by the Soxhiet method (Osborne and Voogt 1978), ash
was determined by combusting dried samples in a muffle furnace
at 550 °C for 12 h, and gross energy content of dried samples was
determined by bomb calorimeter (Cui et al. 1996). Analyses of
each sample were conducted in triplicates.

Calculation of Data

Specific growth rate (SGR), feed intake (Fl), and food conver-
sion efficiency (FCE) in terms of wet weight were calculated as
follows:

SGR„ (%/day) = 100 x (In W-, - In W.j/T

Fl„ {% body weight/day) = 100 x C/[T x (W, -i- W,)/2]

FCE^ (%) = 100 X (W, - W,)/C

Where W, and W, are the final and initial wet weight of the
shrimp, T is the duration of growth period in days, and C is the
weight of food consumed.

SGRs, Fls, and FCEs in terms of dry matter (SGR^, FI^. and
FCEj), protein (SGR^,, FIp. and FCEp), and energy content (SGR^.,
Fl^., and FCE^,) were calculated similarly.

Energy content of protein and lipid in shrimp was calculated
using factors 18.075 and 39.581 kj/g, respectively (Schafer 1968).

Statistical Analysis

Statistics were perfortned using SYSTAT statistical software
(SYSTAT 1992) with possible differences among groups being
tested by one-way ANOVA. Duncan's multiple range test was
used to lest the differences between treatment groups. Differences
were considered significant at a probabilils level of 0.05.

TABLE L

The chances in body weijjht (f;) of I'enaeus chincnsis during the course of experiment (mean ± SE)'.

Al dilTerent times of

recovery growth (day)

(i roups

Initial

starvation

8

16

24

32

S4

2.201 ±0.11.^

2.176 ±0.111"'

.V24.'i ± 0.0.56

4.541 ±0.229

.5.867 ± 0.387

7.212 ±0.461

S8

2. 152 ±0.021

2.0.'i() ± ().02()''''

.^.0IX±O.O4.'i

4.2.54 ± 0.263

5.6.56 + 0.444

7.302 ± 0.732

S12

2.I.S6± 0.040

1 .966 ± ().()4()''

2.8.'5I +0.216

3.900 + 0.263

5.201 +0.288

6.594 ± 0.277

C

2.146 + 0.021

2.146 + 0.02 1'

3.074 ±0. I.S.I

4.266 ± 0.302

5.553 ±0.5 14

6.846 ±0.7 15

Values with elilferenl Icllcrs in ihc same column arc siiinilicanllv clilTcrcnl Iniin each mhcr {P < 0.05).

COMPKNSATOR'I' GROWTH RESPONSE IN PENAEUS CH/NENSIS

719

RESULTS

C ■S4 nS8 HS12

Growth

The mean body weight changes in the shrimp of the all treat-
ment groups during the course of the experiment are shown in
Table 1. During the period of food deprivation all the shrimp in
star\ed groups lost weight and showed characteristic patterns of
the mean body weight lost in proportion to the length of starvation
periods. At the end of starvation, however, no significant differ-
ences in body weight were found among all the experimental
groups, except that the shrimp starved for 12 days were signifi-
cantly lighter than the controls receiving satiation feeding continu-
ously. At the end of refeeding the shrimp previously starved for 4
and 8 days (Groups S4 and S8) were slightly heavier than those fed
on full ration throughout, while the most severely starved shrimp
were slightly lighter than the controls.

During the course of refeeding the dynamics of SGR„ for all
the treatment groups exhibited a similar pattern: the SGR^^ tended
to decreased with the time of experiment (Fig. 1). There were
found to be no significant differences in SGR„ among all groups.
either in each 8-day interval of the refeeding period or during the
whole course of recovery feeding (Fig. 1; Table 2). By contrast,
during the refeeding period, SGRj, SGRp, and SGR^. appear to
depend on the length of previous starvation periods. It is shown in
Table 2 that during the course of refeeding, the SGRj, SGR^, and
SGR^, did not differ significantly between Group S4 and the con-
trol group, but all of these growth rate indexes of Groups S8 and
SI 2 were significantly higher than those of the control.

FI

Changes in feed intake in terms of wet weight (FI^J for all
groups during the refeeding are presented in Figure 2. Compared
with the control shrimp, those previously starved shrimp displayed
a hyperphagic response to a switch from starvation to satiation
feeding. Data in Figure 2 also demonstrate that in the first 8-day
interval of refeeding. there were significant differences in FI,^
between the starved groups and the control, showing a tendency
that the feeding intensities were positively in proportion to the

0.00

0-8

8-16

16-24

24-32

Time of refeeding (days)

Figure 2. Changes in FI„ of Penaeiis chinensis during the period of
refeeding. Means with different letters within each interval are signifi-
cantly different iP < 0.05) and bars Indicate standard errors of the
means.

duration of previous food deprivation. However, the appetite of all
the starved shrimp dropped rapidly to the level of the controls
during the second 8-day of refeeding. Such a case lasted to the end
of the experiment, although the most severely starved shrimp
showed a slightly higher FI„ than those of the other three groups.
The results on FIs are summarized in Table 2. It is shown that
during the course of refeeding, the FIs of Group SI2 were signifi-
cantly higher than those of the other three groups, while the FIs of
the other two starved groups were slightly higher than those of the
control group.

FCE

Upon realimentation all the previously starved shrimp exhib-
ited a lower FCE^^, than that of the controls, with a trend showing
that the FCE^ decreased with the duration of the starvation periods
(Fig. 3). Different dynamics and extents of restoration in FCE„ of
the starved shrimp during the refeeding period are also shown in
Figure 3. Within the second 8-day period of refeeding, the shrimp

6.00

5.00

■a

4.00
3.00

o

C/3

2.00
1.00

0.00

C HS4 nS8 11SI2

0-8

■16 16-24 24-32

C HS4 nS8 11S12

0-8

8-16

16-24

24-32

Time of refeeding (days)

Figure 1. Changes in SGR„ of Penaeus chinensis during the period of
refeeding. Means with different letters within each interval are signifi-
cantly different (f < 0.05) and bars Indicate standard errors of the
means.

Time of refeeding (days)

Figure 3. Changes in FCE„ of Penaeus chinensis during the period of
refeeding. Means with different letters within each interval are signifi-
cantly different iP < 0.05) and bars indicate standard errors of the

720

WU ET AL.

TABLE 2.
The specific growth rate, feed intalie, and food conversion efficiency in Penaeus chinensis during the course of experiment (mean ± SE)'.

Groups

S4

S8

S12

C

SGR,,

SGR,

SGRp

SGR,.

FIw

Fid

FIp

Fie

FCE,

FCE,

FCE^

FCE,

3.74 :
4.26:
4.25:
4.52:
13.66:
13.99:
14.36:
15.70:

24.55 :
26.47 :
25.77 :
24.66 :

0.06

0.04'

O.IO"

0.1 3-'

0.28"

0.14"

0.30"

0.33"

0.78

0.43

0.78

0.73

3.96 :

5.15:

5.32:

5.67:

13.87:

14.04:

14.52:

15.36:

25.31 :

30.26 :

29.83 :

29.33 :

0.32

0.42"

0.32"

0.32"

0.83"

0.99"

0.90"

0.96"

2.34

3.86

2.37

2.27

3.78

±0.13

5.14

± 0.25''

5.56

±0.12"

5.70

±0.13"

16.65

± 2.30"

17.39

± 3.07"

17.86

±2.53"

19.63

± 2.78"

20.53

±3.37

24.76

±3.68

25.13

±3.89

23.25

±3.60

3.61 :

4.06:

4.05:

4.24:

12.93:

12.67:

13.37:

13.65:

25.14:

28.13:

26.65 :
26.98 :

0.35

0,28"

0.22"

0.25"

0.32"

0.29"

0.30"

0.29"

1.37

1.58

1.21

1.14

' Values with different letters in the same row are significantly different from each other (P < 0.05). SGR„. SGRj. SGRp. and SGR^.: specific growth rates

expressed in terms of wet weight, dry matter, protein, and energy content (%/day). FI„,, FIj, FI^,, and FI^.: feed intake expressed in terms of wet weight,
dry matter, protein, and energy content (%/day). FCE^^, FCEj, FCEp. and FCE^.: food conversion efficiency expressed in terms of wet weight, dry matter,
protein, and energy content (%).

previously starved for 4 days rapidly recover tlieir FCE„, to the
level of the controls, while those starved for 8 days displayed an
improved FCE^^ slightly higher than the controls. This pattern of
restoration for the two starved groups was maintained to the end of
refeeding period. In contrast, the most severely starved shrimp
(Group SI2) took a longer period (24 days) to restore the FCE„
approximate to that of the controls.

During the whole course of refeeding, FCEs did not signifi-
cantly differ among all groups, and it seemed that the shrimp
previously starved for 8 days displayed slightly improved FCEs
compared with the controls.

Chemical Composition and Energy Content

Chemical composition and energy content of the food (poly-
chaete worms) were determined to be as follows: moisture was
74.74%, lipid was 2,37%, protein was 19.36%-, ash was 1.66%. and
energy content was 5.424 kJ/g wet weight.

Data on body composition and energy content are presented in
Table 3. At the end of food deprivation water content tended to
increase, while lipid, protein, and energy content decreased with
the prolongation of the starvation periods. Ash content was not

significantly affected by starvation. At the end of refeeding period
there were no significant differences in water, protein, ash, and
energy content between the starved and control groups, except the
case that lipid content was still lower both in Group S4 and SI2
than that in the control group.

DISCUSSION

Previous studies of compensatory growth have shown the abil-
ity to elicit above normal growth rates in fish and other animals
(Wilson and Osbourn I960. Russell and Wootton 1992). The re-
sults for rainbow trout. Oiicorliyiuhiis inykiss (Walbaum) reported
by Quinton and Blake (1990) showed that the fish fed on the "3
weeks starvation and 3 weeks feeding" cycle out-performed the
control group and that it was during the last week of refeeding that
the great increase in growth associated with compensatory growth
response occun'ed. The work by Miglavs and Jobling ( I989h) on
juvenile Arctic charr. Salvi-liiiKs tilpimis (Linnaeus) indicated that
shortly after transfer from a restricted to a satiation feeding regime,
growth rates of the fish increased markedly and were significantly
higher than those of the control. In the present study the previously
food-deprived shrimp did not display significantly higher specific

TABLE 3.

I'he chemical composition and energy ccmtent in Penaeus chinensis at different times of the experiment in relation to duration of starvation

in the food deprivation period (mean ±SE)'.

Time

Groups

Water-

Protein^

Lipid-

Ash^

Energy'

At Ihc end of starvation

Al the end of recovery growth

S4

78.33 ± 0.67"

16.20 ±0.54"

1 .02 ± 0.02"

2.83 + 0.18

3.876 ±0.1.36"

S8

81.70 + 0.53"

1 2.99 ± 0.66"

0.86 ± 0.03"

2.95 ±0.1 7

3.125 ±0.1 96"

S12

82.92 ± 1.06"

1 1 .38 ± 0.75"

0.86 ±0.07'

3.12 ±0.23

2.784 ±0.205'

C

76.92 ± 0.82"

16.79 ±0.68"

1 .46 ± 0.05"

2.87 + 0.09

4.394 ±0.1 18"

,S4

74.43 ± 0.50

19.08 ±0..39

1.77 + 0.0.5"

2.79 ± 0.08

4.975 + 0.088

S8

73.15 ± 1.74

20.(M± 1.24

2.53 ±0.1 6"

2.73 ± 0.26

5.-395 ± 0.372

S12

73.60 ± 1.13

20.03 ± 0.88

1.66 ±0.08"

2.78 ±0.22

5.143 ±0.247

C

73.37 ± 0.64

19.34 ±0..55

2.55 + 0.12"

2.60 ±0.14

5.371 ±0.121

' Values with different letters in the same column are significantly ditforcnl from each mher {P < 0.05).
" Values were expressed as the percentage of wet weight.
' Values were expressed as kJ per gram of wcl weight.

Compensatory Growth Response in Penaeus chinensis

721

growth rate in terms of wet body weight (SGR^, ) than the controls
during any of the 8-day intervals of refeeding, but this cannot
preclude the fact that the compensatory growth response occurred
in Chinese shrimp. As Jobling (1994) pointed out. "within the
franicw ork of the energy balance equation, growth is defined as an
increase in the energy content of the fish body," which means that
growth is dependent on two aspects, wet body weight and body
composition of the fish. The results of the present study showed
that SGR in terms of dry matter, protein, and energy content
(SGRj, SGRp. and SGR^.) of the shrimp previously starved more
than 8 days were significantly higher than that of the control dur-
ing the course of refeeding (Table 2). This obviously indicates that
Chinese shrimp displayed compensatory growth response when
changed from starvation to satiation feeding.

Wieser et al. ( 1992) reported that the growth rate after refeed-
ing increased in proportion to the length of starvation periods in
three cyprinid species. Similar results for the European minnow.
Phoxiniis phoxiniis (Linnaeus) were observed by Russell and
Wootton (1992) and for Procamharus clarkii (Girard) by Bost-
woilh and Wolters (1995). The results of this study appear to be
consistent with the above findings in which the strength of com-
pensatory growth response depends on the length of the starvation
periods. Table 2 shows that during the course of refeeding, the
shrimp previously subjected to food deprivation for 4 days just
displayed a slight compensatory growth response, while those pre-
viously starved for more than 8 days showed noticeable compen-
satory growth responses.

In agreement with previous studies performed on fish and do-
mestic animals (Wilson and Osboum 1960, Russell and Wootton
1992, Hayward et al. 1997), Chinese shrimp also responded to a
switch from food deprivation to satiation feeding by exhibiting
hyperphagia. The extent of the hyperphagic response generally
depends on two variables, namely the feeding intensity and the
duration of appetite elevation. There are two patterns of appetite
dynamics following various periods of food deprivation: The du-
ration of hyperphagia is similar for the animal starved for different
periods, but the feeding intensity increases in proportion to the
length of starvation periods (Russell and Wootton 1992), and the
initial appetite is similar, but the duration of hyperphagia is vari-
able (Russell and Wootton 1993, Bull and Metcalfe 1997). The
results of the current experiment indicated that the feeding inten-
sity increased with the length of starvation periods, but the hyper-
phagic response in all starved groups just occurred within the first
8-day interval of refeeding (Fig. 2), which appear to provide an
evidence for the first pattern of appetite dynamic. The above find-
ings implied that the patterns of appetite dynamics seem to be
dependent on animal species.

Improved food conversion efficiency is also an aspect fre-
quently highlighted in studies of compensatory growth and may be
of practical implications in the production of fish and domestic
animals (Wilson and Osboum 1960; Dobson and Holmes 1984.
Quinton and Blake 1990). The evidence for the improved food
conversion efficiency may be caused by the reduced basal metabo-
lism occurred during starvation extending into the initial stage of
realimentation (Boyle et al. 1981. Yambayamba et al. 1996) or by

the differences in the composition of weight gain between animals
displaying compensatory growth and tho.se fed continuously, the
former often tending to deposit fewer proportions of the gain as
body fat (Yu et al. 1990. Jobling et al. 1994). In the present study
there were no significant differences in food conversion efficien-
cies between the shrimp previously starved for various periods and
the controls during the course of refeeding, with the exception of
the case that Group S8 showed slightly higher food conversion
efficiencies in terms of dry matter, protein, and energy content
(FCEj, FCEp. and FCE^.) than those of the control group (Table 2).
Furthermore, Figure 3 clearly depicts that during the first 8 days of
refeeding the previously starved shrimp displayed lower food con-
version efficiency in terms of wet weight (FCE„) than the controls,
and that the longer the shrimp remained without feeding, the lower
the FCE„ was. The results of this study based on the analyzing of
the changes in body composition and the FCEj, FCEp, and FCE^,
during the course of refeeding seem to provide an evidence that the
lower FCE„ may be attributable to the changes in body water
content. In other words, if the same amount of dry matter, protein,
or energy content was synthesized, the starved shrimp will rela-
tively absorb less amount of water than the controls during the first
8 days of refeeding.

In fish (Miglavs and Jobling 1989a, Wang et al. 1999) and
other crustaceans (Barclay et al. 1983. Stuck et al. 1996). starva-
tion generally leads to an increase in water content and to reduc-
tions in lipid, protein, and energy content. A similar pattern was
observed in the current study. There was a trend for the shrimp that
the extent of increase in water content and decreases in lipid,
protein, and energy content depends on the length of starvation
periods (Table 3). There were different conclusions drawn from
the previous studies on the order of utilization of major energy
reserves in crustaceans (reviewed by Whyte et al. 1986). In the
present study the results calculated from the data of Tables 1 and
2 show that protein contributed 24.84% and lipid contributed
31.68% of total metabolized energy during 4 days of starvation,
while in 12 days of starvation, protein contributed 62.49% and
lipid contributed 14.45%, which was in agreement with the sug-
gestion by Cuzon et al. (1980) and Barclay et al. (1983) that
protein was the major source of energy used during prolonged
starvation. After 32 days of refeeding there were no significant
differences in water, protein, and energy content between the star-
vation-satiation shrimp and the controls, except that lipid content
in the shrimp previously subjected to starvation for 4 or 12 days
was still significantly lower than that of the controls (no obvious
explanation on this case is available). This indicates that Chinese
shrimp have the ability to withstand and recover from relatively
prolonged starvation.

ACKNOWLEDGMENTS

This work was supported by funds from the Chinese National
Science Foundation for Talent Youths (grant no. 39725023) and
the Project under the Major State Basic Research of China (grant
no. G 1 9990 1201 1 ). We thank Fengcheng Shrimp Farm, Qingdao,
People's Republic of China for providing the shrimp used in this
experiment.

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Joiirihil of Shvllfish Ren-anh. Vol. 19. No. 2, 723-729, 2000.

THE USE OF POULTRY MORTALITIES AS AN ALTERNATIVE BAIT FOR THE HARVESTING
OF BLUE CRABS CALLINECTES SAPIDUS (RATHBUN, 1885)

TEENA F. MIDDLETON,' PETER R. FERKET,'

HARRY V. DANIELS,- LEON C. BOYD,'

LARRY F. STIKELEATHER,^ AND ROBERT J. MINES''

^Department of Poultry Science,

North Carolina State University,

Raleigh. North Carolina 27695
"Department of Zoology,

North Carolina State University.

Plymouth, North Carolina 27962

Department of Food Science.

North Carolina State University,

Raleigh, North Carolina 27695
^Dept. of Bio. and Ag. Eng..

North Carolina State University,

Raleigh, NC 27695
^North Carolina Sea Grant,

Morehead City. North Carolina 28557

ABSTRACT Alternative bait products for the harvesting of blue crabs are needed because traditional baits are becoming increasingly
expensive and more difficult to acquire. Poultry mortality carcasses and poultry mortality silage were compared to menhaden for their
ability to attract blue crabs using on-shore attractanl trays as well as off-shore crabpot protocols. When product stability in water was
eliminated as a variable in a preliminary investigation, there was no significant difference (P > .10) in the total number of blue crabs
harvested using the alternative poultry silage baits versus a menhaden bait control. Attractant tray evaluations indicated an increased
preference {P < .035) of female crabs versus male crabs for the alternative poultry baits relative to a menhaden control but failed to
demonstrate any significant differences (/*> .152) in the preferences of blue crabs for the pH. binder, betaine addition, or poultry meat
form used in the alternative bait formulations. In contrast, a marine evaluation of the alternative bait treatments indicated an increased
preference {P < .005) for poultry bait products containing no supplemental betaine and a decreased preference iP < .045) for poultry
baits adjusted to a pH = 8. A consumer sensory evaluation of the crabmeat harvested from crabs with the alternative baits demonstrated
that no off flavors are associated with the use of the alternative poultry baits. Properly formulated, poultry mortality could be utilized
as alternative bait for the harvesting of blue crabs and has potential for use as bait for other aquatic species.

KEY WORDS: blue crab, poultry mortality, alternative bait products

INTRODUCTION In an effort to develop alternative bait products to meet the
The blue crab fishery is the largest commercial fishing industry demands of this industry, many researchers have investigated com-
in the United States, with average landings exceeding 95,0{X) met- pounds that elicit positive responses in the chemoreceptor organs
ric tons per year. (United States Department of Commerce 1995). of various aquatic crustaceans (Laverack 1963, Levandowsky and
Dockside values for the blue crab fishery are second only to the Hodgeson 1965, Lenhoff and Lindstedt 1974, Zimmer-Faust
snow crab, with 1994 dockside values exceeding $137 million 1987, Rittschof 1992). Since 1897, it has been known that
(USDC 1995). chemoreception plays an important role in the food-.seeking be-
Harvesting of blue crabs occurs mainly by trapping in baited havior of crabs (Bethe 1 897). Detection occurs from a distance, so
wire cages known as crabpots. Individual blue crab fishermen it is evident that a highly soluble chemical is carried by water
generally work some 200-300 crabpots set across inland bays, currents and is detected by the Crustacea (Laverack 1963). Sub-
rivers, and estuaries. Millions of pounds of fish by-catch, river stances commonly present in crustacean foods that might readily
herring, and menhaden are used annually to harvest the blue crabs. leach out of damaged tissues are trimethyl amine oxide in tlsh and
Unfortunately, these traditional bait products are becoming in- betaine in invertebrates (Laverack 1963). Unfortunately, previous
creasingly expensive and more difficult to acquire. Mandatory use efforts to identify compounds as alternatives to traditional baits
of by-catch reduction devices, recent net bans, and an increased have met with limited success. The attractant qualities of baits
demand for menhaden for fish meal production have increased the cannot be totally accounted for by any one major component ex-
prices for traditional baits and forced watermen to use less- tracted from natural baits (Shelton and Mackie 1971). There is a
effective products as alternative baits (e.g., shrimp heads, gar, and need for complex mixtures of compounds in specific concentration
gizzard shad). The overall cost of bait for the harvesting of blue ratios to mimic the stimulatory effect of whole tissue (Mackie
crabs has risen more than 300'^ in the previous 5 y (Johnson et al. 1982).

1996). Continuing prosperity of this major coastal business re- Poultry mortality silage is a biosecure. semi-solid product re-
quires that a plentiful, cost-competitive bait supply be available. suiting from lactic acid fermentation of ground poultry carcasses

723

724

MiDDLETON ET AL.

(Murphy and Silbert 1990. Cai and Sander 1995). While the acidic
silage product was not accepted by the blue crabs, preliminary test
results indicated that neutralized silage products were readily de-
tected in the aquatic environment and rapidly consumed by this
species. Therefore, once pH adjusted, this material does appear to
contain the stimulatory components in the proper ratios necessary
to attract the crabs to the bait (Middleton and Hines. unpubl. data).
The objective of this research was to evaluate the effectiveness of
poultry mortality silage as alternative bait for the harvesting of
blue crabs. The u.se of this material as a bait could reduce the
demands placed on our rivers and estuaries by the mass harvesting
of bait fish and provide an economical, environmentally friendly
alternative bait source for the crabbing industry. In addition, a
major recycling/reutilization outlet for poultry mortality will have
been developed.

MATERIALS AND METHODS

Bait Manufacture
Preliminary Evaluation

A preliminary evaluation was conducted to evaluate poultry
mortality silage as a potential alternative bait for the harvesting of
blue crabs. Turkey mortality was deplumed, ground, and stabilized
by lactic acid fermentation for 4 wk to produce a silage (Murphy
and Silbert 1990, Blake et al 1992, Cai and Sander 1995). Bait
quality menhaden was obtained locally (Craven Crab Company,
New Bern. NC 28560). Immediately prior to bait manufacture, the
poultry silage was neutralized using solid sodium hydroxide
(Fisher Scientific, Fairlawn, NJ 07410). Anhydrous betaine (Be-
tafin BT®, Finnsugar Bioproducts, Naantali, Finland) was added
at 150 mg/lOO g silage material to one half of the neutralized
poultry silage. To eliminate variation due to structural stability, all
bait products were prepared by grinding in a commercial meat
grinder (Model A-200, Hobart Mfg. Co., Troy, OH 45373) and
gelled using a sodium alginate binder (Keltone HV®, Nutrasweet
Keico Co.. Chicago. IL 60661) according to manufacturer's rec-
ommendations. Calcium sulfate hemihydrate (Plaster of Paris) was
used as the source of calcium ions (Fisher Scientific. Fairlawn. NJ
07410). Bait products were pressed into 1.5-inch collagen sausage
casings (Coria®. Devro Teepak®. Summerville, SC 29483) and
refrigerated at 5 °C for 12 h to allow for solidification of the bait
material. Baits were then individually packaged in polyethylene
storage bags (Ziplock Freezer Bags. Dowbrand. L. P.. Indianapo-
lis. IN 46268) and frozen at -20 °C until used.

Attraction Tray Trial I

A 2 X 4 X 4 factorial design was used to evaluate the different
experimental bait treatments that included: two poultry meat forms
(fresh frozen versus fermented): four pH levels (5.5. 7.5. 9.5. and
1 1.5); and four binding agents (Keltone HV®. Mannugel®. wheat
gluten with soy protein, and Gelcarin MH91 1 1 ). Poultry mortality
silage, prepared by lactic acid fermentation (Murphy and Silbert
1990. Blake et al. 1992. Cai and Sander 1995). and frozen ground
poultry mortality, both from Ross 308 43-day-old male broilers
(Ross Breeders. Inc. Huntsville, AL 35805) were obtained as raw
materials for poultry bail manufacture. Aliquols of the raw mate-
rials were adjusted with solid sodium hydroxide (Fisher Scientific.
Fairlawn. NJ 07410) to a pH of 5.5, 7.5, 9.5. or 1 1 .5. Each of these
eight treatments were then formed according to supplier's recom-
mendations into bait products using either Keltone HV® (Nu-
trasweet Kelco Co, Chicago. IL 60661 ). Mannugel® (Nutrasweet

KeIco Co. Chicago, IL 60661 ), Gelcarin ME 91 1 1 (EMC Corp.,
Philadelphia. PA 19103). or 3% wheat gluten and 3% soy protein
(Vital Wheat Gluten. Midwest Grain Products. Inc., Atchison, KS
66002 and Promine DS, Central Soya Company, Inc., Fort Wayne,
IN 46802) as binding agents. Encapsulated calcium lactate pen-
tahydrate (Cap-shure®. Balchem Corp.. Slate Hill. NY 10973) was
used as a source of calcium ions to cold set the sodium alginate
binders (Keltone HV® and Mannugel®). The latter two binding
systems were heat set in 140 °C ovens to an internal temperature
of 85 °C. Molds were utilized to manufacture 100-g replicates of
each bait treatment. Replicates were refrigerated at 5 °C for 12 h
to allow for solidification of the bait products. One hundred-gram
fillets were prepared for use as control baits from locally obtained
bait quality menhaden (Craven Crab Co.. New Bern. NC 28560).
Baits were then individually packaged in polyethylene storage
bags (Fisher Scientific. Fairlawn. NJ 07410) and frozen at -20 °C
until utilized.

Attraction Tray Trial 2

A 2 X 2 X 4 factorial design was used to evaluate the different
experimental bait treatments that included: two poultry meat forms
(fresh frozen versus fermented); the presence or absence of betaine
in the bait formulation; and four pH levels (6. 7. 8. and 9). Raw
materials for bait manufacture were mortality silages prepared by
lactic acid fermentation (Murphy and Silbert 1990. Blake et al.
1992. Cai and Sander 1995) or frozen ground poultry mortality.
both from 25-week-old Arbor Acres Yield male broiler breeders
(Arbor Acres Inc.. Gla.stonbury. CT 06033). Anhydrous Betaine
(Sigma Chemical Co, St. Louis MO 63178) was added at 250
mg/lOOg silage (250 mg%) to one-half of the fresh as well as the
silage material. Aliquots of each of the tour treatments were pH
adjusted with solid sodiuin hydroxide (Fisher Scientific. Fairlawn,
NJ 07410) to a pH of 6. 7, 8. or 9. Cheesecloth squares were used
to contain lOO-g replicates of each bait treatment. One hundred-
gram fillets were prepared for use as control baits from locally
harvested bait quality menhaden. Baits were then indi\idually
packaged in polyethylene storage bags (Fisher Scientific. Fair-
lawn. NJ 07410) and fro/en at -20 "C until utilized.

Ocean Evaluation

Poultry bait treatments were prepared from raw materials as
used in the second attraction tray study and gelled into bait prod-
ucts using Gelcarin ME 9111 (EMC Corp.. Philadelphia. PA
19103). To increase structural stability of the poultry baits, treat-
ments were pressed into 1 .5-inch diameter clear fibrous sausage
casings with three 0.44 mm diameter holes per square inch (Vista
International Packaging. Inc. Kenosha. WI 53141 ) to produce sau-
sage shaped bait products averaging 350 g. Poultry bait sausage
products were heated in a 140 °C oven to an internal temperature
of 85 °C and then refrigerated at 5 °C for 12 h to allow for
solidification. Bait quality menhaden fish were harvested locally.
Baits were individually packaged into polyethylene storage bags
(Ziplock Freezer Bags. Dowbrands L. P.. Indianap<ilis. IN 46268)
and frozen at -20 °C until used.

Consumer Sensory Panel Evaluation

Mortality silage prepared by lactic acid fermentation (Murphy
and Silbert 1990, Blake et al. 1992. Cai and Sander 1995) and
frozen ground poultry mortality, both from 25-wk-old Arbor Acres
Yield male broiler breeders (Arbor Acres Inc.. Glastonbury. CT
06033) were obtained as raw materials for bait manufacture. The
poultry products were adjusted to pH = 7.5 using solid sodium

Alternative Poultry-Based Baits for Blue Crabs

725

hydroxide (Fisher Scientific. Fairlawn. NJ 07410) and gelled into
400-g bait products using Gelcarin ME 9111 (FMC Corporation.
Philadelphia. PA 19103). Bait products were heated in a 140 °C
oven in glass beakers used as molds to an internal temperature of
85 '^C and allowed to set under refrigeration for 12 h at 5 °C. Bait
quality menhaden were harvested locally. Individual baits were
stored in polyethylene storage bags (Fisher Scientific. Fairlawn.
NJ 07410) and frozen at -20 °C until used.

Experimental Design

Preliminary Evaluation

The effectiveness of each silage bait product (as measured by
the number of crabs harvested/pot/day) was compared to that of
the bait product manufactured from menhaden fish. Empty pots
were used as negative controls. Fifteen four-funnel crabpols were
baited and harvested daily for 11 days (September 24 through
October 4. 1997) in Pettiford Creek, located in Carteret County
North Carolina. Baits and negative controls were randomly as-
signed and rotated every 3 days. The number of blue crabs/pot as
well as sex determination information was recorded for each day's
harvest.

Attraction Tray Evaluations

Attraction trays were constructed similar to the mazes devel-
oped by Shelton and Mackie (1971) to study the feeding prefer-
ences of the shore crab [Carcinus maenas (L.)], but appropriately

scaled for the larger size and greater motility of the blue crab (Fig.
I ). The trays consisted of three chambers, a large main chamber
and two smaller test chambers that open into the main chamber by
entrances with small ramps. These ramps permitted the crabs to
enter the test chambers if attracted by the bait product, but re-
stricted them from leaving. Entrances to the test chambers were
equipped with sliding perforated partitions that restricted crab en-
try to the test chambers until permitted to do so by the experi-
menter. Each lest chamber contained a small perforated bait com-
partment through which incoming water flowed from a common
tank. The outlet flow from the trays was through an overflow pipe
set in the main chamber to a height of four inches. In contrast to
the multiple crab testing protocol of Shelton and Mackie (1971).
aggressive behavioral characteristics of the blue crabs required that
the responses to the various baits be evaluated using only one blue
crab per trial. Therefore, the protocol proposed by Shelton and
Mackie (1971) was modified to compare test bait products to men-
haden tlsh fillets rather than to seawater as the control. The attrac-
tiveness of the various bait preparations relative to the fish fillets
was evaluated by chi-squared protocols to reflect a positive-
negative outcome of the individual crabs to the test bait products
or the fish fillet controls.

Husbandry practices similar to those employed in crab shed-
ding operations were used during testing protocols. Male and fe-
male crabs were separated and held for a minimum of 3 days prior
to testing to allow acclimation to captivity and to decrease re-

Figure 1. Attraction tray design. M.C., Main Chamber: T.C., Test Chambers; B.C. Bait Compartment; O.P., Overflow Pipe; R., Ramp at
entrance to test chamber: W.L., Water Level. Design modified from experimental maze of Shelton and Mackie, 1971. (Drawing courtesy
HammondAaughan, Inc. Cad Designs, Garner, NC 27529).

726

MiDDLETON ET AL.

sponse times to bait. Any crabs exhibiting signs of molting were
removed from the holding tanks. Water temperatures of between
23.9 and 29.4 °C with dissolved oxygen levels of greater than 4 mg
Oj/L were maintained in the holding tanks as well as the water
flowing through the attraction trays. Flow rates were kept constant
and equal in each test chamber of the attraction tray and water
levels were greater than 4 inches at all times.

Individual crabs were placed in the main chamber of the baited
attraction trays near the outflow pipe and allowed to acclimate
until escape behaviors moderated. When acclimation was com-
plete, the partitions between the large main chamber and the
smaller test chambers were removed in a manner that did not
disturb the subject crab. Crabs could then move, depending on crab
preferences, into the chamber containing the test or control bait
(fish fillet). Test and control baits were utilized for five replicate
trials with good stimulatory responses. Response was recorded and
subsequent trials initiated. Test and control baits were randomly
assigned to the test chambers and chambers were thoroughly
cleaned and flushed with fresh water prior to the initiation of
subsequent trials.

Trial One

A2x2x4x4 factorial design was employed to evaluate the
various bait formulations. Male and female crabs were each used
to evaluate the preferences of blue crabs for baits manufactured
using the two poultry meat forms, the four different pH levels, and
the four different binding agents. Two replicate sets of five male
and five female crabs were used to evaluate each of the bait for-
mulations. The probability of choice relative to 100-g menhaden
fillets (Craven Crab Company, New Bern, NC 28560) was deter-
mined for each product.

Trial Two

A2x2x2x4 factorial design was used to further evaluate the
various alternative bait formulation possibilities. Male and female
crabs were each used to evaluate the preferences of blue crabs for
baits manufactured using the two poultry meat forms, the presence
or absence of betaine, and the four pH levels. Three replicate sets
of five crabs each were used for each bait formulation for both
male and female crabs. The probability of choice relative to locally
harvested menhaden fillets was calculated as in Trial One.

Ocean Evaluation

Each of the poultry bait products, as well as the menhaden fish
control utilized during the second attraction tray trial, was evalu-
ated for its ability to attract blue crabs in their natural environment
using traditional four-funnel crab pots. Two replicate crabpots of
each bail treatment or treatment combination were set for six 48-h
harvesting periods (November 7 through November 19 19981 in
Pettiford Creek, Carteret County NC. The number of male and
female crabs harvested using each bait product per pot per day was
recorded.

Consumer Sensor}' Panel Evaluation

Female blue crabs were held for a period of ."i days in traditional
crab shedding tables and allowed access to either a control bait of
locally harvested menhaden, or an alternative bait product manu-
factured from either freshly frozen poultry mortality or poultry
mortality slabilized by lactic acid lermentalion. Crabs were then
cooked and cleaned under commercial conditions (Luther Lewis
and Son Crab Co., Davis, NC 28524) and lump and backfin crab-

meat was combined and stored in polyethylene storage bags (Zip-
lock Freezer Bags, Dowbrands L. P., Indianapolis, IN 46268) for
14 h at 5 °C pending consumer sensory panel evaluation.

Immediately prior to the initiation of the sensory panel evalu-
ation. 12-g portions of lump and backfin crabmeat were placed in
preheated glass baby food jars set in preheated sand, and main-
tained in a 375 °F convection oven for 1 5 min. The consumer panel
consisted of 31 individuals chosen from the Departments of Food
Science and Poultry Science, North Carolina State University, Ra-
leigh, NC. Panelists evaluated the degree of differences in aroma,
flavor and texture between crabmeat from crabs consuming the
menhaden control and crabmeat from crabs that consumed the
alternate bait treatments using a seven-point scale. Samples were
presented simultaneously in a balanced, random order from each
bait treatment group with a blind control included as one of the
samples. Panelists worked in individual booths equipped with red
lights and no discussion took place during the evaluation. Ran-
domization of the order of presentation was used to control for
contrast and carry-over effects of the various samples (Meilgaard
et al. 1991).

Statistical Analysis

All data obtained during the taste panel as well as the prelimi-
nary and ocean evaluations were tested for significance using the
General Linear Model (GLM) procedures of SAS (SAS Institute,
1996). Treatment effects were considered significant at P < .05.
The LSMeans procedure of SAS was used to determine significant
differences among treatments. Attraction tray data were tested for
significance by chi-squared analysis using the GENMOD proce-
dures of SAS (SAS Institute, 1996). Probability of choice was
calculated using the formula: X = In (P/1 - P). where X =
Genmod probit value estimate (Genmod device for the unrestricted
calculation of probabilities) and P = probability of choice. Dun-
nett's T means procedures of SAS were also employed for the taste
panel data to compare each treatment mean to the control.

RESULTS AND DISCUSSION

Preliminary Evaluation

Highly significant {P < .001 ) bait effects were observed in the
harvest values (Table 1 ). The crabs were significantly more at-
tracted to all bait products than to the negative controls (P < .04).
Similar preferences {P > .05) were demonstrated by the female
crabs for the poultry silage baits in comparison to the menhaden
fish bait. However, they were significantly less attracted (P< .035)
to the poultry silage bait when betaine was included in the bait
formulation. Male crabs demonstrated a significant preference {P
< .034) for the menhaden bait over that of either poultry silage
preparation. However, when total blue crab harvest values were
analyzed, there were no significant dilferences {P > 0.10) in the
total number of blue crabs harvested using the poultry silage bait
versus the menhaden bait. The addition of betaine to the poultry
silage bail did not significantly (P > 0.56) affect total harvest
values when compared to the poultry silage alone. The increased
number of male crabs harvested during this trial is presumably
indicative of the population dynamics of the area surveyed during
the e\aliiation and is not likely a reflection of bait treatments.

.{(traction Tray Trial I

There was a significantly (P < .035) greater probability of
preference for the poultry bait relative to fish demonstrated by
female crabs than bv male crabs; in agreement with the results of

Alternative Poultry-Based Baits for Blue Crabs

727

TABLE 1.
Average daily blue crab harvest": preliminary evaluation.

Male

Female

Total

Treatment

Crabs

Crabs

Crabs

Negative control (empty pot)

1.333'

.1212'

1.454'

Menhaden bait

3.659'

.6818'-

4.341'

Poultry silage (PS) bait

2.727-

.909'

3.636'-

PS + betaine bait

2.864-

.5227-

3.386-

Statistic

s (pooled)

Treatment effects {P values)

.001

.001

.001

SEM"

.303

.148

.349

' ' Means within columns with no common superscript differ significantly

(P< .05).

" Average number of blue crabs harvested/potyday.

"^ SEM = Standard Error of the Mean with 121 degrees of freedom.

the preliminary evaluation (Table 2). No significant differences
were found in the probability of choice for pH. binder, or meat
form used in the bait treatments {P> A 80). No significant two-way
factor interactions were demonstrated [P > .0751). Significant
three-way interactions were demonstrated for pH*binder*meat and
pH*meat*se.x (P < .010 and P < .039. respectively); however,
nothing meaningful was discerned by the evaluation of these in-
teraction patterns. The poultry bait was chosen consistently less
often than the fish controls by both sexes of crab throughout the
experiment. The probability of choice of poultry bait product ver-
sus menhaden fillet was s 48.65%.

Attraction Tray Trial 2

No significant differences (/■ > .152) or interactions (P > .085)
in any of the parameters were demonstrated relative to sex, pH.

TABLE 2.

Probability of choice by blue crabs of poultry bait formulations
relative to menhaden in attraction tray trial".

Factor Category

Factor

Probability of
Choice"'^^

P Values'

meat form, or the addition of betaine (Table 3). The poultry baits
were chosen consistently more often than the fish fillet controls
throughout the course of this experiment. The probability of choice
of poultry bait product versus menhaden fish fillet was <66.72%.

Ocean Evaluation

Difficulties were encountered in this trial due to the casings
utilized to form set the bait products. Although the casings were
manufactured with three 0.44 mm pinpoint openings per square
inch to allow flavor cotnpounds from the mortality silage contents
to readily disperse into the seawater. these openings apparently
were sealed during the cooling process and impeded flavor release
during the ocean evaluation. In order to attract blue crabs, the
casings of the alternative bait products were slit repeatedly to
facilitate seawater contact with internal contents for flavor com-
pound release. Therefore, the available attractant surface area in
the alternative bait products was limited to areas exposed by cut-
ting the casings. While the attractant surface areas among the
alternative bait products were comparable (and therefore compari-
sons between the attractant qualities of alternative products would
be valid), the attractant surface area of the alteiTiative bait products
relative to the traditional menhaden fish bait was compromised.
Physical stability of the alternative bait products in water was
demonstrated to be <5 days; therefore, casing materials on poultry
bait products were unnecessary, resulted in reduced harvest yields,
and are to be avoided in future trials.

The number of blue crabs harvested from the ocean using tra-
ditional menhaden fish as bait was significantly greater (P < .001)
than the number of crabs harvested using either of the alternative
poultry bait products (Table 4). There was no significant difference
(P > .381 ) in the average number of blue crabs harvested between
the fresh and fermented alternative bait products. A significantly
greater (P < .001 ) number of feinale crabs were attracted to the
poultry bait than were male crabs (Table 5). However, a signifi-
cantly larger (P < .003) number of female crabs were also attracted

TABLE 3.

Probability of choice by blue crabs of poultry bait formulations
relative to menhaden in attraction trav trial 2".

Sex

Male
Female
Keltone HV
Mannu2el

40. 1 8%-
48.65%'
51.84%
40.88%

.0346
.1807

Binder

Factor Category

Factor

Probability of
Choice""

P Value"

SovAVheat

42.60%

Sex

Male

66.72%

.7737

Gelcann

48.65%

Female

67.93%

Meat

Fresh

51.15%

.5402

pH

6

58.60%

.1995

Fermented

48.65%

7

55.90%

pH

5.5

42.78%

.1982

8

57.59%

7.5

53.75%

9

67.93%

9.5

43.86%

Meat

Fresh

61.60%

.1525

11.15

48.65%

Betaine

Femented

No

Yes

67.93%
67.09%
67.93%

' ' Means within

nif;,.Qnt1v IP ^ c

factor categories with

no common superscript

differ sig-

.8449

"Probability of choice of treatment main effects. No significant or mean-
ingful two-way or three-way treatment interactions demonstrated.
"" Probability of choice of poultry bait formulation relative to menhaden
fish fillets.

^ Probability of choice (P) was calculated using the formula: X = ln(^/l
- P). whereas X = Gemmod probit value estimate.
"" P values were determined by x"-squared analysis using the Genmod
Procedures of SAS (SAS Institute. 1996).

■' Probability of choice of treatment main effects. No significant (p < .085)

two-way or three-way treatment interactions demonstrated.

'" Probability of choice of poultry bait formulation relative to menhaden

fish fillets.

^ Probabillity of choice (p) was calculated using the formula: X = IntP/l

- P). where X = Genmod probit value estimate.

'' P values were determined by x'-squared analysis using the Genmod

Procedures of SAS (SAS Institute. 1996).

728

MiDDLETON ET AL.

TABLE 4.
Average blue crab harvest in ocean evaluation"

TABLE 5.
Effect of crab sex on combined barvest: ocean evaluation"

Bait Product

Male
Crabs

Female
Crabs

Total
Crabs

Category of Crab

Poultry Bait

Fish Bait

Fermented poultry bait 1.187'

2.292

Fresh poultry bait 1.375'

2.365

Fish bait 3.979"

8.104

Statistics (pooled)

Treatment effects iP values) .001

.001

SEM" .416

.625

3.479-

3.739-

12.083'

.001
.490

- Means within columns with no common superscript differ significantly
iP < .05).

" Average number of blue crabs harvested/pot/day.
"^ SEM = Standard Error of the Mean with 237 degrees of freedom.

to the fish bait products. Therefore, the sex preferences demon-
strated in the previous trials cannot be confirmed by these data due
to the larger population of female crabs in the survey area.

When alternative poultry bait formulations were compared, sig-
nificant differences were demonstrated in the preferences of blue
crabs for specific pHs of bait products as well as the inclusion or
exclusion of betaine in the formulation. While no linear regression
trend was established for pH (P > .05). significant differences {P
< .045) were demonstrated in the total number of blue crabs har-
vested with the various pH formulations, with a reduced preference
indicated for baits of pH = 8 (Table 6). In addition, baits that did
not contain betaine were significantly (P < .005) more attractive to
the crabs than were bait products containing the betaine supple-
mentation (Table 7). No significant differences {P > .090) were
demonstrated in either of these parameters when the blue crab
harvest was analyzed by sex (data not shown). No two-way or
three-way treatment interactions were demonstrated among the
bait treatment formulations for male, female or total crabs har-
vested (P > .174).

This research failed to demonstrate that attractant trays are
effective in predicting harvest value tendencies for blue crabs in
their natural environment. Ocean testing is capable of detecting
significant bait formulation preferences of blue crabs that were not
differentiated {P > .05) using the attractant tray protocol (Table 8).
Modifications of the attractant tray design sample size alterations,
and/or additional replicates of each treatment evaluated might im-
prove the predictive nature of this alternative protocol. Additional
research with corresponding ocean testing is required.

Coiisumir Sensory Panel Evaluation

No significant differences {P > .242) were demonstrated in the
aroma, flavor, or texture of lump and backfin crabmcal harvested
from crabs consuming the various bail products (Table 9). When
direct onc-on-one comparisons were made (Dunnetl's T means
procedures of .SA.S). no significant differences (P > .05) were
demonstrated in these parameters between crabmcat harvested
from crabs consuming either of the alternative bait products and
those consuming traditional menhaden fish (data not shown).

CONCLUSION

Poultry mortality silage has demonstrated potential as alterna-
tive bait for the harvesting of blue crabs. When given a choice
between fish and a poultry bail product (in the attractant tray).

Male
Female

1.281-
2.329'

Statistics (pooled)

Treatment effects (P values)
SEM"

.001
.179

3.979"
8.104'

.003
.949

' - Means within columns with no common superscript differ significantly

{P < .05).

■■ Average number of blue crabs harvested/pot/day.

"^ SEM = Standard Error of the Mean with 382 and 94 degrees of freedom,

respectively.

TABLE 6.
Effect of pH of poultry bait: ocean evaluation".

pH

Male Crabs

Female Crabs

Total Crabs

6

1.479

2.500

3.979'

7

1.312

2.562

3.875'

8

1.667

1.729

2.896-

9

1.667

2.521

3.687'-

Statistics (pooled)

pH Effects (P values)

.8359

.3883

.045

SEM''

.278

.398

.297

- Means within columns with no common superscript differ significantly
(/>< .05).

" Average number of blue crabs harvested/pot/day.
'^ SEM = Standard Error of the Mean with 181 degrees of freedom.

TABLE 7.
Effect of betaine: ocean evaluation".

Factor

Male Crabs Female Crabs

lotal Crabs

Betaine

1.198 1.989

3.187-

No betaine

1..364 2.667
Statistics (pooled)

4.031'

Betaine effect (P

values)

.550 .090

.005

SEM'^

.197 .281

.210

' - Means within foUnims u iih no Lonimon superscript differ significantly

(/• < .051.

" Average number of blue crabs harvested/pot/day.

''SEM = Standard Error of the Mean with 181 degrees of freedom,

TABLE S.

Comparison of protocol's ability to detect slgnillcant treatment
differences: attraction Irav trial 2 and ocean evaluation.

Factor

Attraction Tray Trial 2

Ocean Evaluation

Sex
pH

Meat
Betaine

/' Values

.7735

.001*

.1995

.045*

.1525

.38!

.8449

.005*

Indicates significant differences detected in the parunieler.

Alternative Poultry-Based Baits for Blue Crabs

729

Bait Treatment

TABLE 9.
Effect of baits on the organoleptic indices of crabmeaf'''.

Perceived Mean Difference
from Control'

Perceived Mean Difference
from Blind Contror"

Menhaden (blind control)
Fresh poultry
Fermented poultry

Bail trealmeni effeet [P value)
SEM"

Minimum Signifieant Difference from
Control Required for Dunnett's T

Aroma

Texture

Flavor

Aroma

.753

.841

.815

.8.39

Texture

.767

Flavor

2.387

2.710

2.677

.000

.000

.000

2.548

2.226

2.129

.161

-.484

-.548

2.22fi

2.387
Statistics (pooled)

2.645

-.161

-.323

-.032

.630

.423

.242

.689

.357

222

.237

.264

.256

.264

.241

.249

.791

' Lump and backfin crabmeat obtained from crabs consuming various baits.
' Mean of 3 1 consumer panelists.

Rankings of perceived differences based on a seven-point .scale.
' Blind control presented as sample.
' SEM = Standard Error of the Mean with 90 degrees of freedom.

crahs will choose a poultry bait product between 40% and 67% of
the time, depending on the bait formulation. Poultry mortality or
poultry mortality silage can be used directly as bait or can be used
as a base component to which a variety of flavor enhancements,
aquatic products, or aquatic waste products could be added to
increase the overall attractant quality of the material. Binding
agents utilized and poultry meat formulation (fresh versus fer-
mented) had no effect in any trial on the ability of the bait products
to attract blue crabs. Preferences for bait pH, preference differ-
ences between male and female crabs, as well as a reduced pref-
erence for supplemental betaine were documented in one or more
trials. No off flavors in the crabmeat are associated with the use of
the alternative poultry baits. Bait durability of at least 5 days in the
aquatic environment has been achieved with current binding sys-

tems. This level of product durability allows realistic ocean testing
of various alternative poultry bait formations versus traditional bait
products to proceed. In addition, the potential for use of poultry
based products as baits for other aquatic species is possible and
requires further investigation.

ACKNOWLEDGMENTS

The authors wish to thank North Carolina Sea Grant for pro-
viding financial support of the project. The authors would also like
to thank Dr. Francis G. Giesbrecht for his invaluable assistance
with statistical design and data evaluation as well as Jonathan
Bridges, Robin Doxey, Russ Howell, and Tyre Lanier for provid-
ing facilities, resources, and technical assistance.

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SAS Institute Inc., Gary, NC.
Shelton. R. G. J. & A. M. Mackie. 1971. Studies on the chemical prefer-
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7: 41-49.
United States Department of Commerce. 1995. Fishery Statistics of the

United States, 1994. In: O'Bannon (ed.). Fishery Statistics No. 9300.

B.K. US Department of Commerce. National Marine Fisheries Service,

Washington, DC.
Zimmer-Faust. R. K. 1987. Crustacean chemical perception: towards a

theory on optimal chemoreceplion. Biol. Bull. 172: 10-29.

Journal of Slwlljhh licsi'arch. Vol. 19, No. 2, 731-739, 2000.

THE FIRST LARGE-SCALE FISHERY-INDEPENDENT SURVEY OF THE SAUCER SCALLOP,
AMUSIUM JAPONICUM BALLOTI IN QUEENSLAND, AUSTRALIA

CATHY M. DICHMONT,* MIKE C. L. DREDGE, AND
KATE YEOMANS

Queensland Department of Primary Industries
Southern Fisheries Centre
P.O. Bo.x 76
Deception Bay, 4508
Queensland. Australia

ABSTRACT The saucer scallop, Amusium japonicum ballon, is a valuable component of a multispecies trawl fishery off the
Queensland east coast. In recent years, a decline in catch rates resulted in the closure of small areas within the fishing grounds
(preservation zones) and the allocation of funding for a large-scale fishery-independent survey. The first survey based on a stratified
random survey design is reported in this paper. The survey found relatively low densities within the strata as compared with similar
species in other parts of Australia and elsewhere in the world. The position of two of the three preservation zones was extremely
appropriate, because together they contained 20% of the over-all numbers caught in the whole survey. The highest densities in the
remaining fishing ground occurred in the inshore north and central strata. The southern sites were characterized by a high proportion
of very low or zero density sites. If a previously published gear efficiency parameter on this species and gear is used, then absolute
adult abundance values are within the same order of magnitude as the commercial catch, and fishing pressure may be high. The
application of this measure of gear efficiency to calculate absolute adult abundance estimates is discussed. Four methods of estimating
confidence intervals are discussed. The survey was extremely successful in terms of coverage of the major scallop grounds and
production of density estimates with low coefficients of variation.

KEY WORDS: scallop densities, stratified random, tlshery-independent. bootstrap confidence intervals

INTRODUCTION

The fishery for saucer scallops, Amusium japonicum balloli. is
an important component of a multispecies trawl fishery on the east
coast of Queensland. Annual landings average about 1,200 tons of
adductor muscle meat, with a landed value in excess of $25 m
(Williams 1997). The scallop fishery takes place mainly between
21°S and 27°S, in depths ranging from 20 to 60 m (Fig. 1). It is
regulated through input controls, which include entry limitation
(which applies to the entire Queensland east coast trawl fishery)
and minimum legal size limits designed to optimize yield per
recruit (Dredge 1990, Dredge 1994). The fishery was characterized
by 24-h fishing operations until 1988, but was limited to night-time
only operations thereafter. Three 10 by 10-minute areas were
closed to trawling to act as broodstock reserves in 1989. but were
repealed 15 months later because of policing difficulties. Similar
closures were again introduced in 1997 as a response to serious
declines in catch rates and were still in place at the end of 1999.

Saucer scallops have been shown to spawn in winter and
spring, coinciding with water temperature changes. It is probable
that saucer scallops are serial spawners, with females spawning
more than once in a season (Dredge 1981 ). Growth is rapid, with
most animals attaining sexual maturity at a shell height of 90 mm
or toward the end of their first year of life (Williams and Dredge
1981, Dredge 1981). Natural mortality rates of adults are high,
with an instantaneous rate between 0.020 and 0.025 week^'
(Dredge 1985a), suggesting that few saucer scallops survive more
than .3 years (Heald and Caputi 19811. It is assumed that the bulk
of each year's catch and spawning population comes from a single
year-class which is fished at late O-i- and l-i- animals.

*Present address: CSIRO Marine Research. P.O. Box 120, Cleveland,
4163, Australia.

Queensland's saucer scallop stock was first fished in the mid-
1950s, when prawn trawlers working out of Hervey Bay took
appreciable quantities (Ruello 1975). Although annual landings
have not shown the spectacular variation often associated with
scallop fisheries (Hancock 1979), catch rates declined by an order
of magnitude in the period 1980-1988 (Dredge 1994) and declined
further in the mid-1990s (Fig. 2 and Williams 1997). The fishery
is seasonal. Maximum catches and catch rates occur in early sum-
mer months, when young of year (YOY) animals first recruit into
the fishery, and adductor meat condition is at its peak (Williams
and Dredge 1981 ). Variable minimum legal size limits apply to the
fishery, with size limits being reduced from 95 to 90 mm shell
height in November each year, for a 6-month period to maximize
yield per recruit. This has the effect of amplifying the early sum-
mer effort pulse (Dredge 1994).

Average catch rates observed in late 1996 and early 1997 were
less than half of the 1988-1995 average for that time of year (Fig.
2). This decrease in catch rates was of sufficient concern to man-
agers and fishers to generate support for a gazettal of emergency
broodstock closures. Resources were then allocated for a large-
scale survey designed to collect data on scallop densities, size
composition, distribution, and estimated abundance of saucer scal-
lops in the main fishing grounds. The data were to be collected to
establish baseline information on the state of the saucer scallop
resource.

Many bottom trawl surveys conducted to estimate fish stock
sizes use a stratified random design with stratum boundaries de-
fined by depth-ranges, species-specific distribution, or manage-
ment areas. Confidence intervals for stock size estimates made
from data collected in such surveys are usually estimated on the
basis of sample error being normally distributed, which has been
shown to be the limiting distribution for the stratified mean (and
total) when the central limit theorem is applied to sampling a finite

731

732

DiCHMONT ET AL.

A'-

S28

T28

\

VP

>\,

S29

^4

GBRMPA

xT30

U30 ^X,

BH

V31

X^

"^

\ U31

V \

HB

Bund^eig^^^

\

V32

Figure 1. Map of saucer scallop survey area as well as nearest local
towns and strata areas and codes within the survey. The arrow indi-
cates the Capricorn-Bunker reserve area of the Great Barrier Reef
Marine Parl< Authority (GBRMPA) that was not included in the sur-
vey. Shaded strata (YP. BH, and HB) are the scallop preservation
zones. Inset shows survey position within Australia. T28 is 30 x 30 min
grid.

population (Cochran 1977). Sampling strategies that involve rela-
tively small sample numbers per stratum when sample catches
have skewed frequency distributions may result in biased confi-
dence intervals. Several authors have suggested modeling the dis-
tribution of estimates from surveys using bootstrap resampling
methods (e.g., Effron 1982). Bootstrap confidence intervals do not
require a distributional assumption for their construction and, thus,
can be used to evaluate the standard normal distribution theory
intervals. In this paper, estimates of saucer scallop stock densities
and associated confidence limits have been analy/.ed using the
classic Cochran approach, the bootstrap-t method (Effron 1982)
and two skewness adjusted methods suggested by Hall (1992).

1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998
Year
Figure 2. Commercial saucer scallop catch rate (ba.skets per day) and
effort (day) statistics for the survey area from IM88 to 1998.

METHODS

A survey of adult and juvenile saucer scallop abundance was
undertaken in the period from 5 to 16 October 1997 using four
chartered commercial scallop trawlers. A total of 6.700 n. miles"
(23,000 km") offshore from north of Yeppoon to southern Hervey
Bay was surveyed (Fig. I , Table 1 ). The Great Barrier Reef Marine
Park Authority (GBRMPA) Management Area B in the Capricorn
Bunker area (a conservation zone covering some 5Q0 n. miles" or
1,800 km") on the eastern edge of the survey area was not sur-
veyed. The dates of the survey were chosen so that the survey was
centered around neap tides (beginning just after the new moon and
ending before full moon) to minimize the effects of tide on scallop
catchability. October was chosen to optimize O-l- recruit catch be-
fore the main fishing season, when the size limit reduces from 95
to 90 mm shell height, and to minimize the probability of adverse
weather affecting the survey. The area covered by this survey has.
on average, corresponded to more than 90% of the Queensland
landings in the past 10 years. Trawls were of constant duration (20
min) at constant speed, and, as best could be arranged, in a straight
line. Distance covered and exact locations were recorded using
onboard Global Positioning Systems (GPS).

Design

This is the first large-scale scallop survey of its type conducted
in Queensland waters (Fig. 1 ). As a result, no estimates of density
variance over the whole area were available. However, two sepa-
rate smaller (unreported) surveys had previously been undertaken.
Sampling in one, which took place in 1989, was not sufficiently
randomized to provide estimates of variance in saucer scallop
abundance in the survey area. A better designed survey of the two
preservation areas within the survey area off Bustard Head (BH)
and Hervey Bay (HB) (Fig. I) was completed in early 1997. Fur-
ther historical infomiation on catches and catch rate variation from
the fishery was available in terms of commercial saucer scallop
catch (baskets) and effort (number of days fished) from commer-
cial logbooks. These data can be summarized spatially in 30 x
30-min grids.

The survey area was divided into 12 strata loosely based on the
30' X 30' grids. The three 1997 broodstock preservation zones
(Fig. I ), labeled HB. BH, and YP, were treated as separate strata.
Sample inlensily within the strata was based upon a weighting
process, using a range of commerical catch rates (catch per unit
effort, CPUE) multiplied by stratum area (Table 1 ). Various forms
of CPUE were considered. They included:

1. mean annual CPUE from 1988-1996;

2. mean CPUE between October to December from 1988-
1996;

3. variance of mean annual CPUE from 1988-1996; and

4. variance of mean CPUE between October to December from
1988-1996.

In all cases, the relative weights were extremely similar and
melhod 1 was chosen. The final weights (Tabic I ) were modified
to ensure that no stratum weight allowed l\ir less than 2'; ol the
total sampling effort.

The lolal number of sites to be sampled was limiled hy finan-
cial resources, which allowed four boats to be chartered for 12
days. It was estimated that an average of 10 sites per boat per night
could be sampled. This translated to a total of 480 sites that could
be sampled, of which 4.'S v\ould be set aside for a preliminary

Fishery-Independent Survey of Saucer Scallop

733

TABLE 1.

Description of strata, sampling intensity, and tlie survey results in terms of mean densities and stratum variance for U+ and 1+ age group

saucer scallops.

Mean Relative

Densities

Number
of Sitp<:

(Number ■

m--)

Stratum Variance

Identifier

Area 11.000 m')

Weight

Sampled

0

1 +

0

1 +

S28

3284747

0.17

65

0.0073

0.0057

0.000279

0.000068

T28

3092947

0.17

46

0.00259

0.0036

0.000012

0.000123

S29

2349482

0.10

44

0.0022

0.001 1

0.000004

0.000002

T29

2362447

0.07

29

0.00271

0.0026

0.000013

0.000006

T30

2079926

0.10

37

0.0053

0.0057

0.00003 1

0.000029

U30

1292695

0.05

19

0.00356

0.0036

0.000017

0.000040

U31

293695

0.12

52

0.0017

0.0030

0.000017

0.000022

V31

1938751

0.07

26

0.00138

0.0003

0.000020

0.000001

V32

2881528

0.10

45

0.0016

0.0012

0.000006

0.000002

YP

347998

0.02

11

0.0018

0.0027

0.000001

0.000006

BH

441462

0.02

13

0.0079

0.0260

0.000030

0.000486

HB

347998

0.02

10

0.0053

0.0100

0.000062

0.000174

Total

23356933

i.no

397

calibration experiment and subsequent calibrations. The final num-
ber of sites completed in each stratum is given in Table 1 .

Previous work has shown that saucer scallops occur in beds
with a maximum density of about one per m". Beds are separated
by areas of zero or extremely low densities (Dredge 1988). The
only known saucer scallop beds that have been mapped in detail
have ovoid spatial distributions, with a width across the beds of up
to 4 km (Dredge 1985b). Given this information, sites were se-
lected on the basis of being 4 km (2 nm) apart or more. Subject to
this rule, the sample sites were randomly chosen within each stra-
tum. Because very little detailed knowledge of the grounds was
available to research staff, several backup sites were randomly
chosen to replace sites that fell on untrawlable grounds.

Description of Boats and Gear

The four commercial trawlers and their skippers had extensive
histories of involvement in the Queensland scallop fishery. Table
2 summarizes basic specifications of the vessels and the gear they
used. Small mesh trawls (ca. 50-mm stretched mesh) were used for
all sampling to capture animals much smaller than the commercial
scallop fleet nonnally takes.

Calibration Experiment

Because four vessels using different gear configurations were
to be invohed in the survey, it was necessary to calibrate their

TABLE 2.
Survey vessel characteristics.

Rated Main

Vessel

Engine

Number

Length

Power (hp)

Trawl Gear Used in Survey

1

18,10m

350

2 by 14-m and 1 by 18-m head
rope length

2

15.84 m

350

4 by 10-m head rope length

3

15.66 m

300

5 by 8-ni head rope length

4

15.24 m

300

2 by 9-m. 2 by 7-m head rope
length

relative fishing power. It was originally intended that the vessels
undertake 10 side-by-side trawls on the first night of the survey.
The calibration experiment was actually completed on the survey's
second day. during daylight hours, as a consequence of poor
weather conditions experienced on the first night. The calibration
work took place over a bed of scallops in the general area of
151°38.50'E and 23°27.70'S. Calibration involved having boats
trawl side by side, undertaking 20-min shots, and having all scal-
lops counted and measured at the completion of each shot. Start
and end fishing points for each trawl were recorded from GPS. The
relative (port-starboard) position of each boat was randomly de-
termined for each trawl. Because the variability between the boats
was not known, it was not possible to calculate the number of shots
needed for a statistical expression of the difference in fishing
power between vessels.

To investigate the possibility that the vessels' power changed
during the survey, it was anticipated that some form of recalibra-
tion would occur during or after the survey. In practice, this proved
impossible, because the vessels were widely separated throughout
most of the survey.

Data Collection

The survey proper was conducted by having each vessel work-
ing in an area that approximated about a quarter of the over-all
survey area. Survey vessels steamed to each survey site sequen-
tially, and the skipper determined if it could be trawled. If the
ground was workable, a 20-min trawl shot was undertaken into the
prevailing tidal flow when possible, at fixed speed. The starting
and finishing positions were recorded using GPS (accuracy ± 60
m). The number of saucer scallops taken in all nets was counted at
the conclusion of the trawls. Scallops from either one or all nets
(depending on sample size) were measured to the nearest mm. Site
characteristics were identified on the basis of: unique shot number,
site number, date, time, starting trawl latitude and longitude from
GPS. end trawl latitude and longitude, distance covered, bearing,
depth, and trawl duration. All sampling was done from sunset to
sunrise between the hours of I8h00 and 07hOO.

734

DiCHMONT ET AL.

Analysis

A generalized linear model was undertaken of the natural log of

the catch rates from the calibration experiment incorporating, as
factors, the different vessel, sample sites, and their position relative
to each other using the PROC GLM module of SAS (SAS 1991 ).
A power test of the resultant ANOVA was undertaken to test what
sample size would be needed in the future to detect a possible
difference between vessels (Thomas unpubl.).

The swept area for each trawl was calculated from the distance
trawled and the swept width estimate for each vessel, assuming
gear spread of 60-70% of the full headrope length for the four
vessels, and using the experience and observations of individual
vessel skippers to make estimates for each boat. Relative densities
(numbers ■ m"") at each site were estimated using numbers caught
and the area swept.

The size-frequency plots for scallops from each 10-min by
10-min block within the region and for the over-all pooled data
(Fig. 3) are clearly bimodal. The modes and size distributions are
consistent with known growth rates of scallops (e.g., Williams and
Dredge 1981), with the first mode being in the size range expected
of young of bear saucer scallops (i.e., scallops spawned in the
winter immediately preceding the survey) and the second mode
representing I -I- and older saucer scallops. For analysis purposes,
animals smaller than 78 mm were assumed to be less than 1-year
old (0-1- year class) and those greater and equal to 78 mm more than
I -year old ( l-i- and older year classes, hereafter referred to as "l-i-"
only). The bimodal size frequency distribution was consistent
throughout the region.

Survey data used to estimate relative population densities were
analyzed initially according to Cochran (1977). The initial design
stratum weights (w,,) were used in the analysis. The stratified mean
and its standard error is therefore given by:

a:.,

EW'/A.

and

where

X„ is the stratified mean (numbers • m"');

.V('(X„) is the Studentized version of the estimated standard

error (Cochran 1977),

^1, ~ 'hJ^ '^ 'he weigh! for stratum h with n,, the number of

stratum sites

20 40 60 eo 100 120 140
Size (mm)

Fiiiure i. Si/.i'-t'requvncy plot <>r all animals measured within the sur-
vey.

planned to be sampled out of a total of N sites; and
5,^ is the stratum variance.

Ninety-five percent confidence intervals were calculated using
four different techniques:

1. assuming the population abundance has a near normal dis-
tribution within each stratum. An approximate 95% confi-
dence interval (i.e., a = 0.05) for the population mean |x is,
therefore, given by:

X„ ± t{df.a)se(XJ

with (//' being the appropriate degrees of freedom for the
t-distribution (Cochran 1977)

2. a general alternative of the above method through the use of
bootstrapping procedures. This was conducted by resam-
pling the Studentized version of the stratified mean; that is,
use the bootstrap-t method of calculating confidence inter-
vals (Effron 1981). Ten thousand bootstraps were used in
this process.

3. an alternative to these two methods of Studentized confi-
dence intervals is a skewness corrected cubic transformation
of the bootstrap-t method (Hall 1992), and

4. bootstrapping, again with 10,000 bootstraps, the above
skewness corrected bootstrap-t method (Hall 1992). The lat-
ter two methods have been shown to be less biased than the
first two methods through simulation modeling.

These methods of calculating confidence intervals are well dis-
cussed and explained in such texts as Manly (1997) and Fletcher
and Webster (1996).

RESULTS

Calibration Experiment

The full general linear model with vessel, relative position, and
trawl site explained 51% of the variance between the vessel's catch
rates. However, only the factor "sample site" was significant {P <
= 0.05) and explained more than 45% of the variance. Vessel as
a factor was not significant, because there was very little difference
in catch rates between vessels, and the "between vessel" variance
was extremely small. A posleri power tests suggest that 77 cali-
bration trawls would have been needed to detect a possible differ-
ence between vessels at a power of 0.6. The residuals ot the mod-
els were normally distributed.

Survey Analysis

Samples were taken from 397 o'i the planned 480 sites. Sites
were nol sampled either through unsuitable bottom conditions or
restrictions on vessel time. The shortfall in sampling did not skew
the planned sampling frequency between strata. Scallop densities
lor the total, commercial, and preservation areas are given in
Tables 3 and 4. The total area has been defined as the sum of the
preservation and commercially accessible areas. Average trawl
speed was 2.38 ± 0.38 knots.

The frequency distribution of densities per stratum for both O-l-
aiid I -I- year olds are given in Figures 4 and 5, respectively. There
is some suggestion of a change in frequency distribution of den-
sities from north to south. The northern sites tended to have a
greater range of densities including some high values; whereas, the
southern sites, especially V3I, had many sites with few or no
saucer scallop. The preservation zone, BH, also had a large pro-
portion of high density sites for bolh O-i- and l-h year olds.

Fishkry-Independent Survey of Saucer Scallop

735

TABLE 3.
Relative densities of 0-f year old saucer scallop for the total survey area, the commercial areas only and the preservation areas alone.

Total Area

Commercial Areas

Preservation Area

Mlmii density (numbers • m"^)

Standard error

Coefficient of variation

Lower and upper 95% confidence limit: NT

Upper and upper 959c confidence limit: BT

Lower and upper 95% confidence limit: NH

Lower and upper 957c confidence limit: BH

0.0035

0.0004
11.4%
0.00275:0.00435
0.00295:0.00475
0.00290:0.00485
0.00295:0.00505

0.0034

0.0004
12.2%
0.00250:0.0040
0.00265:0.0045
0.00260:0.0046
0.00265:0.0077

0.0050

0.0009
19.5%
0.00305:0.00685
0.00340:0.00760
0.00330:0.00780
0.00345:0.00945

Confidence limits were calculated using four methods: NT analysis using Cochran's ( 1977) t-distribution method; BT. the bootstrap-t method; NH, the
skewncss corrected cubic transformation of the bootstrap-t method; BH. the bootstrap version of the skewness corrected bootstrap-t method.

Densities per site for both O-l- and l-i- year olds are shown in
Figures 6 and 7 respectively. To provide contrast in the data, the
classification scales are not linear. Most of the densities are very
low with a few patches of much higher values. In some cases, these
large densities coincide with the preservation areas.

DISCUSSION

Production levels of saucer scallop from Queensland waters
have been relatively stable compared with most natural fisheries
for scallops (see reviews in Shumway 1991), although catch rate
have varied considerably (Dredge 1994). The dramatic decline of
catch rates in 1996 triggered a series of responses. The two sig-
nificant decisions made were the creation of three preservation
zones and financial support for a large-scale independent survey.
The survey was designed to develop baseline data on scallop popu-
lations on the main saucer scallop commercial fishing ground and
is the first of its kind undertaken in Queensland waters. Its main
aim. to produce a relative recruitment index, requires that these
surveys be continued over time and that estimates of abundance
are obtained with narrow confidence intervals. The survey covered
an area from which more than 90% of Queensland saucer scallop
landings are, on average, taken. Estimates of population size are,
therefore, highly relevant to the management of the fishery.

The survey was undertaken in October to optimize the size of
recruits (and, therefore, their catch) before the main fishing season,
when the size limit changes from 95 to 90 mm. It was designed to
give data on relative density and relative abundance.

The preservation zones together contained 20% of the esti-
mated animals in the survey. Most of these were within the central
(BH) and southern (HB) preservation zones, with the highest den-
sity of 0-1- and \+ year olds being recorded in BH. However, the

northern zone (YP) contained few scallops. Subsequent to this
survey and as a consequence of information obtained from the
survey, the YP preservation area was moved to a higher density
area within stratum S28. Overall, therefore, if the animals within
these zones do seed surrounding areas and illegal catches are mini-
mized, they are well situated to offer an effective mechanism for
protection of broodstock and reduce the risks of recruitment over-
fishing.

There is a widespread, but little published belief that recruit-
ment of scallops is of such irregularity and unpredictability that
there is little purpose in attempting to manage broodstock levels to
maintain recruitment. Orensanz et al. (1991) discussed scallop
stocks in the context of four recruitment categories — steady stocks,
cyclical stocks, irregular stocks, and spasmodic stocks, and stated
that most scallop stocks belong in the latter group, with irregular
pulses of high abundance followed by periods of scarcity or col-
lapse. There are, however, examples of scallops recruitment levels
being related to parent stock levels. McGarvey et al. (1993) de-
scribed a stock-recruitment relationship for Placopecten magel-
lanicus in the Georges Bank area, and there is clear graphical
evidence of increased recruitment with increased parental popula-
tion size in Platinopecten yessoensis (Ito and Byakuno 1990).
Mace and Sissenwine (1993) refer to Atlantic stocks of Pla-
copecten magellanicus as requiring relatively low spawner per
recruit levels to minimize the risk of overfishing.

The introduction of broodstock preservation areas when catch
rates of saucer scallops were depressed well below those histori-
cally observed were consistent with a belief that recruitment over-
fishing was a possibility in this species. Results from this survey
show that about 20% of the population are protected from fishing.

However, little is known of the dynamics of larval transport and

TABLE 4.
Relative densities of the l-i- and older saucer scallop for the total survey area, the commercial areas only and the preservation areas alone.

Total Area

Commercial Areas

Preservation Areas

Mean density (numbers ■ m~-)

.Standard error

Coefficient of variation

Lower and upper 95% confidence limit: NT

Upper and upper 95% confidence limit: BT

Lower and upper 95% confidence limit: NH

Lower and upper 95% confidence limit: BH

().()03X

0.0004

9.9%
0.00315:0.00465
0.00330:0.00500
0.00300:0.00495
0.00325:0.00555

0.0033

0.0087
1 1 .2%
0.00245:0.00385
0.00260:0.00435
0.00255:0.00420
0.00260:0.00685

0.0129

0.0025
19.2%
0.00795:0.01760
0.00860:0.01860
0.00840:0.01975
0.00875:0.01915

Confidence interval codes as in Table 3.

736

DiCHMONT ET AL.

S2e

_ 0.8

s«

I'O.e

0.2

^L—^

,

S29

_OB

s«

&0.6

1-04

11.
0_2

'-

°

T30

~0.8

!S

3-0.6

|0.4

02

J

^08

S-0 6

c
a

D-04

it
02

L

U31

■i

— 0.8

S-oe

S-04

u.
02

V32

_0.8

I'o.e

1-0.4

ijT

02

0

1- «-

BH

.1

8 .8

8 8

Density (numbers. m )
Figure 4. Density-frequency plots of catch (numbers • m"*) of 0+ year olds per stratum.

recruitment within Queensland waters and so the long-term value
of the preservation zones is hard to estimate. The abundance pat-
tern in Shark Bay, Western Australia of 1990 had a core of very
high abundance surrounded by an area of relatively high abun-
dance, suggesting that larvae were contained within a well-defined
eddy at settlement (Joll 1994). Dredge (1988) suggested that a gyre
in Hervey Bay, which falls in the survey area described in this
paper, might act to trap larvae. Caddy (1979) hypothesized that
recruitment to the Bay of Fundy fishery was positively inlluenccd
by the degree of retention of larvae within a gyre. Further study
into the oceanography of the survey region would be required to
understand the dynamics of larval transport fully, settlement and
recmitment.

Within the main fishing grounds, the highest relative density
strata within the survey were the inshore north and central areas.
Apart from a few high relative density sites, the southern strata
contained very few saucer scallops. This over-all low relali\c den-

sity within a stratum is most notable in V3I where more than 80%
of the sites had densities of less than 0,001 saucer scallops per m"
of 0-1- and \+ year olds. This contrasted with the two highest
relative density sites for juvenile and adults (S28 and T30) in
which more than 10 and 20%, respectively, of the sites contained
relative densities higher than 0,01 scallop per nr of 0+ and l-i- year
olds, respectively.

The availability of gear efficiency estimates for capture of sau-
cer scallops from another study (Joll and Penn 1990). makes it
possible to estimate absolute abundance values of legal size ani-
mals, assuming 100% selectivity for animals >90 mm and no
swept area changes during the survey. The relative density from
the survey of animals >9() mm in the commercial areas were
0.0010 scallops • m~". Given the range of values estimated in Joll
and Penn ( 1990), efficiency values of 0..S and 0.6 were tested and
give legal size animal abundances in the commercial areas of
0.0020 and 0.0017 scallops - m"'. respectively.

Fishery-Independent Survey of Saucer Scallop

737

Density (numbers. m")
Figure 5. Density-frequency plots of catch (numbers • m"") of 1+ year olds per stratum.

Because the survey estimates relative densities, it is difficult to
compare results with other studies. Minchin and Mathers (1982)
found densities of up to eight Pecten maxinnis scallops per m" in
Ireland. In the same region, commercial fishable concentrations
were considered to be around 0. 1 to 0.2 scallops per m"" (Gruffydd
1972). although improvements in fishing gear efficiency may have
lowered this threshold slightly. Buestal et al. (1985) found average
densities from dredge samples to be about 0.6 scallops • m^" in the
Bay of St. Brieuc. Within-bed densities have been well published
with a summary of some of these in Brand (1991 ) and Oresanz et
al. ( 1991 ). Most of the densities of those scallop species recorded
in these texts are a few orders of magnitude higher than we ob-

served and were directed at known high-density beds. Surveys in
the 1980s in Port Philip Bay. Australia also produced estimates of
densities ranging from 0.01 to 0.6 scallops • m~" (e.g.. Gwyther
and McShane 1985). These variations in abundance may reflect
intrinsic differences in behavior and density tolerances between
species of Amiisium and less mobile pectinids. Joll (1994). how-
ever, reported estimated Amusiiim densities of scallops in the area
of highest abundance in the 1990 and 1991 surveys as 6.7 scallops
m^" and general densities at a time of very high stock abundance
of 0.57 1+ scallops m"" and 1.42 O-i- scallops m"-. Joll and Penn
(1990) reported densities of scallops of 0.08-0.09 m"' in an area
of Shark Bay in 1986. These studies were on beds of normal

738

DiCHMONT ET AL.

w

V •• -••■ ^- ■■•. ■■••• •

Gladstocfe

0 -0.004

.

0.004

0.012

•

0.012

- 0.020

•

0.020

0.028

•

0.028

0.107

■•

• •
•

' ml
< •

1

^^

iN>-v;-:\

• ^S?

AV""-'

• . x^

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Bundaber^J\

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•

Figure 6. Densities (numbers ■ ni "I of the 0+ year old saucer scallops
caught in the survey.

abundance within the area at the time. In Wilson and Brand (1995),
the over-all preseason density of commercial size Pecteii maximiis
was around three scallops • 100 m"" based on survey areas cover-
ing six fishing grounds.

Generally, variances of mean densities of Q+ and 1+ scallops
within strata were low. This suggests that stratum borders were
well situated. However, this low variance was also influenced by
the over-all low densities. The over-all coefficient of variation of
1 1 .4 and 9.97f of O-i- and l-i- year old densities was also very low.

0 -0 004

.

0 004 • 0,012

•

0 012 -0 020

•

0 020 ■ 0 028

•

0 028-0107

figure 7. Densities (numhers • m "I of the \+ and older year old sau-
cer scallops caught in the survey.

suggesting relatively uniform (but low) densities. Confidence in-
tervals of estimates are consequently fairly small, and there was
little difference between the different methods of estimating inter-
vals. An exception is the upper limit from the bootstrapped version
of Hall's cubic transformation method. Simulation tests of the data
would be the only method of explaining this difference, but were
not done in this study. In other studies, simulation of groundfish
trawl surveys of Georges Bank and the Scotian Shelf data indicate
that the bias-corrected and accelerated confidence limits may over-
correct for the trawl survey data and that the percentile limits were
closer to expected values (Smith 1997). These methods were there-
fore not applied to these data.

Several biases and sources of variance need to be assessed for
future surveys. Most importantly is the swept width of each ves-
sel's trawl gear, which is based on sparse information. Large bi-
ases can result, and uncertainties may be underestimated if scien-
tists treat catchability coefficients as constants without error and
subsequently use survey biomass values as absolute estimates of
biomass (McAllister and Pikitch 1997). Because the gear effi-
ciency parameter used in this study was based on Leslie and De-
Lury methods (Leslie 1952, Delury 1947), their assumptions
should be noted. These are that the target species has constant
catchability over the sampling period, the fishing effort is distrib-
uted uniformly over the fishing ground, the fishing methods do not
change, the target population is closed, and the landings and effort
are reported correctly. Biases in estimates of fishable biomass
cannot be corrected without knowing the cause (Miller and Mohn
1993).

A further source of bias is mesh selectivity. Experiments made
in Shark Bay on the relative selectivity of prawn and scallop mesh
was undertaken in November 1985 (Joll 1987). Prawn mesh of 50
mm and scallop mesh of 100 mm (stretch mesh) were used. The
length frequency data show that prawn nets catch scallops as small
as 30 mm, but efficiency of capture for such small scallops is
unknown. Scallop and prawn mesh have a similar selective effi-
ciency for scallops of 90 mm or greater. The scallop mesh selec-
tivity is negligible below 70 mm or less (Joll 1987), This means
that juveniles below a certain size were not being caught by the
gear used in this survey and that there is a selectivity ogive of
unknown slope and configuration above this size. It may, there-
fore, be difficult to determine whether a change in estimated sur-
vey density between years is attributable to a change in true num-
bers or a change in recruitment timing. Further work on selectivity
and recruitment timing is, therefore, justified.

The survey reported in this paper offers some insight into dis-
tribution, recruitment, and density variation of saucer scallops
throughout their major fishing ground. The results, however, will
be of real value as the survey is repeated over time, and a history
of fishery-independent recruitment processes, w ith their linkage to
climate and other variables, is developed.

ACKNOWLEDGMENTS

This survey was funded by the Queensland Departmcnl of Pri-
mary Industries. The authors thank all those who went on the
survey and collected the data, often working long hours. Also, the
skippers and their crews for their cooperation and hclptulncss, and
Dr. David Mayer for his useful comments on the design and sub-
sequent analysis.

Fishery-Independent Survey of Saucer Scallop

739

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Cochran. W. G. 1977. Sampling techniques. .Vd ed. John Wiley. New
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DeLury. D. B. 1947. On the estimation of biological populations. Biomet-
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Dredge, M. C. L. 1981. Reproductive biology of the saucer scallop Amu-
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Dredge. M. C. L. 1985a. Estimates of natural mortality and yield-per-
recruit for Amusium japoniewn balloti Bemardi (Pectinidae) based on
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Dredge. M. C. L. 1985b. Growth and mortality in an isolated bed of saucer
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Dredge. M. C. L. 1988. Recruitment overfishing in a tropical scallop fish-
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Effron. B. 1981. Nonparametric .standard errors and confidence intervals.
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Fletcher. D. & R. Webster. 1996. Skewness-adjusted confidence intervals
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130.

Gruffydd. L. L D. 1972. Mortality of scallops on a Manx scallop bed due
to fishing. / Mar Biol. Assoc. U.K. 52:449-455.

Gwyther. D. & P. E. McShane. 1985. Port Phillip Bay scallop population
assessment and catch forecast for the 1985 season. Ministry for Con-
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Hall. P. 1 992. On the removal of skewness by transformation. J. Royal Stat.
Soc. .54:221-228.

Hancock, D. A. 1979. Population dynamics and management of shellfish
stocks, pp. 8-19. In: H.J. Thomas (ed.). Population Assessments of
Shellfish Slocks. Rapports et Proces-VerbaiLX des Reunions. V. 175.
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Heald. D. I. & N. Caputi. 1981. Some aspects of growth, recruitment, and
reproduction in the southern saucer scallop Amusium balloti Bernardi
1861 in Shark Bay. Western Australia. Fish. Bull. W. A. 25:33 pp.

Ito. S. & A. Byakuno. 1990. The history of scallop culture techniques in
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ing of the Australasian Scallop Workshop. Tasmanian Government
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Joll, L. M. 1987. Fisheries Management Paper No 11: The Shark Bay
Scallop Fishery. Fisheries Department. Perth. 123 pp.

Joll, L. M. & J. W. Penn. 1990. The application of high resolution navi-
gation systems to Leslie-DeLury depletion experiments for the mea-
surement of trawl efficiency under open-sea conditions. Fish. Res.
9:41-55.

Joll. L. M. 1994. Unusually high recruitment in the Shark Bay saucer
scallop (Amusium balloti) fishery. Mem. Queensl. Mus. 36(2):26l-267.

Leslie. P. H. 1952. The estimation of population parameters from data
obtained by means of the capture-recapture method II. The estimation
of total numbers. Biometrika. 38:369-392.

Mace, P. M. & M. P. Sissewine. 1993. How much spawning per recruit is
enough? In: S. J. Smith, J. J. Hunt and D. Rivard (eds.). Risk Evalua-
tion and Biological Reference Points for Fisheries Management. Can.
Spec. Piibl. Fish. .Atpiat. Sci. 120, pp. 101-118.

Manly. B. F. J. 1997. Randomization, bootstrap, and Monte Carlo methods
in biology, 2nd ed. Chapman & Hall. London. 399 pp.

McAllister. M. K. & E. K. Pikitch. 1997. A Bayesian approach to choosing
a design for surveying fisheries resources: application to the eastern
Bering Sea trawl survey. Can. J. Fish. Aquat. Sci. 54:301-311.

McGarvey, R., F. Serchuk & I. A. McLaren. 1993. Spatial and parent-age
analysis of stock recruitment in the Georges Bank {Placopecten ma-
gellanicus) population. Can. J. Acpiat. Sci. 50:564-574.

Miller, R. J. & R. K. Mohn. 1993. Critique of the Leslie method for esti-
mating sizes of crab and lobster populations. N. Am. J. Fish. Matmg.
13:676-685.

Minchin. D. & N. F. Mathers. 1982. The escallop, Pecten maximus (L.), in
Killary Harbour. Ir. Fish. Investig. Ser. B (Mar). 25:13 pp.

Orensanz. J. M.. A. M. Parma & O. O. Iribarne. 1991. Scallops: Biology.
Ecology, and Aquaculture. pp. 625-713. In: S. A. Shumway (ed. ).
Elseiver. Amsterdam.

Ruello. N. V. 1975. An annotated bibliography of prawns and the prawning
industry in Australia. In: P. C. Young (ed.). First Australian Prawn
Seminar. Australian Government Publishing Service. Canberra.

Shumway. S. A. (ed.). 1991. Scallops: biology, ecology, and aquaculture.
Elsevier. Amsterdam. 1095 pp.

Smith. S. J. 1997. Bootstrap confidence limits for groundtlsh trawl survey
estimates of mean abundance. Can. J. Fish. Aquat. Sci. 54:616-630.

SAS. Statistical Analysis Software version 6.12. SAS Institute Inc.. Carey,
North Carolina.

Thomas. M. V. unpubl. Power calculations: an introduction for fisheries
biologists. CSIRO IPP&P Biometrics Unit Course Notes. 39 pp.

Williams, L. E. (ed.). 1997. Queensland fisheries resources: current con-
dition and recent trends 1988-1995. Department of Primary Industries,
Brisbane. 101 pp.

Williams, M. J. & M. C. L. Dredge. 1981. Growth of the saucer scallop,
Amusium japonicum balloti Habe in central eastern Queensland. Aust.
J. Mar. Freshw. Res. 32:657-666.

Wilson. U. A. W. & A. R. Brand. 1995. Variations in commercial scallop
{Pecten maximus) density in a seasonal fishery. Abstract in 10th Inter-
national Pectinid Workshop, Corl Island, p. 13.

Joiinwl of Slwlltlsh Rcsetirch. Vol. 19. No. 2. 741-747, 2000.

SEASONAL VARIATIONS IN CHEMICAL COMPOSITION OF THE FEMALE GONAD AND

STORAGE ORGANS IN PECTEN MAXIMUS (L.) SUGGESTING THAT SOMATIC AND

REPRODUCTIVE GROWTH ARE SEPARATED IN TIME

TORE STROHMEIER," ARNE DUINKER.* ' AND 0YVIND LIE"

lustitute of Marine Research
P.O. Bo.x 1870, Nordnes
581 7 Bergen. Norway
'Institute of Nutrition
Directorate of Fisheries
P.O. Bo.x 185 Sentrwu
5804 Bergen, Norway

ABSTRACT Glycogen, protein, and lipid coniposilion were determined in the female part of the gonad, striated adductor muscle, and
digestive gland during 1 year in great scallops {Pecten maximu.^). The scallops were sampled from Raunetjorden outside Bergen on
the west coast of Norway. From the spring phytoplankton bloom in March to June, gonad growth, spawnings, and rebuilding took place;
whereas, no somatic growth was seen during this period. A marked increase in digestive gland protein during the spring bloom may
have represented an increase in digestive capacity induced by the increased food levels. Between June and August, a concentrated
period of somatic growth and increased storage was seen: whereas, the female gonads showed no signs of rebuilding, because they
decreased due to spawning activity. Gonad rebuilding look place between October and December. Reproduction in many populations
of P. maxinnts is regarded as more or less continuous, since individuals with tilled gonads can be found all year. However, for the
population in this study, it seemed that no energy was allocated to the gonads in the period starting in June, with somatic growth and
storage, until the gonad rebuilding in October, even though the gonads were still containing presumably growing oocytes. From these
results, we suggest that switches in energy allocation divide the sea.son in two parts in these scallops: ( 1 ) priority of reproductive growth
from October to June: and (2) priority of somatic growth and storage from June to October.

KEY WORDS:

reproduction

adductor muscle, chemical composition, digestive gland, energy storage, female gonad, growth, Pecien ma.ximii.'^.

INTRODUCTION

Scallops, as do other marine bivalves, exhibit cyclic changes in
both reproductive and somatic body components as a consequence
of the seasonality of environmental conditions in temperate areas
(Barber and Blake 1991), Reproductive cycles are based on the
build up of gametes and release at a time with favorable conditions
for larval growth and survival. The seasonality in temperature and
food conditions results in annual cycles in build up of somatic and
storage material that is later utilized during periods of food short-
age or also to support gametogenesis. In scallops, it has been
demonstrated that energy is stored in the adductor muscle and
digestive gland, since weights of these tissues increase during
periods with excess food and decrease during periods with strong
gametogenesis or low food levels (Ansell 1974. Barber and Blake
1981, Comely 1974, Pazos et al. 1997, Robinson et al. 1981.
Taylor and Venn 1979). Chemical analyses of the storage organs
have revealed that glycogen and protein in the adductor muscle
and lipid in the digestive gland are the major energy reserves
(Barber and Blake 1991).

Timing of reproduction and the relationship between gonad
growth and variations in the storage organs have been studied in
several scallop species. Generally the storage organs increase in
size and accumulate energy throughout spring, summer and au-
tumn (Ansell 1974, Comely 1974, Mackie and Ansell 1993, Sun-
detandVahl, 1981, Taylor and Venn 1979). Different reproductive

'Corresponding author. Current address: Institute of Nutrition, Directorate
of Fisheries, P.O. Box 185 Sentrum, 5804 Bergen, Norway.
E-mail: Ame.Duinker@nutr.fiskeridir.no

strategies require different use of this storage material versus avail-
able food for fueling gametogenesis, as discussed by Ansell
(1974), Mathieu and Lubet (1993), and Taylor and Venn (1979).
Gonad build up during winter is fueled by stored reserves whereas,
gonad build up during spring is fueled by available food. In both
cases, the food shortage in winter requires the use of stored energy
for maintenance metabolism. Energy storage may also be inter-
rupted by rapid gametogenesis in summer at the expense of energy
stored earlier in the season (Barber and Blake 1981. Robinson et
al. 1981).

In many populations of Pecien ma.ximus, gonads with some
degree of fully grown gametes can be found all year, although the
gonad indexes vary because of differences in spawning activity
and gonad growth (Comely 1974. Mackie and Ansell 1993. Mason
1958. Paulet et al. 1988, Strand and Nylund 1991 ). Somatic growth
and reproduction are competing for resources, and the partition of
energy between these two processes is an important trait of the life
history of any species. The reproductive effort generally increases
with age (Thompson and MacDonald 1991). but the pattern of
energy allocation to the gonads within a season has not been given
particular attention in studies of scallops.

Better understanding of the proces.ses of growth, storage, and
reproduction is a keystone in understanding the general biology of
a species and also important for aquaculture, harvest, and man-
agement of scallops. This study was conducted with special em-
phasis on reproduction related to conditioning and spawning in
hatchery production. The aim of this .study was to describe the
variations in weights and content of protein, lipid, and carbohy-
drates in the female parts of the gonads and in the storage organs
during 1 year in a defined population of P. maximus in Western

741

742

Strohmeier et al.

Norway. Histology and visual characterizations of the female go-
nads are described elsewhere (Duinker unpublished data).

MATERIALS AND METHODS

Three hundred great scallops (Pecten maximtts) with shell
heights between 95 and 120 mm, shell weights between 90 and 160
g, and ages between 4 and 7 years were obtained from commercial
harvest by diving from the area around Bergen. The scallops were
individually tagged (Hallprint Pty. Ltd, Australia, ref. no. T1625)
and reseeded at the sampling site (N 60 ° 15' 36", E 5 ° 05' 00")
in Raunefjorden south of Bergen on December II, 1996. The
sampling site was a naturally bounded area with shell sand and
small gravel at 1 1-14 m depth. Temperature was recorded by a
Tinytag® temperature logger (Intab Interface-Teknik AB, Swe-
den) attached 50 cm above the seabed at 13 m depth. Samples of
15 scallops were collected by diving and transported in a cooling
box with seawater from the site at monthly intervals.

From each scallop, the adductor muscle, digestive gland, go-
nad, and dried shell were weighed. Samples from the striated ad-
ductor muscle, digestive gland, and female part of the gonad were
taken for analysis of total glycogen, protein, and lipid. For each
organ, samples were pooled into three groups with tissue from five
individuals in each and stored at -80 °C.

All samples were freeze dried before analyses (Virtis Genessi
25 SE. Holem, USA), and dry weight percentages were calculated.
Glycogen was analyzed using an enzymatic and spectrophotomet-
ric method described by Hemre et al. ( 1989). Protein was analyzed
as total nitrogen after total combustion using a Nitrogen-Analyser
(Perkin-Elmer, 2410 Ser. II, Norwalk Connecticut, USA). The
protein was calculated by the assumption that protein contains
16% N. Lipid was analyzed using a gravimetrical chloroform/
methanol method modified from Bligh and Dyer ( 1959) according
to R0nnestad et al. (1995).

The content of glycogen, protein, and lipid in the subsamples
were calculated to represent the content per organ as follows:
content of constituent = percentage constituent of dry weight x
dry weight percentage x average wet weight of the organs in the
subsample. Wet weight for the whole hermaphroditic gonad di-
vided by 2 was used for calculating the content of the female
gonad constituents, because wet weight was not taken for the
female part separately. Hence, the data were not used for evalu-
ating energy flow between the female gonad and the storage or-
gans, but the .sea.sonal variations could still be described. All
weights and data for the chemical contents were standardized with
shell weight to represent a standard scallop of lOO-g shell weight
according to the formula: standardized measure = original mea-
sure X lOO/shell dry weight. Energy from glycogen, protein, and
lipid were calculated using conversion factors of 17.38, 23.66, and
35.17 k/g '. respectively (Beukema 1997), and total energy for
each organ was calculated as the sum of energy from glycogen,
protein, and lipid.

Wet weights were tested for deviations from the normal distri-
bution using the Kolmogorov-Smirnov test (Zar 1999). For the dry
weight and chemical content data from the pooled subsamples,
normal distribution was assumed according to the Central Limit
Theorem (Bhattacharya and Johnson 1977, Zar 1999). because the
data were considered as mean values for five indi\ iduals. Devia-
tions from homogeneity of variances were tested using the Levene
F test (Brown and Forsythc 1974) together with considerations of
F-max. Because of pronounced heteroscedaslicity in many of Ihc

time series, ranks of the data were used in further analyses. Dif-
ferences between the sampling points were tested with one-way
analysis of variance (ANOVA) followed by Newman-Keuls mul-
tiple comparison tests when significant differences were found.
The results from these tests were compared with results from para-
metric Newman-Keuls tests and from manually performed non-
parametric Newman-Keuls tests (Zar 1999). Statistica version 5.0
(Stalsoft inc.. Tulsa, OK, USA) was used for all statistical analy-
ses. The significance level (a) was 0.05.

RESULTS

Temperature

Temperature was close to 6 °C from February until the middle
of April (Fig. 1). It then increased gradually throughout May and
fluctuated between 9 and 12 °C in June, followed by further in-
creases and several sharp drops until the maximum of 19.6 °C in
late August. Temperature then dropped to 13 °C in September,
gradually decreased until December and then dropped from 8 °C to
the winter temperatures of 4 to 6 °C.

Female Gonad

Protein content, lipid content, and dry weight of the female
parts of the gonads (Figs. 2 and 3) all followed the same pattern
throughout the period of sampling, and the following changes were
significant in either one or more of these parameters. Between
February 15 and June 8, a decrease was followed by an increase to
a new maximal level. A sharp decrease from June 8 was followed
by a gradual decrease with stable standard deviations. From mini-
mum levels in September and October, a rapid increase occurred
between October 18 and December 16 to maximum levels for the
season, and no significant changes in dry weight, protein content.
or lipid content were seen on February 7 (Newman-Keuls test on
ranks of the data, 0.4 > P > 0.5).

The glycogen concentration in the female gonads varied be-
tween 3 to 5% of dry weight during the period of sampling, and it
did not exceed 4% of the average total energy in the female gonad.
Protein was the major constituent in the female gonad, with con-
centrations ranging from 62 to 67% and energy percentages be-
tween 67 and 83% during the year. Energy from lipid varied from
29'^ in February 1997 to 15% in September, corresponding to
concentrations between 8 and 19% of drv weiuht. Maximum con-

20
18
16
14

■D

1?

ra

Q)

F

10

OJ

8

6

4

If,

'if^'^4

FMAMJJASONDJ

Kij-ure. I. Teniptrature ri'C()rdin)>s fnini .lanuary 30 1997 lo February
7 1998 ill M meters depth.

Seasonal Variations in Great Scallops

743

30

a) 1

-1

25

I

/

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2 20

a>
u
m

1 15

10

ji

4

1 ■

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1

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t

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1

1

{

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0

!

\

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7

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6

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5

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\

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0.5

Figure. 2. Seasonal variations in a) wet weight and b) dry weight of the
female gonad (— • — ). adductor muscle ( — D — ), and digestive gland
( — V— ) for a standardized scallop with a 100 g shell weight. See
Materials and Methods for note on the calculation of the female gonad
dry weights. Vertical bars indicate standard deviations, n = 15 for wet
weights, and « = 3 from pooled samples for dry weights.

centration of protein and minimum concentration of lipid corre-
sponded with the minimum degree of filhng of the gonads in
September.

Adductor Muscle

The dry weight (Fig. 2) and content of protein and glycogen
(Fig. 3) in the adductor muscles were low at the end of winter with
minimum values on March 31. Glycogen increased slowly from
March 31 to June 8, with 5.3 mg per day. and then more rapidly
until August 13. with 19 mg per day. The increase in glycogen
concentration between March and August was from 2 to 25% of
dry weight, corresponding to an increase in energy from glycogen
from 1.5 to 30 kJ. Protein content was stable with no significant
variation until July 3 (Newman-Keuls tests on ranks of the data. P
£ 0.34) but then increased rapidly between July 3 and August 13
(P = 0.01 1. Glycogen content decreased from September 15 with
a significant decrease between October 18 and December 16 (P =
0.02) and a larger decrease from December 16 to February 1 (P =
0.04). Protein content was stable between August 13 and February
7 (P < 0.28).

Lipid content in the adductor muscle (Fig. 3) varied signifi-
cantly (ANOVA on ranks of the data. P = 0.006). but no clear
seasonal trends were seen. The lipid level remained low and varied
between 3.1 and 4.9<7f of adductor muscle dry weight.

Digestive Gland

Minimum levels in dry weight, lipid content, and protein con-
tent in the digestive glands were found at the end of winter on

March 12 (Figs. 2 and 3). Between March 12 and May 8. the
protein content increased rapidly with 79% (Newman-Keuls test
on ranks of the data, P < 0.001 ). Lipid was accumulated slowly
between March 1 2 and July 3 at 2.8 mg per day. but then increased
to 7 mg per day between July 3 and August 13. In August and
September, the digestive glands had the maximum levels of lipid
for the season, and lipid was the largest constituent in this period.
The lipid percentage of dry weight varied from 1 3.5% on March 1 2
to 44% on September 15, and energy from lipid varied between 3
and 25 kJ in the same period. Protein content decreased gradually
from May 8 to February 1 (P < 0.001). Lipid content decreased
gradually between September 15 and February 1 (P = 0.001).
Glycogen remained low and varied between 0.8 and 5% of diges-
tive gland dry weight. Peaks in glycogen content were found in
May and in August/September. The loss in total energy from both
the adductor muscles and digestive glands between October and
December was 1 1 kJ; whereas. 27 kJ were lost between December
and February.

Female gonad

o> 0.6.

c
Si
c
o
u

i 0.3
o

0.0

5.4

^- 4.9

c

01

0 4.4
u

c

1 3.9
a.

3.4

2.9
0.8

Ol

•i 0.6

c
o
" 0.4

T3

a

■g 0.2
n

_c

OJ

o 0.0

Muscle

0.3

0.2

0.1

0.0 U

2.0

1.5 S

1,0 .s-

0.5

CO

Digestive gland

Figure. 3. Seasonal variation in the content of lipid (—>—), protein
(—•—), and glycogen ( — D — ) in the female gonads, adductor
muscles, and digestive glands. See Materials and Methods for note on
the calculation of the female gonad data. Values are standardized to a
scallop of 100 g dry shell weight. Vertical bars indicate standard de-
viations, n = i from pooled samples.

744

Strohmeier et al.

The different multiple comparisons tests that were compared
gave largely the same conclusions as to detecting significant dif-
ferences or not, but the P-values varied.

DISCUSSION
Constituents of the Organs

The major constituent of the female gonad was protein, fol-
lowed by lipid, and glycogen remained low throughout the period
of sampling. This agrees with other studies of pectinids (Couturier
and Newkirk 1991. Pazos et al. 1997. Taylor and Venn 1979).
Female gonads in scallops mainly consist of oocytes and collagen-
rich acinus walls (Beninger and Le Pennec 1991 ), and there are no
specialized storage cells as found in other bivalve gonads (Mathieu
and Lubet 1993). Hence, the composition of scallop female gonads
reflects the composition of bivalve eggs (Gabbot 1976). The pro-
tein and lipid content followed the filling of the female gonad, with
relatively constant composition despite large variations in filling,
which is consistent with the findings of Comely ( 1974). This prob-
ably reflects that growth of the female gonad is propagated by
gradual accumulation of increasing numbers of large oocytes. The
development of individual eggs is of short duration relative to the
period of gonad growth, and several different sized cohorts of
oocytes are present at any one time, with recruitment of new
cohorts on several points (Paulet and Boucher 1991 ). A different
scenario would be seen if one or a few cohorts were developing
synchronously throughout the period of gonad growth, with com-
position of the female gonad following the development of the
individual oocyte. However, at the point of minimum filling of the
female gonads in September, the peak in protein percentage and
minimum lipid percentage reflect the increasing proportion made
up by protein from the acinus walls as the female gonads are
emptied.

The adductor muscles consisted mainly of protein but also con-
tained highly variable amounts of glycogen and low lipid levels.
Protein represents the major structural part of the scallop adductor
muscles, although it can also be utilized to some extent to supply
gametogenesis or for maintenance purposes, especially in the se-
melparous species (Barber and Blake 1991 ). Protein in the muscles
is also the best indicator of somatic growth, that is, increase in size
of the whole animal, among the parameters measured in this study
(see also Barber and Blake 1981). The fluctuations in glycogen
concentration between 2 and 25% are comparable to the 20-fold
increase reported in Peclen maximus by Comely (1974) and dem-
onstrate the importance of this substrate in energy storage. The
importance of glycogen storage in the muscle can be related to the
monomyarian condition with reduction in the foot and visceral
regions in scallops (Ansell 1974), and to the need for a readily
mobilizable carbohydrate reserve for swimming and predator
avoidance (Ansell 1974, de Zwaan et al. 1980). The lipid concen-
tration fluctuated between 3 and 5'/r of dry weight during the
sampling period. Giese ( 1966) concluded thai lipid levels less than
5.2 '/(' of dry weight are not to be considered as reserves. The low
lipid content in the adductor muscle was, therefore, considered to
be structural lipid that made little contribution to the energetics of
somatic growth.

In the digestive gland, protein and lipid were the main con-
stituents, with lipid dominating during late summer and protein
dominating throughout the rest of the year. Protein represents the
enzymes related to the digestive activity of this organ (Beninger
and Le Pennec 1991, Henry et al. 1991 ) and also structural parts.
The rapid increase in protein content between March and May

coincided with a marked reduction in visibility (own observations),
which was attributable to the spring phytoplankton bloom that
normally occurs at this time (pers. comm. B. Heimdal, University
of Bergen, Norway, see also Erga and Heimdal 1984). The in-
crease in digestive gland protein was probably reflecting an in-
crease in digestive capacity during the spring bloom, a period with
high food levels at low temperatures. Hence, it is not regarded as
somatic growth in the same context as increase in muscle protein.
The decrea.se in digestive gland protein between May and February
may be attributable to reduced needs for high digestive capacity
after the spring bloom. A similar increase in digestive gland pro-
tein level during the spring bloom occurs in great scallops in the
Bay of Brest, France (pers. comm. C. Saout, University of Brest,
France). Lipid showed large variations and high maximum levels
of more than 40% in late summer. This is consistent with the
second role of the digestive gland as the most important site of
lipid storage, as suggested from histological studies (Henry et al.
1991) and from losses during gainetogenesis (Barber and Blake
1981. Barber and Blake 1991. Robinson et al. 1981 ). With the low
glycogen content, the variations may be attributable to variations
in food content in the digestive gland diverticula following varia-
tions in feeding activity. The peaks in May and in August/
September may correspond to periods with favorable combinations
of teinperature and food levels giving high feeding rates, and this
may reflect conditions similar to those found by Mason (1957)
when he reported peaks in filling of the stomachs also in May and
August/September.

Because of a low number with the three subsamples. heterosce-
dasticity in the data, and a high number of groups being compared,
the P-values from different varieties of the multiple comparison
tests were not perfectly consistent. Hence, the statistics alone
should not be decisive when considering the seasonal changes, but
rather should be used as rough measures of differences between
samples related to the variation in the data, in combination with
biological considerations. This was preferred to omitting statistics.

Somatic versus Reproductive Growth

The increases in the different constituents observed in this
study represent various anabolic processes that can be categorized
into: (1) reproductive growth, involving increase in the gonad
constituents; (2) somatic growth, represented by increa.se in ad-
ductor mu.scle protein: (3) increase in digestive capacity, observed
as protein increase in the digestive gland: and (4) energy storage
with accumulation of glycogen in the adductor muscle and lipid in
the digestive gland. The apparently underlying seasonal patterns of
these processes suggest a strategs' of dividing the year into periods
with different priority of reproduction versus somatic growth and
storage.

From February to .luiie. gonad growlli dominated. Somatic
growth was not obser\ed, because adductor muscle protein was
stable, and some storage occurred, but at low rates. The scallops
displayed an extended period with spawnings of varying intensity
between late March and September, and the drops in gonad con-
stituents coincided with evidence of spawning activity (Duinker
unpublished data). The net increases and decreases observed in
gonad constituents would depend upon the relative contribution of
gonad growth versus losses from spawning. Between February and
June, it seemed that energy allocation to the gonads was continu-
ous, with rebuilding tollov\ing the spawnings.

Between .lune and .Auizust. somatic srowth and storage domi-

Seasonal Variations in Great Scallops

745

nated, and no increase in gonad constituents was observed. The
llireet'old increase in storage of glycogen in tine adductor muscle
starting in June suggested that more energy was available for stor-
age from this point. Protein in the muscle showed significant and
marked increase only during the narrow period between July and
August, with no significant changes during the rest of the year.
During the same period, there was also a marked increase in the
rate of accumulation of lipid in the digestive gland. Preliminary
analvses of daily growth rings were conducted on left valves from
the present study according to Chauvaud et al. ( 1998). This indi-
cated that also shell growth was limited to a period of 3^ months,
depending upon age. that started in June (pers. comm. L. Chau-
vaud. University of Brest. France). The large drop in gonad content
between June and July led to decreased standard deviations and
may have been attributable to spawning in large parts of the popu-
lation. Between July and September, gonad content decreased
gradually, with relatively stable standard deviations. This suggests
that the gonads were all emptied in steps more or less at the same
rate and that no gonads increa.sed in size during this period. His-
tology showed no signs of massive resorbtion of the oocytes (Du-
inker unpublished results), so the decrease was probably caused by
spawning activity. It seemed that no energy was allocated to the
gonads during this period. If correct, this suggests that all available
energy was directed to the somatic growth and storage that was
observed. Oogenesis seemed to continue as long as the ovaries
contained some degree of oocytes of various sizes, but this may
have been fueled by recycling of the energy already present in the
gonads, in cycles of oocyte growth and atresia (Duinker unpub-
lished results). Verification of such a hypothesis, with filled go-
nads and ongoing gametogenesis but no input of "new" energy.
will cast new light on what seems to be continuous gametogenesis
in many populations of P. maxinnis. where filled gonads can be
found all the year (Comely 1974. Mason 1958. Paulet et al. 1988.
Strand and Nylund 1991. Wilson 1987). Between August and Oc-
tober, neither adductor muscle protein, gonads, or storage in-
creased. A good explanation for this cannot be given. Undoubtedly
there was no allocation of energy to the gonads until October.
Between October and December, energy was again allocated to the
gonads, because the gonads were rebuilt to a new maximum during
this period.

Several models developed for plants and animals using dy-
namic optimization technique have led to the conclusion that so-
matic and reproductive growth should be separated in time in order
to maximize reproductive output, both for semelparous species
(Cohen 1971. Mirmirani and Oster 1978. Vincent and Pulliam
1980) and for iteroparous species (Ziolko and Kozlowski 1983). In
some situations, however, a model with gradual shifts can be ben-
eficial, although in the end 100% priority of either one of the
processes is the result also from such shifts (see discussion in
Kozlowski and Ziolko 1988). We suggest that reproductive and
somatic growth in the present study were separated in time, with
the increase in adductor muscle glycogen storage from June mark-
ing a more or less complete shift from reproductive growth to
somatic growth and storage and with the start of gonad rebuilding
around October marking the shift to again allocating energy to the
gonads.

Kozlowski and Uchmanski (1987) assumed instant shifts be-
tween reproduction and somatic growth when discussing data from
Chlamys islandka. although data for the growth pattern within a
season were not given in the original data by Vahl ( 1981 ). Similar
discussions have not been given in studies of other scallop species

(e.g.. Barber and Blake 1981. Comely 1974. Mackie and Ansell
1993, Pazos el al. 1997, Robinson et al. 1981. Sundet and Lee
1984, Taylor and Venn 1979). However, in a study of Placopecten
mageUankus, shell and somatic tissue growth took place only in a
limited period after spawning (Couturier and Newkirk 1991 ). Also,
the finding of changing and strong negative relationship between
RNA/DNA in adductor muscle and gonad during conditioning of
P. magellaniciis (Paon and Kenchington 1995) may reflect similar
strategies with separate periods of reproductive and somatic
growth. Pearse et al. ( 1986) suggested for the sea urchin Strongy-
locentrotus purpiiratus that the seasonal change in growth and
gametogenesis was attributable to competition for energy and re-
sources. Furthermore, they suggested a physiological switch, pos-
sibly under photoperiodic control, that determined which process
was favored at any one time.

In this study, we did not obtain data on the composition of the
male gonad, and it is unknown to us if there were any differences
in the relative distribution of energy between the female and male
part. Visually, the development of the two parts seemed to follow
the same temporal pattern, and there did not seem to be a differ-
ence in the timing of growth of the testis and ovary, as has been
described for C. islandica (Sundet and Lee 1984). However, until
proper data have been found, this remains uncertain for P. maximiis.

Storage and Fueling of Gametogenesis

The decrease in stored energy from September to February
coincides with two energy demanding processes: the gonad re-
building between October and December; and maintenance of the
somatic and, eventually, large reproductive tissue. Numerous re-
ports exist on fueling of gonad growth in pectinids by stored re-
serves and the transfer of substrates from storage organs to the
gonad (review by Barber and Blake 1991). In the present study,
however, there seemed to be no gonad growth between December
and February, and considerably more stored energy, 27 kj, was lost
during this period as compared to the 1 1 kJ lost during the gonad
rebuilding between October and December. This suggests that for
the scallops in this study, the gonad rebuilding in autumn was
fueled largely by food available during this period and that the
more important role of the storage organs is to support mainte-
nance energy demands during winter. Maintenance of a large re-
productive mass may be costly as compared to somatic tissue
(Bruce 1926). Various strategies for fueling gametogenesis have
been reported among pectinids. including gonad rebuilding in win-
ter fueled by stored energy in P. maximus (Comely 1974) and
Chlamys opeirulahs (Taylor and Venn 1979), gonad growth in
spring fueled by available food in Chlamys septemradiata (Ansell
1974) and rapid gametogenesis in summer fueled by reserves ac-
cumulated the same spring together with available food in Pla-
copecten magellaniciis (Robinson et al. 1981) and Argopecten ir-
nidians irradians (Barber and Blake 1981 ). Furthermore, P. maxi-
mus from Galicia in Spain displayed two periods of gonad growth,
with fueling by stored reserves in winter and by available food in
summer (Pazos et al. 1997). In the present study, also the reini-
tiation of gonad growth in spring was probably fueled by available
food, since energy reserves were low and slowly increasing.
Hence, these suggestions represent yet another strategy of fueling
gonad growth largely by available food, both in autumn and in
spring, with a cessation of gonad growth during the period of food
shortage in winter. The gonad growth in autumn contrasts with the
observation of a resumption of gonad growth in winter in another

746

Strohmeier et al.

study of a population of P. maxinuts south of Bergen (Strand and
Nylund 1991 ). A good explanation for these differences cannot be
given at present. Population differences inay be possible, but the
population structure of P. maxinuts along the coast of Norway is
not known, and within the area around Bergen, the exact origin of
the scallops used in the present study could not be given by the
company that supplied them.

Energy storage and utilization was observed in both the adduc-
tor muscles and the digestive glands. Comely (1974) concluded
that the adductor muscle was the most important storage organ
when considering the energy from both protein and glycogen in P.
maximus. However, in the present study, adductor muscle protein
was stable during autumn and showed only a tendency to decline
between December and February, while the other storage sub-
strates decreased from September. This supports the view for some
scallop populations that this substrate is preferably not catabolized,
and if it is, not until the other substrates are depleted (Barber and
Blake 1991). Hence, when comparing the range in energy avail-
able from glycogen in the adductor muscle of between 1 .5 and 30
kj with between 3 and 25 kJ for the digestive gland lipid in the
present study, the importance of the two storage organs can be
regarded as equal. Muscle protein is frequently used, though, and
Pazos et al. (1997) found utilization of equal amounts of energy
from muscle glycogen, muscle protein, and digestive gland lipid.
Considerably more energy was stored during the period of somatic
growth in summer than during the period of gonad growth in
spring. This may indicate a strategy with controlled somatic
growth and opportunistic reproduction as suggested by Mac-
Donald and Thompson ( 1 985 ) for P. inageUaniciis.

The apparent cessation of gonad growth during winter contrasts
with studies of P. maximus in the bay of Seine (Lubert et al. 1991 )
and in the Fosen area in Norway (Strand and Nylund 1991), where

gonad growth also started in the autumn but continued throughout
the winter. In the present study, both temperature and food may
have been limiting factors causing the stoppage. The cessation of
gonad growth coincided with observation of clear water (own ob-
servations) indicating the reduction to low food levels of winter, at
the same time as temperature dropped from 8 to 5 °C. However, it
is likely that with elevated food and temperature levels, for ex-
ample, in hatchery conditioning, gonad growth could have oc-
curred also during this period. A picture then emerges with a
seasonal pattern in energy allocation consisting of two parts if food
and temperature are not limiting: priority of reproductive growth
from about October to June and somatic growth and storage from
June to October.

The hypothesis of separate periods of somatic and reproductive
growth remains to be tested experimentally, and comparison be-
tween populations with differences in their reproductive cycles
could provide increased understanding of underlying strategies. As
a next step, regulation of timing of the shifts should be investi-
gated. For a given population of scallops, the possible existence of
a period without allocation of energy to the gonads would obvi-
ously set restrictions on which periods of the year it is possible to
induce gonad growth with normal elevated food and temperature
conditioning.

ACKNOWLEDGMENT

We thank 0ivind Strand for critical reading and comments on
this manuscript, and Sigurd Stefansson who was one of the super-
visors for the cand. scient. thesis upon which this manuscript is
based. Thanks to Kari Elin Langeland and Edel Erdal for super-
vising the chemical analyses at the Institute of Nutrition. This work
was supported by the Norwegian Research Council, project
1 1 1388/100 and the Norwegian State Education Loan Fund.

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for an asexual annual plant model. Theoret. Popul. Biol. 17:215-231.

Wilson. J. H. 1987. Spawning of Pecten maximus (Pectinidae) and the
artificial collection of juveniles in two bays in the west of Ireland.
Aquaculture. 61:99-1 1 1.

Zar, J. H. 1999. Biostatistical analysis. 4th ed. Prentice Hall. Upper Saddle
River. New Jersey.

Ziolko. M. & J. Kozlowski. 1983. Evolution of body size: an optimization
model. Math. Biosc. 64:127-143.

Journal oj Shellfish Hc.minh. Vol. 19, No. 2, 749-755, 2000.

SETTLEMENT SITES OF JUVENILE SCALLOPS ARGOPECTEN PURPURATUS
(LAMARCK, 1819) IN THE SUBTIDAL ZONE AT PUERTO ALDEA, TONGOY BAY, CHILE

MARCELO AGUILAR AND WOLFGANG B. STOTZ*

Uiiiversiilad CatoUcci del Norte

Facultad de Ciencias del Mar

Departamento Biolngia Marina Casilla 117, Coquimho, Chile

ABSTRACT A study was made of settlement sites of the Chilean scallop Argopeclen purpunitiis on a small natural bank at Puerto
Aldea, Tongoy Bay. Chile (30 "17 'S). Four distinct habitats in the area were surveyed for juvenile scallops. Including seagrass,
sand-gravel, fme sand with polychaete tubes, and muddy bottom. The highest densities of juvenile A. purpuraius were found In areas
of fine sand dominated by polychaete tubes (Diopaint sp.), followed by areas with sand-gravel and seagrass. No juveniles were found
on the muddy bottom. Settlement was found to occur in sites having habitat complexity such as the worm tubes and seagrass. because
these provided protective refuge for the scallops, particularly in areas of soft bottom.

KEY WORDS: Chile, scallop, Argopeclen piirpiiratus. settlement site, natural bank

INTRODUCTION

Some of the keys to the understanding of population and com-
munity dynamics of rnarine benthic organisms are found in knowl-
edge of the settlemenl and postsettlement processes of their earliest
life stages (Connell 1985. Gaines and Roughgarden 1985. Menge
and Sutherland 1976. Menge and Sutherland 1987, Roughgarden
et. al. 1985. Rowley 1989. Underwood and Fairweather 1989).
This knowledge is of practical importance regarding marine spe-
cies subject to exploitation or mass culture. Knowledge concerning
the intensity of settlement and survival of settled individuals may
allow prediction of future population structure, and through this,
prognostications on the sustainability of stocks where the resource
is exposed to fisheries pressure.

The northern Chilean scallop. Argopeclen purpuraius, has in
the past been exposed to intense exploitation that has depleted its
stocks. However, recently it has been cultivated using Japanese
technology, which has significantly increa.sed the stock of this
species and resulted in the repopulation of natural banks (Stotz in
press), A small natural bank at Puerto Aldea on Tongoy Bay
showed good recuperation when placed under management by
local fishermen and showed potential for sustained exploitation.
Data on growth and production of the scallops were obtained from
this bank to develop a management plan (Stotz and Gon/alez
1997). However, to understand better the dynamics of the bank and
avoid its overexploitation. information concerning the processes of
their settlement and recruitment was required. As a first step, data
were required concerning the primary settlement sites of the ear-
liest benthic life stages of these scallops.

Information on the settlement process of this species is scarce,
Hogg (1977) found small juveniles on the red alga Rhodymenia sp,
in Herradura Bay, and DiSalvo et al. (1984) mentioned finding a
few recently settled postlarvae on ramose bryozoans {BiiguUi sp.)
in Tongoy Bay.

In general, pectinids show a strong tendency to settle on a large
variety of algae. Argopeclen irradians juveniles have been asso-
ciated with algae where they attach to fronds and algal thalli using
byssal threads, and thus avoid predation by epibenthic predators
(Thayer and Stuart 1974), Juveniles of this species were also ob-
served attached to the .seagrasses Zostera marina (Eckman 1987,

Pohle et al, 1991) and Halodule wrighlii (Irlandi and Peterson
1991),

Juveniles of other scallop species have also been observed at-
tached to algae. For example, Mason and Drinkwater (1978) found
juveniles of Peclen maximus and Chlamys opercularis byssally
attached to Lilholhamnium calcareum. These data suggest that
subtidal areas with high vegetational density are favorable zones
for scallop settlement. An example of such a zone in northern
Chile is the bed of the seagrass Helerozoslera lasmanica located
off the fishing village of Puerto Aldea.

This bed. measuring about 0.5 by 1 km. has a high density
(2.250-4.850 shoots/nr. Phillips et al. 1983) and is a potenfial
environment for the settlement of juvenile Argopeclen purpuraius
(Gonzalez 1990). The present study recorded the presence of
juveniles of A. purpuraius in different habitats within the sub-
tidal area around Puerto Aldea and evaluated the importance of
the Helerozoslera bed in the settlement process, relative to sand-,
eravel-. and silt-dominated habitats.

MATERIALS AND METHODS

Study Area

*Corresponding author. E-mail; wstotz@socompa.ucn.cl

The study was carried out near the small fishing village of
Puerto Aldea (30 °17 "S, 71 °36 "W ) in the SB extremity of
Tongoy Bay. about 60 km south of the city of Coquimbo. Chile,
SA. (Fig, I ). The physiographic location of Puerto Aldea on the
east side of Lengua de Vaca peninsula protects it from prevailing
winds and waves, although it may be exposed to (rare) storms from
the north. The fishing community of the village has protected the
small natural scallop bank (ca. 100 ha) from excessive exploitation
since 1991 as a "management area" under Chile's Law of Fisheries
and Aquaculture in return for exclusive fishing rights to the bank.
The subtidal environment in the area has a sandy to muddy bottom,
in which four distinct habitats may be recognized as follows (Fig 2 ).

Fine Sand with Seagrass

This area occurs in the western part of the management area,
extending from the coastline to approximately an 8-m depth, hav-
ing a bottom consisting of fine sand. Biologically, the community
is dominated by the seagrass Helerozoslera lasmanica in great
abundance (2.250-4,850 shoot.s/nr. Phillips et al, 1983); the habi-

749

750

Aguilar and Stotz

CHILE

Pacific

1

Ocean ,^^Coquimbo

^^ 30° S

. / 4 Guanaqueros
/\-^ Bay

/ L,^ 4 Tongoy Bay
( 71020' W

N

f'\ Tongoy Bay *

lengua!

DE VACA,
POINT '

h
Puerto A ]

Aldea *W:

(. — i.

Study site ^^

45' S

Antarctica

Figure. 1. Location of study area at Puerto Aldea, Tongoy Bay.

tat is homogeneous tliroughout, also showing scattered presence of
sand-constructed tubes of the gregarious polychaete Diopatra sp.

Sand-Gravel

This sector is located in the central part of the management
area, extending from 5-IO-m depth, and is characterized by coarse
sand and shell particles plus gravel, rock fragments, and boulders
to 1.5-m height. Algae characteristic of rocky substrates ari.se here,
including Dendrymenia sp., Cryptomenia obovata. and Closso-
phora kiinihii. In some sectors, there are aggregations of the as-
cidian Pyiini chileiisis, which may be covered with the red alga
Chondnuantluis chamissoi. which attaches to the ascidian. This
alga is also found in small patches on rocks and shells. This habitat
was the most heterogeneous of those studied.

Fine Sand with the Polychaetes

This area occupies the SE region of the management area. It
extends from the coastline to about a 5-m depth. The sediment is
fine sand, with the dominant presence of the tubiculous polychaete
species Diopaira sp., which produces a generally homogeneous
environment including labyrinthine structures providing extensive
spatial refuges.

Muddy Sand

This sector was located within the NK pari of the management
area , extending from 5- to 1 5-m depth in an ample zone of bare
muddy (fine) sand interspersed with small patches of the alga
Sarcodiotheca gaiidichaudi and small clumps of rhodymenial
rhodophytes.

Rocks

There are just few rocks in the area, most of which are covered
by the ascidian Pyiira chilensis and/or the red algae Clwndracan-
thus chainissoi.

Sampling Design

Timing of Sampling

To determine the best moment for the comparative sampling of
habitats, a survey of newly settled juveniles of Argopecten purpu-
ratus was carried out between February and May 1997. These
months had previously been cited as the setting season for this
species in Tongoy Bay (lllanes et al. 1985, Alarcon and Wolff
1991). During these months, every week, five samples of sedi-
ment, including flora and fauna, using a 95 cm" corer were taken
in different, randomly chosen sites within each of the four mayor
habitat types cited above (rocks were not included, because they
comprise a very small habitat within the area). The presence and
abundance of newly settled scallops was verified in each sample.
Once the first newly settled juveniles appeared on April 29 in some
of the samples signifying the beginning of settlement, the sampling
of habitats was .scheduled to be started 2 weeks later (May 14) to
allow the occurrence of an important ainount of settlement before
sampling. All the samples were obtained between May 14 and 21.

Sampling Strategy

Because the scale of the natural variability of newly settled
juveniles within and between habitats, as well as the scales of
variability of environmental characteristics within each habitat,
were not known (neither apparent nor obvious), a nested sampling

Habitats

Fine sand with
seagrass

Sand-gravel

Very fine sand wilji
polychaetes Diopatra spp

Muddy sand

Figure. 2. Hahitat types in the study area at Puerto Aldea. Depth
contours in meters.

Settlement Sites of the Chilean Scallop

751

design proposed for such cases by Morrisey and Underwood
(1W2) was employed, using four spatial scales. This design in-
volved the collection of 108 samples of flora, fauna, and sediment
using a 95 cm" corer. Sampling was distributed equally on differ-
ent scales within each of the four habitat types described above.
The 27 samples taken within each habitat were taken in such a way
that each scale was nested within each larger scale. Thus, each
habitat was subdivided into three sites at lOO-m distance from each
other and then subdivided again into three parcels at 10 m from
each other, and again into three replicates at distances of 1 m from
each other. The spatial distribution of samples is shown in Fig 3.
Dimensions reported for the different sampling scales may not
have been obtained with exactitude in practice and represent "best
approximations" because of logistical constraints encountered in
the fieldwork.

Collection and Analysis of Samples

Samples were collected on 14 and 21 May 1997 by means of
HOOKAH diving. A hand-held corer having 95cm" area was in-
serted about 1 cm into the bottom, and sediment was cut off into
the corer using a plastic plate. Each sample was then inverted into
a plastic bag that had been affixed to the upper end of the corer

\.

N

LaPiriata

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"-20

t

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A

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Puerto AWoa

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with an elastic band. The plastic bag was then closed with the
elastic band. Samples were returned to shore where they were
fixed with 10 % seawater-formalin for subsequent analyses. At the
laboratory, samples were washed on a 200-p.m mesh nylon screen
and then observed in a stereoscopic microscope where the number
of juvenile scallops was counted, and shell height was recorded for
each specimen.

Statistical Analysis

Given the heterogeneity of the variances, all data were trans-
formed using log (X 4- 1 ) and then a nested analysis of variance
(ANOVA) was used to compare the abundance of scallop juveniles
among the di.stinct habitats sampled, among sites within habitats,
and among plots within sites. This analysis permitted calculation
of the amount of total variation in abundance given by each sam-
pling scale with the total variation of abundance (Morrisey and
Underwood 1992). The nested ANOVA is more robust and pow-
erful when the sampling design is balanced; that is, when each
level of a factor (scale) has the same number of replicates within
it (Morrisey and Underwood 1992). In this way, it was possible to
determine if the potential differences encountered along the scale
of habitats was attributable to intrinsic factors in each habitat or to
the contribution from variances within the smaller scales.

To establish the pattern of spatial distribution of newly settled
juveniles, a chi-square analysis was performed, comparing their
distribution with a Poisson and necative binomial distribution.

RESULTS

Desciption of Juveniles

Figure. 3. Distribution of samples (a I between and (b) within the habi-
tats at the study area at Puerto Aldea. Letters represent parcels within
the sites and i. ii, iii represent replicates within the parcels. Depth
contours in meters.

Juveniles smaller than 1 .53 mm shell height had uniformly
white shells with concentric striae; both valves had circles of dark
color on their dorsal sector. The right valve was smaller in size
than the left valve, with the latter more concave than the former.
Juveniles larger than 1 .53 mm shell height had violet colored shells
and had both radial and concentric striae. The size of the valves
was similar but with the left valve continuing to be more concave
than the right; although, this difference was less notable than in the
small individuals. The shells of these small specimens begin to
resemble adult shells at a very early stage.

Timing of Sampling

No juvenile Argopecten piirpiiratKs were found in the study
area during the first 1 1 weeks of observation; recently settled
juveniles were first observed on 29 April 1997. These first indi-
viduals were found only in the sand-gravel sector at low densities
(4.6 ± 2.41 individuals m~"). Sizes of juveniles recovered from this
area in that moment varied between 0.306 and 4. 1 73 mm in height.
Thus, when the comparative sampling of habitats took place,
settlement had been occurring for at least 2 weeks before sampling.
It is assumed that, considering the small distances between habitats
compared to water movement (several m sec^"), within this time
larvae may have equally reached all sites within the study area.

Patterns of Abundance

Settlement occurred in only three of the four habitats analyzed.
There was no settlement observed in the muddy-sand sector. The
highest density of juveniles (686 individuals m"~) was found in the
habitat dominated by Diopatni sp. tubes followed by the sand-
gravel sector (206 individuals m"~). and finally the seagrass sector.

752

Aguilar and Stotz

l^UU -

^ 1000 -

IN

^ 800

3

1 600-

C

u

>. 400

c
C 200

J

1 »
1 1

L

0

L

•

Seagrass Sand-gravel Djopatra

HABITATS

Muddy

q

Sand-
gravel

Diopatra

Muddy

Seagrass

0,15 ns

2,42 '"

0,82 ns

Sand-gravel

2,27 *"

0,98 ns

Diopatra

3,25 "*

Figure 4. Mean density of juvenile Argopecten purpuratus in different
habitats at Puerto Aldea. Table gives the results of Tukey's test on
pairs. ***significant difference, ns = no significant difference.

with 174 individual m"" (Fig 4). The ANOVA showed a signifi-
cant difference among the habitats, with no significant differences
among sites within habitats or among plots within sites (Table 1 ).
The power of the analysis was 0.97 with a a = 0.01. This result
gave statistical support to the hypothesis that differences among
habitats were attributable to intrinsic characteristics of each and
not to random variation or to variation among sites within habitats
or among plots within sites. Despite this finding, high total per-
centage variation (."^8.7Vf ) was observed at the I- m scale. It was
shown, using Tukey's test, that abundance of scallop juveniles in
the Diopatra sp. -dominated habitat was significantly higher than in
the other habitats sampled. There were no significant differences
between sand-gravel and seagrass areas.

Size Structure

The smallest scallop juveniles were found in the Diopatra sp.-
dominatcd habitat (Fig. ^). The sizes of the juvenile scallops from
all habitats tluctuated from 0.350 to 6.5.35 mm in height (size
classes from 0.25 to 6.75 mm). A size/frequency histogram
showed the most abundant size class to be that measuring 0.75 mm

and 87% of the sampled individuals measured less than 1.75 mm
in height (Fig 6).

Patterns of Spatial Distribution

The distribution of Juvenile scallops in the bank was signifi-
cantly different from random (Poisson), and resembled a negative
binomial distribution (x": 24.3, degrees of freedom (dof).: 1, P >
0.001 ). This observation was supported when utilizing the Morisita
index, which suggested a pattern of aggregated distribution (Table
2). Analysis of distribution of recently set scallops by habitat type
produced a negative binomial distribution; that is, aggregated dis-
tributions within each different habitat (seagrass: x": II. 02. dof:
20, P > 0.05; sand-gravel: x": 25, dof: 20, P > 0.05: Diopatra: x":
29. dof: 20, P > 0.05). The degree of aggregation is similar for the
different habitat types, but the different habitats show different
degrees of environmental heterogeneity. For example, the sand-
gravel habitat is much more heterogeneous than the other habitat
types. This suggests that the aggregation of scallops is an attribute
attributable to a behavior of the scallops, not an attribute of the
habitat. It is probable that new arrivals settle close to indi\iduals
already settled.

DISCUSSION

Larvae of A. purpuratus. cultivated in the laboratory at 14 °C,
reached metamorphosis after 30 days of culture, at a length of
about 231 ± 10 p.m (Bellolio et al. 1993). In the laboratory, post-
larvae were observed in 30 days to attain about the same size as the
smallest specimens obtained by us at the Puerto Aldea site (< 1.75
mm). Once settled, juveniles of A. purpuratus remained attached to
settlement sites by their byssal threads until they reached 8-10 mm
in height (Navarro et al. 1991 ). A similar size ( 1 1 mml was noted
for in A. irradians (Garcia-Ezquivel and Bricelj 1993). Based on
these observations and the small sizes of the juvenile scallops
found in our sampling (< 7 mm shell height), it is probable that
these juveniles had remained at their original sites of settlement. It
was. therefore, assumed that the pattern of abundance and distri-
bution of juveniles collected in the present study reflected the
natural settlement pattern and could be used as an indirect measure
of this, as suggested by Rowley ( 1 989 1. Thus, prevalence of juve-
nile A. purpuratus in habitats dominated by Diopatra sp. tubes, the
seagrass Heterozostera tasmanica. and in the sand-gra\el area
reflect preference for initial settlement in these areas. No settle-
ment was recorded for the muddy bottom in our study, as noted for
other Peclinid species that fail to settle on this type of bottom (e.g.,
Clilaiiixs varia. Pecten maximus, A. opercularis: Burnell 1991).
Although settlement may have occurred on this substrate, followed
by mortality, no empty shells were encountered in our sampling.

TABLE 1.

Analysis of variance of the abundance of newly settled juveniles of Argopecten purpuratus in different habitats at Puerto Aldea (Chile)

(significance level sA P < O.OUI).

Source of Variation

Degrees of Freedom

S,S.

M.S.

F,

Between habiials
Between sites within habitats
Between plots within sites
Error

3

8

24

72

92.70

30.40

m.6***

48.65'7f

12.62

1..SS

3.9 ns

5.84%

9.70

0,40

0.47 ns

6.85%

62.15

O.Sd

38.66%

Total

107

177.18

Settlement Sites of the Chilean Scallop

753

O

c

3

cr

0)

40
30

20 -
10 •

0 •
40
30
20
10

0

60
50
40

30

20

10

0

50
40
30
20
10
0

SEAGRASS
n = 37

SAND-GRAVEL
n = 49

DIOPATRA

n = 167

TOTAL
n = 253

120

100

80

60

40

20

0

0.26 1.25 2.25 3.25 4.25 5.25 6.25

Shell Height (mm)

7,25

o
o

<

Figure. 5. Size structure of juvenile A. purpuratus in each liabitat. Size
structure of all sampled juvenile A. purpuratus at Puerto Aldea (;i =
253) and cumulative frequencies sho»ing the class-mark (arrow) at
which 85 % of the individuals are included.

which would support such a hypothesis. Because this habitat is at
a greater depth (10-15 m depth) as compared to the others, the
larval supply may have been poor, because larvae have been
shown to prefer settlement at lesser depths.

The habitats in which settlement was found to occur have in
common an erect ministructure that may provide important refugia
for scallop settlement, particularly over .soft bottoms. This has
been cited for diverse species of pectinids, where settlement has
been reported on algae (Ambrose and Irlandi 1992, Mason and
Drinkwater 1978, Minchin 1976), sea grasses (Eckman 1987,
Irlandi and Peterson 1991, Pohle et al. 1991, Thayer and Stuart
1974). such metazoans as bryozoans and hydroids (Allen 1979).
and extensive metazoan-produced substrates such as polychaete
tubes iSiibella pavonina) or ascidian tunic {Ascidiella aspersa)
cited by Bumell (1991). Settlement on erect structures favors the
growth of the scallop juveniles, because they are exposed to more
water movement than near the seabed, which gives them a better
quality diet (Eckman and Peterson 1989). Moreover, their elevated
position allows them to evade predation by epibenthic predators
(Thayer and Stuart 1974) as well as to avoid burial in soft sedi-
ments (Ambrose and Irlandi 1992. Pohle et al. 1991). The com-
plexity of the habitat afforded by the erect structures may also
afford some protection from predation, as found by Pohle et al.
( 1991 ). who demonstrated a direct relation between survival of A.
irradians and the density of stems of Zostera marina: a similar
relationship was demonstrated for several species between density
of algae or seagrass and loss to predation. For example, a decrease

Shell height (mm)
Figure 6.

in foraging behavior of the pinfish Lagodon rhoinboides has been
observed as an effect of the presence of benthic macrophytes
(Stoner 1982, Main 1987). Similarly, inhibition of foraging by
macrophytes was also observed in Penaeus duorarum. fishes, and
decapods (Leber 1985), as well as for amphipods living associated
to algae (Nelson 1979). The major occurrence of settlement of A.
purpuratus over a soft bottom inhabited by Dioptra sp. tubes may
indicate a preference for a less complex habitat than the Hetero-
zostera stems in favor of one with more permanence. Seagrass
stems undergo continual death and renovation, increasing the prob-
ability of loss of the scallops (Eckman 1987. Pohle et al. 1991).
The Diopatra sp. tubes, constructed of sand and shell fragments
may represent habitat more secure from perturbation (Woodin
1978). In addition, water flow over the Diopatra tubes may be
better than within a dense .seagrass meadow or algal bed. The tubes
produce little resistance to water flow, because they emerge only
slightly from the bottom. Moreover, selective adaptation for the
Diopatra sp. microhabitat is more probable, because the occur-
rence of Heterozostera in Chile is unusual, with the bed at Puerto
Aldea, and a second smaller one farther north, being its only
known occurrence on this coastline (Phillips et al. 1983, Gonzalez
and Edding 1990).

Settlement of scallops on the algae Chondracanthus chamissoi
in the study area remains problematical, because local fishermen
harvest this alga commercially in spring and summer. If scallop
settlement should occur in periods before the algae harvest, im-
portant mortality of juvenile scallops may occur. Thus, it is im-
portant to carry out studies in the future that provide data useful in
minimizing this potential impact on juvenile scallops.

The pattern of distribution of juvenile scallops at Puerto Aldea
suggested the occurrence of aggregated (patchy) settlement. Al-
though the habitats were different in their degrees of complexity,
with the sand-gravel areas more heterogeneous than the others, the
degree of distribution of scallops within the distinct habitats was
similar. This fact suggested that aggregated settlement was a prop-
erty of the species, wherein the settlement of some individuals
induced further settlement by their congeners.

The areas containing the juveniles were not correlated with
sectors containing high densities of adults (Stotz and Gonzalez
1997). Differential mortality may have occurred between habitats
as observed by Luckenbach (1984) for an estuarine bivalve. Be-
cause detached scallops are active swimmers, they probably dis-
tribute them.selves to habitats most favorably suited to their growth
and survival. This suggests that postsettlement processes were oc-

754

Aguilar and Stotz

TABLE 2.

Chi-square analysis comparing the distribution observed for A. piirpiiraliis juveniles with a negative binomial and Poisson distributions
using data from 105 cores obtained in the study area. (***) expected frequency significant at P < 0.001; ns = nonsignificant.

Negative Binomial Distribution

Poisson Distribution

No Scallops
bv Core

Observed
Frequency

Expected
Frequency

No Scallops
bv Core

Observed
Frequency

Expected
Frequency

0
1
2
3
4
5
6
7 and more

47

12

15

6

3

6

6

10

47.00
16.33
9.90
6.85
5.04
3.84
2.99
12.18

0
1
2
3

4
5
6
and more

47

12

15

6

3

6

6

10

8.04

20.65

26.54

22.74

14.61

7.51

3.22

1.70

9.36 ns

262.19***

Mean: 2.57

Variance: 16.56

No samples: 105

No scallops: 270

Ix-: 2412

K: 0.4016447 to negative binomial

Morisita index (Im): 3.1

curring that later determined the distribution of the adults as sug-
gested by Rowley (1989).

ACKNOWLEDGMENTS

The authors are grateful to the fishermen's organization of
Puerto Aldea for the facilities given during the fieldwork. special

recognition to Sergio Gonzalez, who helped with the diving and
collection of samples, and to Domingo Lancellotti who helped
with the statistic design. Thanks are given also to Louis DiSalvo.
who translated the paper and provided editorial advice. Financial
support was partially received by the Regional Government,
through project BIP 20065131.

LITERATURE CITED

Alarcon. E & M. Wolff 1991. Esludio biologico pesquero sobre el recurso
de osliones {Argopcclen purpiimms) dc bahi'a Tongoy durante el
femimeno El Niiio 1982-83. liwest. Pesq. 32:167-173.

Allen, D. M. 1979. Biological aspects of the Calico scallop. Aiiioitecten
!>ibbus. determined by sp. at monitoring. Nautilus. 94:107-1 19.

Ambrose, W. G. & E. A. Irlandi. 1992. Height of attachment on seagrass
leads to trade-off between growth and .survival in the scallop Ar-
gopecten irnulicms. Mar. Einl. Prof>. Ser. 90:45-51.

Bellolio, G., K. Lohrniann & E. Dupre. 1993. Larval morphology of Ihe
scallop Arffopecteii puipiiiiiius as revealed by scanning microscopy.
VcHiiei: 36 (4):332-342.

Burnell. G. 1991. Annual variations in the spawning and settlement of (he
scallop Chhimys varia (L.) on the west coast of Ireland. /h:S. E. Shimi-
way. (ed.). An Inlenuitional Compendium of Scallop Biology and Cul-
ture. World Aquaculture Society and National Shellfisheries Associa-
tion. World Aquacullure Workshops, Number 1. 357 pp.

Connell. J. H. 1985. The consequence of the variation in initial settlement
vs. poslselllcmenl mortality in rocky intertidal communities. J. E.xp.
Mar. Biol. hcol. 93:11^5.

Di Salvo. L. H., E. Alarcon, E. Martinez it E. Urihc. 1984. Progress in
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Jiiiirmil of Shclllhli Research. Vol. 19. No. 2, 757-764, 2000.

A COMPARISON OF SIZE SELECTIVITY AND RELATIVE EFFICIENCY OF SEA SCALLOP,
PLACOPECTEN MAGELLANICUS (GMELIN, 1791), TRAWLS AND DREDGES

DAVID B. RUDDERS, WILLIAM D. DUPAUL, AND
JAMES E. KIRKLEY

Virginia Institute of Marine Science, School of Marine Science. College of
William and Mary, P. O. Box 1346, Gloucester Point, Virginia 23062

ABSTRACT During August and September 1997 and May 1998. three comparative fishing experiments were conducted aboard
commercial sea scallop trawl and dredge vessels to assess the etTicacy of gear restrictions found in Ainendment 4 to the Sea Scallop
Fishery Management Plan (SSFMP). This amendment involved certain gear restrictions including minimum mesh and ring sizes and
maximum gear widths and was intended to equate the perfonnance of sea scallop trawls and dredges with respect to size .selectivity
and efficiency. Stati.stical analysis indicated that selectivity and efficiency were not equal for the two gear types. While absolute gear
size selectivity could not be estiinated. there was clear evidence of differential relative size selectivity between the two gears. Relative
harvest efficiency values shifted at 90 to 95 mm shell height. Trawl vessels were more efficient capturing sea scallops less than 90 mm,
and dredge vessels were more efficient capturing sea scallops greater than 90 mm. This shift in relative harvest efficiency coupled with
an observed cull size at 70 to 75 mm shell height resulted in the trawl vessels being more dependent on age 3 sea scallops with shell
heights of 70 to 90 mm. Operational differences observed between the two gear types restricted sea scallop trawl vessels to areas of
smooth substrate. Large differences in both relative efficiencies and operational requirements will present considerable impediments
to the desired outcomes of having equivalent performance between gear types.

KEY' WORDS: sea scallops, Ptacopecwn nuigcllaiiicns, fishing gear, relative efficiency

INTRODUCTION

Wild populations of the sea scallop, Placopecten mageUaiiicits,
occur exclusively on the continental shelf of the northwestern At-
lantic Ocean from the Canadian Maritimes to Cape Hatteras. North
Carolina (Posgay 1957). Within the Exclusive Economic Zone
(EEZ) of the United States, the commercial sea scallop fleet is
comprised of vessels using both dredges and modified otter trawls.
During 1998, dredge vessels operating coastwide. accounted for
90% of total landings, while trawl vessels focused operations on
the softer substrates of the mid-Atlantic resource area tallied the
remaining 10%. Total sea scallop landings for 1998 were 5.549
metric tons of shucked meats valued at $74,8 million (NEFMC
1999).

Sea scallop landings peaked in 1990 when a record high 17,500
metric tons of shucked meats worth $149 million were landed
(NEFMC 1999). The sea scallop fishery, however, has historical-
ly been characterized by cycles of high and low production due
to fluctuations in recruitment and varying levels of fishing ef-
fort (Dickie 1955). The onset of more frequent and extreme
fluctuations in landings during the late 1960s and early 1970s.
coupled with dramatic increases in ex-vessel prices, effort, and
capital prompted federal regulatory measures (NEFMC 1982).
Since May 1982. the sea scallop fishery was managed under the
provisions found in the Sea Scallop Fishery Management Plan
(SSFMP).

Regulatory measures found in the SSFMP initially focused on
controlling age at entry in an effort to maximize yield per recruit
(NEFMC 1982). Regulations required an average meat count for
shucked scallop meats and a minimum size for shell-stocked sea
scallops (sea scallops landed in the shell). These regulations, how-
ever, proved to be inadequate and resulted in the continued ex-
ploitation of small sea scallops (>40 meats per pound. MPP), high
levels of fishing mortality (F). and allegations of inequity between
dredge and trawl vessels (Naidu 1987. Shumway and Schick 1987,
DuPaul et al. 1989b, 1990, Kirkley and DuPaul 1989, Schmitzer et

al. 1991), To address these problems. Amendment 4 to the SS-
FMP. adopted in 1994, changed the management strategy to an
effort control program in an attempt to reduce F by 70% over a 7
year rebuilding period (NEFMC 1993), The primary measures of
Amendment Number 4 included the establishment of a limited
access fishery and the institution of days at sea restrictions
(NEFMC 1993). Supplemental measures included gear restric-
tions, crew size limits, vessel replacement restrictions, and catch
limits for non-permitted vessels (NEFMC 1993).

Although the management strategy was changed by Amend-
ment 4, the objective of establishing age at entry was again ad-
dressed. Modifications to the two gear types in the fishery replaced
the meat count and shell height restrictions in an attempt to control
age at entry. These modifications would theoretically allow juve-
nile sea scallops (<70-mm shell height) to escape the gear, rather
than relying on the crew to discard them. Sea scallop dredges were
required to meet specific criteria of ring size, chafing gear, twine
tops, and maximum dredge width. The configuration of sea scallop
otter trawls were restricted on the basis of minimum mesh size,
mesh orientation, and maximum trawl sweep.

The gear restrictions found in Amendment 4 were guided by
the assumption that these modifications would result
in equivalent performance between trawls and dredges with re-
spect to size selectivity and harvest efficiency. Equivalent perfor-
mance of the two gear types addresses a management objective
attempting to control sea scallop age at first capture and a policy
mandate that requires equity between user groups. There are no
data to support the assumption that Amendment 4 gear restrictions
would achieve the desired result. Comparisons of sea scallop
dredge and trawl gear have been conducted by Kirkley (1986) and
DuPaul et al. ( 1989c), however, the gear consisted of smaller mesh
and ring dimensions than required by Amendment 4.

The objective of this study was to examine size selectivity and
relative efficiency of sea scallop trawls and dredges as regulated
under Amendment 4 to the SSFMP. This comparison will establish
whether Amendment 4 gear restrictions are effective in both con-
trolling sea scallop age at entry to the fishery.

757

758

Rudders et al.

and results in the equitable treatinent of user groups operating in
the U.S. sea scallop fleet.

MATERIALS AND METHODS

The study area was located along the continental shelf off the
East Coast of the United States from Sandy Hook. New Jersey to
the Virginia/North Carolina border (Figure I ). Water depths in the
study area ranged from 25 to 45 fathoms (46-82 m). Seabed to-
pography and substrate composition were uniform throughout the
area, dominated by level expanses of mud and sand with scattered
areas of large boulders. This general area is considered a tradi-
tional .sea scallop fishing ground, however, specific areas for the
comparative fishing experiments were located using the local
knowledge of the participating commercial vessel captains.

Gear deployment and vessel design constraints prevented a
dredge and an otter trawl from being towed by the same vessel
simultaneously. The comparison of the two gear types was con-
ducted by sampling with both a commercial dredge vessel and a
commercial otter trawl vessel. Utilizing the parallel fishing method
the two vessels fished the same ground at the same time and
sampled from a single population of sea scallops (Pope et al.
1975). To ensure that the criteria of the sampling design was met,
data from tows which were sampled, but did not occur in the same
area at the same time were subsequently excluded from analysis.
The study consisted of three comparative fishing cruises conducted
as an adjunct to normal commercial fishing trips between August
1997 and May 1998. The only modification to a commercial fish-
ing trip being that both vessels operate in the same area at the same
lime and use Amendment 4 compliant fishing gear.

Figure 1. Map depictint> the location of the three comparative trips.

A description of the New Bedford style offshore sea scallop
dredge is given by Posgay ( 1957) and Bourne (1964). Pursuant to
Amendment 4 restrictions, the chain bags of all dredges were knit
with rings that had an inside diameter no greater than 3.50" (89
mm). Standard 5.50" (140 mm) diamond mesh twine tops were
used on all dredges, and split tire shingles were used on the bottom
of the chain bags as chafing gear.

The sea scallop otter trawl vessels utilized two trawls towed
from separate warps. Wood trawl doors with dimensions of 120" x
40" (3.05 X 1. 01 m) were attached directly to the wings of the nets.
Steel sleds (approximately 400 lb. [181 kg]) in place of trawl doors
were used on the inner wings of the two nets. The bodies and
codend of the trawls consisted of 5.50" (140 mm) diamond mesh.
Varying configurations of sweep chains ranging from 1/2" (12.7
mm) to 5/8" ( 15.9 mm) were used on the footropes of the trawls.
A 1/2" (12.7 mm) tickler chain was also used. Chafing gear con-
sisted of a doubled 1 -m piece of nylon attached to each mesh on
the belly of the codend. The length of warp fished varied with
depth, but generally was held at a warp length/depth ratio of 3:1.

Deck operations were conducted under near normal commer-
cial fishing conditions. For all tows, the catch from each gear was
dumped on the deck, culled, shucked, bagged, and placed on ice or
frozen until the termination of the trip. For comparative tows that
were sampled, the crew culled the catch for sea scallops to be
retained for shucking. A subsample of up to two baskets ( 1 basket
equals approximately 1.5 bushels [53 L]) of retained sea scallops
were set aside for length frequency analysis. Discarded sea scal-
lops were subsampled as appropriate depending on the volume of
trash and number of discards present. A shell height for each
sampled scallop was taken in 5 mm intervals from the umbo to the
ventral margin of the shell using a NMFS sea scallop measuring
board.

Catch data were standardized to reflect harvest per unit area
covered by the fishing gear. Linear distance traveled for each tow
was calculated as the product of towing speed and low duration.
Area swept for each tow was estimated as the product of linear
distance traveled and gear width. Dredge width varied between
trips and was either 14 ft. (4.6 m) or 15 ft. (4.5 m). Trawl mouth
spread was calculated as one-half the average of the headrope
and the footrope (DeAlteris 1998). Kostyunin (1971) reported
the fishing spread of modern trawl nets to be from 45*?^ to 509!- of
the headline length corroborates this estimate. The estimates of
area swept by the gear were then con\ erted to hectares ( 1 ha =
10.000 m-).

Relative harvest efficiency was calculated as the percentage
difference in the number of sea scallops captured per hectare by
the trawl relative to the dredge for each shell height size class.
Relative production efficiency was examined with respect to the
number of sea scallops harvested, production of scallop meats
(grams), and average MPP at both observed cull sizes and at hy-
pothetical cull sizes of 70, 80. and 90 mm shell heights. To esti-
mate production of scallop meats and MPP, a shell height:meat
weight allometric relationship for the mid-Atlantic region was ap-
plied to llie midpoints of ihe shell height intervals (NEFMC 1982):

W = 5.929 X 10 " C^'\

L = shell height and W = meat weight. Statistical differences in
mean number of sea scallops harvested, mean production rates,
and average MPP between the gear types were determined by a
two tailed Student's ; test at the 5'^/( significance level.

Size selecli\ ilv in the sea scallop fishery occurs as two different

Comparison of Sea Scallop Gear Types

759

TABLE 1.
Summary of operational procedures for comparative gear trials.

Trip 1

Trip 2

Trip 3

Date

August 8

through

18. 1997

September 8 through

18. 1997

Area

Virginia Beach

. va

Hudson

Canyon

Vessel

Stephanie B

Triangle 1

Carolina Breeze

Capt. AT

Gear

Dredge

Trawl

Dredge

Trawl

Tows on

trip

199

80

286

99

Comparative

tows

77

34

49

30

Scallops

measured

31,689

47,385

13,685

22.665

May 13 through 18. 1998
Chincoteague, VA
Carolina Clipper Triangle 1

Dredge Trawl

121 48

29 14

24,455 24,929

processes: that imposed by the type and characteristics of the fish-
ing gear and that imposed by the crew culling the catch. Estimates
of relative si/e selectivity and efficiency were inferred for the two
gear types from the numbers of sea scallops harvested and shell
height frequency distributions. The size selection characteristics of
the crew were determined by collecting the data in a manner that
differentiated between sea scallops that were retained for shucking
or discarded. The crew size selection curve was calculated as the
ratio of the number of sea scallops retained by the crew for shuck-
ing to the total number of sea scallops captured for each shell
height. Linear regression of normal deviates versus shell height
was performed to determine the 25%. 509^, 75%, and 100% re-
tention shell heights and selection range. Selection range was de-
tmed as the difference between the 75% and 25% retention shell
heiahts.

RESULTS

Trip Data

Data for the study was collected on three comparative fishing
trips during August and September of 1997 and May of 1998. Each
comparative trip was considered an individual set of trials due to
differences in geographic location and sea scallop abundance and
size composition. Operational procedures for each set of trials are
shown in Table 1 . Sea scallop shell height frequencies for each
individual trip are shown in Figure 2.

Crew Size Selection

The estimated selectivity parameters for sea scallops retained
by the crew for shucking with associated size .selectivity curves are
shown in Table 2 and Figure 3. Although the size composition of
the target species varied considerably over the three trips, crew
size selection remained relatively constant. The shell height at
which a scallop had a 50% chance of being retained for shucking
(L^i,) ranged from 69.3 to 77.5 mm. Scallop sizes from Ly, to L,^
ranged from 3.6 to 12.0 mm, which indicated that the crew selec-
tion process was relatively knife edged. Size selection of sea scal-
lops was complete (L,,,,,) at shell heights that ranged from 79.4 to
109.7 mm. However, larger sea scallops (>90 mmi classified as
discards were probably the result of oversights by the crew.

Relative Efficiency

Relative harvest efficiency for each trip is shown in Figure 4.
The relative harvest efficiencies of the gear types were approxi-
mately equal at a shell height range of 85 to 95 mm. Sea scallop
catch per unit effort at a shell heights of 85 to 95 mm were not

statistically different (P > 0.05) between gears for all three trips.
Trawl vessels harvested sea scallops less than 85 to 95 mm shell
height more efficiently and sea scallops greater than 85 to 95 mm
shell height less efficiently relative to the dredge vessels. Relative
harvest efficiency values for small sea scallops (<30 mm shell
height) and large sea scallops (>130 mm shell height) meant little
as sample sizes were limited.

Catch statistics for each trip calculated using the observed cull-
ing practices of the crew are shown in Table 3. Length frequency
distributions for sea scallops taken by dredges and trawls vessels
differed appreciably. However, the total number of sea scallops
harvested and retained per hectare swept by the trawl gear was not
statistically significant (a = 0.05). Greater numbers of larger sea

120
100

Trip 3

I

jjj.

I Dredge (C. cupper)
) Trawl (Triangle I)

I

► ■-,I-L>-

■y ■y' -B^ i?' c> »v ^-v* ^v i> ^> ^> ^ ^ ^

Shell Height (mm)

Figure 2. Shell height frequency distributions (mean ± SE) for each
comparative trip standardized to one hectare covered by the gear.

760

Rudders et al.

TABLE 2.

Crew size selection lengths for all comparative gear trips. Values represent shell heights in millimeters at which a scallop had a 25%, 50%,

75%, and 100% probability of being retained by the crew for shucking.

Trip I

(August 1997)

Trip 2

(September 1997)

Trip 3 (May

1998

Stephanie B

Triangle I

C. Breeze

Capt. AT

C. Clipper

Triangle I

Dredge

Trawl

Dredge

Trawl

Dredge

Trawl

Selection lengths

L25

73.0

71.8

67.5

68.0

70.5

74.3

^50

76.5

75.9

69.3

71.6

76.5

77.5

L7.

80.0

80.0

71.1

75.2

82.5

80.6

^100

95.9

98.6

79.4

91.3

109.7

94.9

Selection range L75-L25

7.0

8.2

3.6

7.2

12.0

6.3

scallops with larger meats harvested by the dredge vessel resulted
in significanlly higher (P < 0.0.5) production rates during August
1997. Differences in production rates for September 1997 and May
1998 were not statistically significant at the 5% level. MPP from
the trawl vessels were significantly higher (P < 0.05) than meat
counts from the dredge vessels for all trips.

The trawl ves.sel on the May 1998 trip took 35.4^^ more sea
scallops per hectare than the dredge vessel. This difference was
due to large numbers of 70 to 90 mm shell height sea scallops
which constituted 92% and 58% of the catches of the trawl and
dredge boats, respectively. Larger meats from the greater numbers
of 90+ mm sea scallops captured by the dredge boat, however,
resulted in the trawl boat being 8% less efficient relative to the
dredge boat with respect to grams of meats produced per hectare.

During the August 1997 and September 1997 trips. 70 to 90
mm sea scallops were less abundant. Sea scallops in this size range
constituted 57% and 62% of the catch by the trawl boats and 32%
and 28%i of the dredge boats for the August 1 997 and September
1997 trips, respectively. Trawl boats on these two sampling trips
were 6.5% and 0.7% less efficient than those using dredges with
respect to the number of sea scallops caught per hectare due to the
paucity of 70 to 90 mm sea scallops. Differences in the number of
large sea scallops harvested resulted in the trawl boats being 27.47fi

v" s^ .vv ^> >v .y" .-^ ,^^ i> i> ^^ ^,:> ^ ^^ ^i> ^^
Shell Heighl Inimi
Figure 3. .Size selection curves for the crew culling process.

and 25.3% less efficient relative to the dredge boats with respect to
grams of scallop meats produced per hectare.

Relative Efficiency al 70, 80. and 90 mm Shell Heights

Relative production efficiency was also examined by imposing
hypothetical culling sizes of 70, 80, and 90 mm shell heights to
examine the effects of possible changes in scallop age at entry to
the fishery. This analysis further demonstrated the effect that dif-
ferential catch compositions had on the comparison between the

Trip 1

TTT'

h.

■11IIIIIIJIL

■

Trip2

urn) -

1500

1004t

■

500

ol

llll...ll-

400
300

Trip 3

200

100-

lllll

1

•100

1"

•lllllllll

0? .-v'-

jy .pV <.^' ^C^' s^"^- ^i-" ,!^'

Shell Height (mm)

KIguro 4. Relative harvest ofliciencv of the .5.50" (140 mm) diamond
mesh sea scallop otter trawl relallve (n the 3.50" (89 mm) ring sea
.scallop dredge for all comparative trips.

Comparison of Sea Scallop Gear Types

761

TABLE 3.

Mean number of sea scallops harvested, mean grams of scallop meats produced, and average meats per pound (MPP) for all comparative
gear trips. N'alues were calculated using the observed culling practices of the crev» with the data standardized to reflect catch per hectare

covered by the gear.

Trip 1 (August 1997)

Trip 2 (September 1997)

Trip 3 (May 1998)

Stephanie B.
Dredge

(« = 34)

Triangle I
Trawl

(n = 77)

C, Breeze
Dredge

(/I = 30)

Capt. AT
Trawl

(» = 49)

C. Clipper
Dredge

Oi = 29)

Triangle I
Trawl

(n = 14)

Harvest (#/ha.)
Production (grams/ha.)
MPP

69.0 ± 2.4

1.068.4 ±33.5*

35.6 ± 0.4*

64.5 ± 3.7

776.1 ±42.6*

44.4 ± 0.6*

59.9 ± 2.8

908.5 ±44.8

35.9 ± 0.6*

59.5 ± 5.5

687.9 + 61.9

46.8 ± 1.4*

96.4 ± 7.7

1.298.0 ±73.4

45.0± 1.1*

130.5 ± 17.3

1,194.2 ± 141.9

56.3 ± 0.7*

two gear types. Catch statistics for each trip calculated using the
imposed cull sizes of 70. 80. and 90 mm shell are shown in Ta-
ble 4.

During August and September of 1997, the sea scallop resource
consisted of few age 3 sea scallops (70-90 mm sea scallops) and
relatively low numbers of age 3+ (>90 mni)sea scallops. For these
two trips, the total number of sea scallops caught per hectare was
not significantly different {P > 0.05) at the 70 and 80 mm shell
height cull sizes. When the cull size was increased to 90 mm, the
dredge vessels captured significantly more (P < 0.05) sea scallops
per hectare than did the trawl vessels. The dredge vessels were able
to produce significantly more (P < 0.05) scallop meats at all cull-
ing sizes. These results reflected the differing relative harvest ef-
ficiencies and sea scallop abundance and size distribution at the
time of the two trips.

The trawl vessel captured and produced significantly more (P <
0.05) sea scallops and meats than the dredge vessel at the 70 irtm
cull size in May 1998 due to the presence of large numbers of 70
to 90 mm .sea scallops. When the cull size was increased to 90 mm
and 70 to 90 (age 3) sea scallops were excluded from the analysis,
dredge vessels captured and produced significantly more iP <
0.05) sea scallops and scallop meats relative to than the trawl
vessels. Meat counts from the trawl vessels were significantly

higher (P < 0.05) than meat counts from the dredge vessels for all
trips at all culling sizes.

DISCUSSION

The sea scallop resource is in a constant state of flux as a result
of variable recruitment, rapidly growing individuals, and high rates
of fishing mortality. Sea scallop abundance and size distribution
can change dramatically, even during the time scale of this study
(August 1997 to May 1998). Despite the changing resource con-
ditions, two general patterns were observed during the three trips.
The two resource conditions differed with respect to the presence
or absence of an age 3 (70-90 mm shell height) recruiting year
class of sea scallops.

Sea scallops recruit to the fishery at 3 years of age. Three year
old sea scallops, which in the mid-Atlantic region have a shell
height of roughly 70 to 90 mm, represent an important age class in
the fishery. As sea scallops grow to 70 to 75 mm shell height, they
begin to be retained by commercial vessels (DuPaul and Kirkley
1995, DuPaul et al. 1995). Recent high levels of fishing mortality
have reduced the abundance of older sea scallops in the population,
and 3-year-old sea scallops that recruit to the gear each year have
primarily supported the fishery (Serchuk et al. 1979, NEFMC
1993).

TABLE 4.

Mean number of sea scallops harvested, mean grams of scallop meats produced, and average meats per pound (MPP) for all comparative
gear trips. Values are calculated using assumed culling sizes of 70, 80. and 90 mm shell heights, standardized to reflect catch per hectare

covered by the gear.

Trip 1 (August 1997)

Trip 2 (September 1997)

Trip 3 (May

1998)

Stephanie B.

Triangle I

C. Breeze

Capt. AT

C. Clipper

Triangle I

Dredge

Trawl

Dredge

Trawl

Dredge

Trawl

(11 = 34)

(/I = 77)

Ut = 30)

(n = 49)

in = 29)

(« = 14)

Harvest (#/ha.)

Cull at 70 mm

71.7 ±2.5

70.0 ± 4.0

61.5 ±2.9

60.6 ± 5.6

110.1 ±9.7*

264.7 ± 50.5*

Cull at 80 mm

67.9 ± 2.3

59.2 ± 3.4

58.4 ± 2.7

49.6 ±5.1

67.3 ± 3.4

71.3 ±6.8

Cull at 90 mm

46.6 ± 1.5*

26.5 ± 1 .7*

43.2 ± 2.3*

20.9 ±2.2*

40.0 ±2.1*

10.3 ± 1.0*

Production (grams/ha.)

Cull at 70 mm

1.088.6 ±34.3*

816.2 ±44.5*

918.1 ±45.4*

688.7 ±62.1*

1.399.5 ±84.8 2.1 1 1.1 ± 3.54.7

Cull at 80 mm

1.062.0 ±33.3*

743.4 ±41.4*

897.6 ±44.88*

61 1.7 ±60.0*

1.096.5 ±53.3*

768.6 ±70.7*

Cull at 90 mm

834.3 ± 28.6*

397.0 ± 24.9*

734.2 ± 43.3*

31 1.7 ±32.5*

824.0 ±45.1*

170.3 ± 15.4*

MPP

Cull at 70 mm

36.7 ± 0.4*

46.0 ± 0.7*

36.9 ± 0.7*

46.4 ± 1.3*

47.4 ± 1.2*

63.7 ± 1.1*

Cull at 80 mm

34.9 ± 0.4*

41.3 ±0.3*

35.1 ±0.6*

41.0 ±0.9*

35.6 ± 0.5*

48.2 ± 0.2*

Cull at 90 mm

30.0 ± 0.3*

34.0 ± 0. 1 *

3 1 .3 ± 0.4*

33.6 ± 0.4*

25.8 ± 0.4*

31.3 ±0.7*

762

Rudders et al.

Shell height distributions for trips I and 2 portray a population
that was characterized by a low abundance of age 3 sea scallops.
The absence of large numbers of 3-year-old sea scallops had a
large impact on the relative production rates of the two regulated
gear types. The reduced ability of the trawl to capture sea scallops
greater than 90 mm relative to the dredge, coupled with a mini-
mum observed crew cull size of roughly 70 to 75 mm resulted in
trawl boats being dependent upon 3-year-old sea scallops for pro-
duction. In the absence of large numbers of age 3 sea scallops,
production rates of the dredge vessels in terms of numbers of sea
scallops captured per unit area and weight of scallop meats pro-
duced exceeded those from the trawl vessels during the first two
trips.

During August 1997 (trip 1), large numbers of 40 to 60 mm
shell height sea scallops were observed in the catches of both the
dredge and the trawl. Growth of this cohort over the next 9 months
resulted in these sea scallops attaining a shell height range
whereby they were recruiting into the fishery the following spring.
During the May 1998 trip, age 3 sea scallops from this cohort were
captured in numbers 5 to 6 times greater than the previous trips in
1997. The presence of this strong age 3 year class had a profound
effect on the relative production rates of the dredge and trawl
vessels. When age 3 sea scallops were present in large numbers,
the trawl vessels catch per hectare was 35.4% greater than that of
the dredge vessel. The observed shift in relative harvest efficiency
and the resulting ramifications in relation to production rates dem-
onstrated an inherent inequality between the two regulated gear
types.

Irrespective of changing resource conditions, a significant shift
in relative harvest efficiency at 90 to 95 mm shell height was
observed over all three trips. Trawl vessels were more efficient at
capturing sea scallops less than 90-mm shell height relative to the
dredge vessels. At shell heights greater than 90 mm. the trawl
vessels were observed to operate less efficiently relative to dredge
vessels. This shift in relative harvest efficiency had a large effect
on catch compositions and ultimately production rates. The mag-
nitude of the observed differences were dependent on the resource
conditions at the time and location of the study. DuPaul et al.
(1989c) observed similar results in comparing pre-Amendment 4
scallop trawls and dredges. At approximately 90 mm shell height,
the 3 inch (76 mm) ring dredge started to perform more efficiently
relative to the trawl nets used in the study.

The shift in relative harvest efficiency may be explained by
behavioral characteristics of the sea scallop. Sea scallops less than
100 mm shell height have been found to be highly mobile (Caddy
1968. Dadswcll and Weihs 1990), and have been observed to elicit
a night response at the approach of a dredge (Caddy 1968. Worms
and Latienge, 1986). As scallops grow larger than 100 mm, mo-
bility decreases and these larger animals become sedentary, living
in shallow depressions created in the substrate (Bourne 1964). A
dredge which is designed to scrape the substrate may be able to
capture larger sea scallops OlOO mm shell height! found in slight
depressions in the substrate. A liawl thai skims over the substrate
may not be able to capture these larger sea scallops as efficiently
as the dredge.

Size Selectivity

Gear selectivity occurs as a scallop enters a trawl or dredge on
the sea floor. Selection properties of the gear dictate whether a
scallop escapes or is captured, and is primarily a function of scal-
lop si/.e relative to the mesh or ring si/c in the traw 1 or dredge. Sea

scallops that are too small to be retained by the gear pass through
spaces in the meshes, rings, or inter-ring spaces. Selection by the
crew occurs when the catch is dumped on deck and the crew culls
the catch for sea scallops to be retained for shucking. Under
Amendment 4, no meat count restrictions exist and it is up to the
discretion of the captain and crew to establish the size of sea
scallops that are retained for shucking.

Traditional size selectivity studies are based on a comparison
between length frequency distributions from an experimental (se-
lective) versus a control (non-selective) gear. The non-selective
gear provides an estimate of the size distribution of the animals
that pass through the meshes or rings of the experimental gear.
Covered codends. small mesh codends, and small mesh liners rep-
resent some non-selective devices utilized in the literature (Hodder
and May 1965, Pope et al. 1975, Serchuk and Smolowitz 1980,
DuPaul et al. 1989a, Wileman et al. 1996). The length frequency
distribution from the non-selective gear is then compared with the
catch from the experimental gear to generate a size selection curve.

A non-selective gear was not used to determine absolute selec-
tivity in this study. The data collected represented the catch from
two experimental (selective) gear configurations. With no estimate
of the length frequency distribution of sea scallops that passed
through the rings of the dredge and meshes of the trawl, absolute
selection curves could not be generated. Millar (1995) states that
comparative gear selectivity experiments in which no control is
u.sed can not provide conclusive evidence of any selection curve
because any fit to the data can arise from an infinity of selection
curve models. In the absence of an estimate of absolute gear se-
lectivity, relative gear selectivity can be inferred from length fre-
quency distributions, catch composifions, and relative efficiency
estimates.

Results of the crew size selectivity analysis suggest a standard
for minimum retention size. DuPaul and Kirkley (1995) reported
that sea scallops begin to be retained by the fishery at roughly 70
to 75 mm shell height. Our findings corroborate this observation,
as the L51, values over all trips ranged from 69.3 to 77.5 mm.
DuPaul et al. ( 1995) and DuPaul and Kirkley ( 1995) observed that
crew culling practices changed in response to a dominant year
class that grew over the course of the study period. In this study,
however, no shift in sea scallop size selection was observed even
though the size composition of the catch varied widely over the
three trips.

Implications for the Fixbery and Management

Controlling age at entry is one management strategy used to
maximi/e yield per recruit and increase the spawning potential ot
the managed population. Serchuk et al. (1979) estimated that maxi-
mum yield per recruit for sea scallops is attained at an age of first
capture of 8 years. Only minor increases are realized as age at first
capture increases from ages 6 to 8. While it may be unrealistic to
delay the age al first capture to 8 or even 6 year old sea scallops,
significant benefits in terms of yield per recruit can be realized if
sea scallops are allowed to reach age 4 before recruiting to the
fishery. Serchuk et al. ( 1979) estimated an increase of 39% in yield
per recruit for mid-.^llantic sea scallops if harvested at 97 mm as
opposed to 77 mm shell height. Similarly. Caddy (1972) estimated
a 65% increase in yield per recruit if sea scallops were allowed to
grow from 73 to 92 mm shell height. The harvest of 3-year-old sea
scallops compromises the management objective of maximizing
yield per recruit.

Comparison of Sea Scallop Gear Types

763

In addition to increasing yield per recruit, delaying age at first
capture from age 3 to 4 also adds to reproductive potential in terms
of egg production. Age 3 sea scallops produce from 10 to 13.5
million eggs, while 4-year-old sea scallops will produce as many
as 22 to 34 million eggs (MacDonald and Thompson 1985, Lang-
ton et al. 1987). While exact fecundity estimates vary, age 4 sea
scallops can produce 2 to 3 times more eggs than age 3 sea scal-
lops. McGarvey et al. (1993) found a statistically significant
spawner-recruit relationship for sea scallops on Georges Bank, and
determined that age 3 and to some extent age 4 sea scallops did not
measurably contribute to egg production and recruitment on
Georges Bank. The harvest of age 3 sea scallops may at best
represent a large reduction in spawning potential or possibly the
removal of animals before they have had a chance to reproduc-
tively contribute to the population.

Equity

The examination of equity between different regulated gear
types found in Amendment 4 was an objective of this study and
was predicated on relative size selectivity and efficiency. Analyses
of shell height frequencies, catch compositions, and relative har-
vest efficiency indicated that regulated trawls and dredges appear
quite different in relation to both size selectivity and harvest effi-
ciency.

Future attempts at equating dredges and trawls in relation to
size selectivity could be accomplished through comparative gear
research. Studies utilizing differing diamond or square mesh sizes
would result in the escape of greater numbers of pre-recruit (<70
mm shell height). Previous comparative gear studies demonstrated
that modifications such as increasing ring and mesh sizes reduced,
but did not eliminate, the capture of smaller sea scallops, and often
reduced overall harvest efficiency (DuPaul et al. 1989c, DuPaul
and Kirkley 1995).

While size selection properties of sea scallop gear seem to be
broad, the crew culling process has been shown to be very selec-
tive. Assuming the majority of sea scallops that are discarded
survive the capture and culling process, the crew culling process in
combination w ith more selective gear types has the potential to be
an effective tool in controlling scallop size at entry into the fishery
process (Medcof and Bourne 1964, DuPaul et al. 1995, DuPaul and
Kirkley 1995).

Sea scallop trawls were observed to have a reduced ability to
capture sea scallops greater than 90 mm relative to standard sea
scallop dredges. This differential harvest pattern coupled with an
observed minimum culling size at 70 to 75 mm implies that trawl
vessels will depend, in a large part, on age 3 sea scallops for
landings. If the resource consists of large numbers of sea scallops
less than 90-mm shell height, dredge vessels will be at a competi-
tive disadvantage relative to trawl vessels. Management strategies
have clearly pointed to the objective of restoring the abundance
and age distribution of the adult stocks (NEFMC 1982). If resource
composition is restored in the future, sea scallops greater than 90
mm will represent a larger proportion of the resource. The ability
of dredge vessels to more efficiently harvest sea scallops larger
than 90 mm shell height dredge vessels will result in a competitive
advantage for dredge vessels relative to trawl vessels. This gener-

alization is dependent upon the relative abundance of scallop size
classes present in the population.

The reduced ability of trawls to capture sea scallops greater
than 90 mm shell height relative to the dredge may make equating
the two gears difficult. Future trawl design modifications may be
able to reduce the catch of small sea scallops, but results from this
and previous studies suggest that current trawl designs may not be
able to harvest larger sea scallops as efficiently as scallop dredges
(DuPaul et al. 1989e). Once trawl and dredge designs are engi-
neered to have similar selectivity patterns, the issue of harvest
efficiency could be addressed. Harvest efficiency is partly a func-
tion of gear width, or the area over the bottom that the gear can
cover. Currently, gear width is mandated to be a maximum of 30
ft. (9.0 m) of dredge width and 144 ft. (43.2 m) of trawl sweep.
Modifications of gear width could possibly equilibrate the two
gears in relation to relative harvest efficiency.

The comparison of relative efficiency and size selectivity of the
two regulated gear types represents the first comparative level of
analysis on how dredge and trawl vessels operate. To adequately
compare the two gears, a broader view of how dredge and trawl
vessels operate at the fleet level should be examined. Trawl vessels
hold 227c of the total permits in the fishery and account for 10%
to 15% of the annual landings. Trawl landings for the 1998 to 1999
fishing year were 1.29 million pounds, or 1 1% of the total landings
(NEFMC 1999). Trawl vessels tend to operate out of ports in the
mid-Atlantic region and are operationally limited to working in
areas of smooth, clean bottom. As a result of this limitation, trawl
vessels can operate in only a fraction of the area that is available
to the dredge boats. Therefore, only a limited portion of the scallop
resource is subject to harvest by sea scallop trawl gear. Intense
fishing activity by trawl vessels in this limited resource area may
result in dramatic localized effects to incoming year classes of 70
to 90 mm shell height sea scallops.

This study demonstrated that the assumptions that formed the
basis of the gear regulations found in Amendment 4 were not
entirely correct. Clearly, if a management objective is to require
that sea scallop trawls and dredges have equivalent size selection
and relative efficiency, more comparative gear research is a ne-
cessity. In general, quantifying the role that different fishing gears
have on the utilization of the sea scallop resource is an objective
yet to be fully achieved.

ACKNOWLEDGMENTS

We would like to thank the captains, crews, and owners of the
commercial fishing vessels that participated in the study. Without
their cooperation, knowledge, skill, and patience this project never
would have been completed. Individuals who deserve special rec-
ognition are: Mr. Jim Jones, captain of both the FA' Triangle I and
the F/V Capt. AT; Mr. Andy Benavidez, captain of the F/V Steph-
anie B.; Mr. Juan Araiza, captain of the F/V Carolina Breeze: Mr.
George Porter, captain of the FA' Carolina Clipper: and Mr. Car-
roll Tillet, captain of the FA' Triangle I. Thanks also goes to David
Kerstetter and Todd Gedamke for participating in the sea sam-
pling. This study was funded by Saltonstall-Kennedy Fisheries
Development Fund Award No. NA76FD0146. VIMS Contribu-
tion No. 2323.

764

Rudders et al.

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the northeast coast of the LInited States. Proc. Natl. Shellfish Assoc.
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Wileman, D.A.. R.S.T. Ferro. R. Fonteyne & R.B. Millar. 1996. Manual of
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Joimuil ol Shellfish Research. Vol. 19. No. 2. Ibi-lli^. 200U.

A SHIPMENT METHOD FOR SCALLOP SEED

ALFONSO N. MAEDA-MARTINEZ,' ^ MARIA TERESA SICARD,'
AND TEODORO REYNOSO-GRANADOS'

Centra cic Invcstigaciones Biologicas del Noroeste S.C.,

P.O. Box 128, La Paz,

B.C. 5. Mexico 23.000

Centra de Investigacion en Alimentacidn y DesarroUo A.C.,

Km. 0.1 a La Victoria,

Hennosillo, Son. Mexico 83,000

ABSTRACT .\ "sandwich" made of layers of sponge and plywood lids placed inside plastic bags and packed in styrofoam coolers
was evaluated as a device for shipment of catarina scallop {Argi/peclen ventricosus Sowerby II, 1842) spat. Spat survival was measured
to evaluate the combined effect of temperature, emersion time, sponge thickness, spat density, shell gapping, and O, levels. In addition,
temperature inside the coolers with different amounts of ice was measured under different external temperatures. Results indicate that
scallop seed can successfully be shipped out of the water over long periods of time (>37 h). taking advantage of the scallop's capacity
to breathe in air. Survival was greatly enhanced when a pure-0, atmosphere was used. Highest survival was obtained at lower
temperature and at shorter emersion times. Survival was not affected by either sponge thickness or by spat density. Higher rates of
survival occurred when shells were forced to remain tightly closed. Mortality was attributed to a combination of O, shortage and
desiccation, but the possibility of a genotoxic effect of several metabolites and toxicity by nitrogenous compounds as possible causes
are discussed. To maintain temperatures in the coolers, 50 g L"' of ice was found to be optimum.

KEY WORDS: live transport, scallop, emersion, anaerobiosis. Art>opecten veinricosus

INTRODUCTION

Shipment of live aquatic organisms cuirently is performed ei-
ther in tanks with aerated water or out of the water under moist
conditions. The first method is appropriate for only short-term
shipments (a few hours) because of bacterial growth, the presence
of dissolved material voided in the feces (Bayne 1976). 0-, deple-
tion, and the accumulation of toxic nitrogenous excretion products
such as ammonia, which quickly reduce the quality of the water in
which the animals are being transported. In addition, this method
is expensive because of the cost of shipping water together with
the animals. Conversely, shipment without water under moist con-
ditions is comparatively inexpensive but exerts a physiological
cost to the animals, which results in high mortality if shipping
conditions are not appropriate.

Rhodes and Manzi (1988) wrote about a shipping method for
clam and scallop seed at different stages for different emersion
times for up to 6 days. The only information given was that there
was a direct correlation between shipping duration and mortality
of bivalve .seed. The greatest mortality occurred in the smallest
size bivalves shipped over the longest period of time. The highest
survivals were for the largest size bivalves shipped over the
shortest period. Hard clams had greater overall survival than
scallops at almost all size classes and shipping durations. No fur-
ther information on temperature, emersion time, or survival was
given.

To find an adequate out-of-water shipping method for scallops,
background information is needed. Contrary to the majority of
bivalves, scallops are monomiarian bivalves that gap their shells
when exposed to air. causing desiccation. Pecten maxiinus. as all
scallops, initially responds to air exposure by violent adductions of
the shell and tachycardia, followed by gradual bradycardia, ac-
companied by wide gapping of valves. Inability to control air
gapping and consequent vulnerability to desiccation is a charac-
teristic feature of scallops similar to other sublittoral species
(Brand and Roberts 1973). Desiccation could be avoided in trans-

port by forcing the scallops to keep their shells closed while in a
highly moist atmosphere, although loss of water may occur
through the bysal notch of the shell.

If desiccation can be prevented or reduced, how the animal is
going to respire during shipment should be considered. Anaerobic
respiration is a common alternative used by many intertidal
molluscs in the events of anoxia or exposure to air (Shumway
and Scott 1983. Devi et al. 1984. Maeda-Martinez 1987, Aunaas
et al. 1988, Marshall and McQuaid 1989. Vial et al. 1992,
Oeschger and Storey 1993, Wang and Widdows 1993, de Zwaan et
al. 1995, Simpfendorfer et al.l995). However, scallops do not
seem to be adapted to glycolysis pathways for this purpose (de
Zwaan et al. 1980, Thompson et al. 1980). The remaining alter-
native for the scallop to breathe during shipment is to gain oxygen
from the atmosphere, to which the respiratory apparatus is ill
adapted. The air breathing capacity in scallops has not been dem-
onstrated. However in other bivalves such as Mytillus califor-
nianits. Modiolus deinissus, and Cardium edule. air breathing was
found to be 63%-74% of the standard rate of oxygen consumption
in water at the same temperatures (Kuenzler 1961, Boyden 1972a.
Bayne et al. 1975). The median survial time of A/. rfem/,s.vi/.v in air
is proportional to the amount of oxygen present (Lent 1968). and
therefore an atmosphere of pure oxygen might enhance survival.
The use of pure O, for shipping animals has not been reported.
Pure Ot is currently in use in fish and shrimp aquaculture indus-
tries to supersaturate the water in which the animals are trans-
ported.

Temperature is considered the most important factor determin-
ing the level of activity in poikilotherms (Bayne 1976). Therefore,
within limits, a decrea.se in ambient temperature may improve
survival during shipment because the amount of oxygen required
(which is limited inside the shipment device) for the respiration of
the animals under transport will be reduced. Therefore, optimum
temperature for shipment is critical. Optimum temperature for
growth in Argopeclen ventricosus is 19-22 °C (Sicard et al. 1999)

765

766

Maeda-Martinez et al.

but the median lower lethal temperature is not known. From the
literature, it is known that the species may withstand temperatures
as low as 12 °C because they have been captured in the continental
shelf of the Baja California Peninsula at a depth of 180 m, where
a temperature of 12 °C has been measured (Maeda-Martfnez et al.
1993). At this temperature the respiration rate (VO,) was 0.5-1.0
mL Oj g ' h" ' , whereas at 28 °C , VO^ was three times higher (3. 1
mL O, g"' h"') (Sicard et al. 1999).

If the optimum temperature range for shipment of the species is
known, the problem is to produce and maintain the temperature
within that range during the trip. The current method for shipping
live animals uses styrofoam coolers of different shapes and thick-
nesses and employs frozen bricks of blue ice to lower the tem-
perature. The insulation capacity of the cooler can be provided by
the manufacturer, but this is of little use when the amount of ice
bricks are not standardized and large variations in external ambient
temperature occur. In the tropics, a cooler may be exposed to
temperatures from freezing (if the boxes are placed in the nonther-
moregulated compartment of the plane during flight), to 40 °C or
higher if exposed to direct sunlight. Because of this, it is important
to determine the temperature variations inside a styrofoam cooler
containing a known amount of ice and exposed to different ambi-
ent conditions.

We have developed a shipping device and evaluated it for
shipping scallop seed. Scallop survival was measured at different
temperatures, emersion periods, spat densities and shell gapping
levels. The effect of sponge thickness and the effect of a pure-O,
atmosphere against a normal air atmosphere on scallop survival
were estimated. The cooling effect of different quantities of blue
ice was measured inside styrofoam coolers exposed to sunlight and
under shade.

MATERIALS AND METHODS

Experimental Animals

Fourteen thousand catarina scallop juveniles (3. .5 ± 0.4 mm
shell-height and 3.7 mg dry tissue weight) produced at the hatchery
of CIBNOR La Paz. Mexico were used in the study.

Shipping Device

The shipping device was made with three layers of sponge
rubber (20 x 10 cm) placed between two 1/S-inch plywood rect-
angles of the same size (Fig. 1 ). The scallop seed was placed on the
first (bottoir) and second (middle) layers. The top layer served as
a lid. The sponges were soaked with seawater before u.se. A plastic
tie was used to secure the sandwich. The sandwiches were placed
inside 2-L plastic bags, filled with air or pure O, before they were
clo.sed and sealed with a rubber band. The approximate gas volume
inside the bags was 600 mL . The 7-cm high sandwich had a
volume of 1.4 L (20 x 10 x 7 cm).

Experimental Design

Spat survival was measured to evaluate the combined effect of
three different temperatures (17, 22, and 28 '("), three emersion
titiies ( 19, 26, and 37 h), two sponge thicknesses ( I.2.S and 2..'i cm
). two spat densities (2 and 25 scallops/ cm"), two shell gapping
levels (tight and loose), and two O, atmospheric conditions (air-
only and pure-O,). In the pure-O, experiments, only thick sponges
were tested. Hach treatment was made with ojie replicate. The spat

styrofoam cooler

Ice pack

Plywood lid

Rubber sponges

Plastic tie

Figure 1. Device used for shipping catarina scallop (Argopecleii ven-
tricosus} spat.

from each treatment were removed at the emersion times and were
placed in 1-L glass containers with seawater at 37'^r and at the
experimental temperatures to allow the scallops to recover from
exposure to air. Each container received constant aeration and
150,000 cells/mL of a mixture of cultured microalgae (hocluysis
galhcma and Chaelocerus gmcilUs). The spat remained at least 2 h
in these containers before the dead and the live animals were
counted.

The experimental temperatures were achieved by placing the
sandwiches in temperature-controlled rooms at 17, 22, and 28 °C.
Shell gapping (tight and loose) was produced by the strength at
which the sandwiches were secured. In the former, the sponge
maintained a moist atmosphere around the animals but care was
taken not to compress the animals. In the latter, the shells were
forced to remain tightly closed, securing the sandwich as tightly as
possible. To test the density (spat cm^-) effect on survival, each
sponge was marked at the middle with a pen marker. About 100
seed were spread over one-half of the sponge at a density of two
scallops cm~", while in the other half the seed was heaped up
covering only 4 enr at 25 scallops cm"-. Each sandwich then held
400 spat.

The pure-O, experiments were done replacing the air in the
plastic bags with medicinal O, from a cylinder. Each bag holds
approximately 2 L of gas.

Styrofoam Cooler Temperatures

The temperature changes inside 27.3-L styrofoam coolers (39
cm long X 28 cm wide x 25 cm deep and 2.2 cm thickness)
containing 4 or 8 0.35-kg blue ice bricks (50 or 100 g of blue ice
per liter of cooler) were monitored over 48 h with a data logger set
to record temperature every 0.5 h. This allowed continuous read-
ings with 0.01 "C precision. The coolers were similar to those used
commercially for shipping live animals, such as shrimp postlarvae.
One set of coolers was exposed to direct sunlight, another was
placed in the shade, and a third was kept in a temperature-
controlled room at 22 "C. The temperature fluctuations in the
coolers of the last treatment were only tested using eight blue-ice
bricks. These results were contrasted against ambient temperature
in the shade, which was simultaneously recorded by the meteoro-
logical station at CIBNOR.

A Shipment Method for Scallop Seed

767

RESULTS

Styrofoam Cooler Temperatures

Air-only Expermients

In the air-only experiments, survival was higher at shorter
emersion times and at lower temperatures. The highest survival
was 617r at 17 °C, using thick sponge, at low density, with tight
shell gapping, and at the shortest emersion time tested (19 h).
Negligible survival was obtained after 26 h in all air-only treat-
ments. A Tukey multiple-range test indicated significant differ-
ences between survival at 17 "C-\9 h and at warmer temperatures
and longer emersion times.

To evaluate differences among sponge thickness, spat density,
and shell gapping treatments, Tukey multiple-range tests were
made. Results show that survival was not affected either by sponge
thickness or by spat density. However, a signit~icant difference
between shell gapping levels was found at P > 0.01. Higher sur-
vival was obtained when shells were forced to remain tightly
closed.

Pure-02 Experiments

Figure 2 shows the comparative results of pure-O-, experiments
against their corresponding air-only treatments. From this, survival
in pure-O, was significantly higher than their corresponding air-
only treatment, which confirms the scallop capacity for air respi-
ration and the advantage of using a enriched O, atmosphere. Av-
erage survival in pure-O, experiments under the most adverse
conditions of highest temperature (28 °C) and longest emersion
time (37 h) was 20%. At 17 °C and 19 h of emersion, average
survival was 97%. A significant effect of shell gapping was ob-
tained here as in the previous air-only experiments at f > 0.01.
Survival was greater when the shells of the scallops were tightly
closed.

Variations in ambient shade temperatures and inside styrofoam
coolers with 50 and 100 g L' of ice exposed to direct sunlight, in
the shade, and in a temperature controlled room at 22 °C for 48 h
are shown in Figure 3. Average ambient temperature was 29.4 °C
with a maximum of 38.2 °C and a minimum of 21.4 °C. Indepen-
dently of the quantity of ice employed, temperature inside the
coolers dropped from ambient temperature (=25 °C) to the lowest
value of 5.9 and 14.5 °C in the 100 and 50 g L" treatments within
the first 4 h of the experiments. Using 100 g L~' of blue ice. a
temperature shock of 5 °C h" ' was produced, which could prob-
ably be lethal to scallops. In addition, the low temperature reached
(5.9 °C) may exceed the lower thermal limit of the species. With
50 g L~' of blue ice. temperature also fell in 4 h, but the lowest
temperature was only 14.5 °C. which is higher than the lowest
temperature (12 °C) at which the catarina scallop has been cap-
tured (Maeda-Marti'nez et al.l993). Once the lowest temperature
was reached, it began to increase steadily (Fig. 3). and the velocity
of the increase was a function of the quantity of ice employed and
of the external conditions. In the 50 g L^' treatment, temperature
increased to about the average external ambient temperature of
29.4 °C (Fig. 3a) 25 h from the start of the experiments, whereas
in the 100 g L"' treatments, temperature never reached equilibrium
with the external medium in the 48 h of the experiment (Fig. 3b).

a

40 -,

35

"^^ y^\^ r-

o

30

\ / •''''^^\r'''''''"^- / '

0}

25

\ ^^^. l-'X ^^- /

3

«

20

V ..--''^^,1-*-^

il

Q>

V"-- -''"' .^^

Q.

15

>^ ' -^^

E

0)

1-

10

In the shade

5

Exposed to sunlight

^^^Ambient shade temp.

0

20 25 30

Time (h)

50

Room at 22 "C
in the shade
Exposed to sunglight
Ambient shade temp.

Figure 2. Survival of catarina scallop (Argopeclen ven(ricosus) spat
(3.5 mm shell height: 3.7 mg dry tissue weight) at different tempera-
tures and emersion times, and incubated in the shipment device under
a pure-O, (gray columns) and air-only (white columns! atmo.spheres.
a = high density-loose, b = high density-tight, c = low density-loose, and
d = low density-tight.

20 25 30

Time (h)

Figure 3. Temperature variations inside 27.3-L styrofoam coolers con-
taining 4 (a) and 8 (b) blue ice bricks, each weighing 0.35 kg, over 48
h. Coolers were exposed to direct sunlight, kept in the shade, and in a
temperature controlled room at 22 C. This figure also shows the
ambient temperature in the shade.

768

Maeda-Martinez et al.

To determine the temperature conditions that would be expected at
the same emersion times as the previous experiments, the extreme
temperatures were obtained from the data logger at 19, 26. and 37
h (Table 1). In the 100 g L"' treatment, extreme temperatures
remained the same at the different emersion times regardless of the
external variations in temperature (Fig. 3b). The cooling effect of
the blue ice bricks lasted for more than 19 h. In the 50 g L"
treatment, the maximum temperature exceeded the median lethal
temperature (29 °C) of the species (Sicard et al. 1999) 26 h from
the beginning of the experiment under both external conditions
tested (Table 1).

DISCUSSION

The results indicate that scallop seed can be successfully
shipped out of water over long periods of time (>37 h), taking
advantage of the scallop's capacity to breathe in air. This capacity
was not expected because many invertebrates exposed to air show
different responses because of their different behavioral strategies
and physiological tolerances. These tolerances are certainly greater
in intertidal than in subtidal species, because intertidal species
might experience short-term exposure to air on a daily basis at low
tide. Subtidal species, such as A. ventricosus. are rarely exposed in
their natural environment.

It may seem odd that many intertidal invertebrates enter
anaerobiosis at low tide when there is access to atmospheric oxy-
gen. But for essentially all aquatic invertebrates, the desiccation
stress when exposed to air may be so severe that many species
isolate themselves within closed shells. At low tide the facultative
anaerobe Myrilns must cease gill irrigation and remain with its
valves tightly closed, shifiting to anaerobiosis (Bayne 1976). As an
advantage, Mytilus does not produce lactic acid as an end product
but the less toxic alanine and succinate following a modified gly-
colytic scheme (Wells 1980). Although anarobiosis has been dem-
onstrated to play an important role in intertidal mollusks during
exposure to air, it is unlikely that this pathway would be used by
the scallop. Anaerobic respiration in scallops is predominantly
used for energy production during sudden bursts of activity such as
swimming or the valve-snapping escape response (de Zwaan et al.
1980. Thompson et al. 1980). Phosphoarginine is used as the main
energy source and octopine is produced as an end product (Bricelj
and Shumway 1991). The scallop adductor muscle, which is the
main storage organ, only contains 18 to 25 % of glycogen, whereas
in Mylilliis ediilis. an intertidal bivalve that commonly uses anaero-

TABLE I.

Maximum and minimum temperatures (°C) recorded inside 27.3-L

slynifoam coolers containing 1.4 and 2.8 k^ <>f blue ice

(50 and 10«glr')

19 h 26 h

(n = 38) (H = 52)

37 h

(H = 74)

Experimental conditions Max Min Max Min Max Min

.^0 g 1. ' in the sliadc 2.S.9 14.8 29.4 14.8 31.2 14.8

50 gL-' exposed to sunlight 26.1 16.5 297 16.5 32.5 16.5

lOOgl.-' inaroomal22°C 22.0 5.9 22.0 .5.9 22.0 .5.9

UK) g L'' in the shade 24.7 7.7 24.7 7.7 24.7 7.7

KM) g 1.-' exposed to sunlight 27.3 8.4 27.3 8.3 28.5 8.3

Coolers were exposed to direct sunlight, in the shade, and in a temperature-
controlled room at 22 "C at different iiKiibation times.

bic pathways during prolonged valve closure, attains high maxi-
mum seasonal glycogen levels of 42 to 53<7f in the mantle, the
principal long term storage organ in mytilids (de Zwaan and
Zandee 1972. Gabbott 1983).

On air exposure. Lent (1968. 1969) and Boyden (1972a) re-
ported that groups of Modiolus Jeniissiis (337f) and Cardiitm spp.
(42%) tolerated those loses of weight as water before mortality
occurred. We did not measure water loss but direct evidence of this
was the significant difference found between survival of tight and
loose shell-gapping treatments. The shipping method described
minimized desiccation by forcing the shells of the juveniles to
remain closed. Water may have been lost through the bysal notch
of the shells. Although the rubber sponge helped to maintain a
moist environment around the seed, this does not seem to be an
optimum material because the upper layers of the sandwich were
dryer than the bottom layers at the end of the experiments. This
explains the lower survival observed (not quantified) from upper
layers than from bottom layers in all treatments. If this is so. the
method can be optimized either with the use of a spongy material
with higher hydrophilic properties or by placing a layer of absor-
bent paper between the seed and the sponge.

No differences in spat survival were found at different densi-
ties. This indicates that the seed could be piled up during shipment,
which will reduce the number of shipping containers needed and
costs. However, this does not indicate the number of scallops that
can be shipped per unit of bag volume. In both treatments (2 and
25 spat cm~"). the same volumetric density of 400 spat /600 mL
was tested, assuining that only 30% of the bag volume was occu-
pied by gas (2.0 L bag-1.4 L sandwich). The volumetric density
used was 0.66 spat mL"' or 2.4 mg dry tissue weight (dtw) mL"'.
if the dtw of a 3.5 mm shell-height spat was 3.7 mg. From this, a
total of 5.600-3.5 mm spat or 20.2 g dtw can be shipped success-
fully with results similar to our experiments, using a 28-L com-
mercial styrofoam cooler. These numbers and biomass per cooler
are low and probably not economically useful. Further research is
needed to find the optimum volume density for shipment.

The differences in survival between air-only vs. pure-O, ex-
periments may suggest that mortality in the former was because ol
a shortage of oxygen. In Modiolus demissus, the median survival
time in air is proportional to the amount of oxygen present (Lent
1968). Boyden (1972b) showed that by preventing Cardium edule
from gapping, its survival in air was significantly reduced. How-
ever a straight forward explanation like this cannot be given for the
mortality in the pure-O, treatments because survival from some
air-only and pure-O, experiments were similar, as in 17 °C-19 h
air-only and 17 C-37 h pure-O, low density-tight treatments (Fig.
2d), though a much higher oxygen content in the pure-O, bags
would be expected than in the air-only experiment. To test this
hypothesis, final O, available in the bags at the different treatments
has to be determined. For this, the oxygen consumption of the spat
has first to be estimated. LInforlunately the O, consumption in air
was not measured during the experiments, but a good approach
could be made if the O, uptake in air was assumed to be about 70''/;
the standard respiration rates (VO, J in water determined by Si-
card et al. (1999) in this species, as in other bivalves (Kuenzler,
1961, Boyden. 1972a. Bayne et al. 1975). In the catarina scallop,
the relationship between V(K , and temperature is described by the
equation (« = 6; r =0.98):

VQj, = 0.047 e"'-"'''"''C

From this, the hypothetical oxygen concentrations at the end of

A Shipmknt Method for Scallop Seed

769

the air-only and the pure O, experiments at different temperatures
and emersion times, were calculated (Table 2). In this table, the
total amount of oxygen consumed (TVO,) on emersion was ob-
tained with the formula:

TVO,

(VO, J(0.7)(dtw)(t)

where dtw was the total biomass incubated in the bags (400 spat x
3.7 mg dtw = 1,48 g) and t was the emersion time. For the
estimation of TVO,. it was assumed that VO, varied indepen-
dently from the available oxygen (PO,). In the catarina scallop,
VOt has been found to remain independent of PO, only in the
range between 100 and 76% O, saturation (Sicard et al. 1999). At
lower oxygen concentrations. VO, became dependent on PO, and
therefore TVO, overestimated the oxygen consumption in the ex-
periments, reflecting the maximum O, uptake possible by the scal-
lops during emersion. With these results, we conclude that in pure-
0-, experiments there was sufficient oxygen and that mortality was
produced by a combination of other factors.

The other factors that may have contributed to the scallop's
mortality besides shortage of O, and desiccation, could have been
the genotoxic effect of emersion (Brunetti et al. 1992) and to a
minor degree the accumulation of toxic nitrogenous compounds.
Brunetti et al. (1992) found that on emersion and exposure to
anoxic seawater. the frequency of micronuclei in gill tissues of
Mytiliis gaUopr<n'incialUs rose as a function of time. They sug-
gested that the genotoxic agent may be a product of anaerobic
metabolism such as propionic or acetic acid. For the toxicity by
nitrogen compounds, Bayne et al. (1975) found that the rate of
production of ammonia in Mytillus californianus was only about
5% of the immersed rate during exposure to air.

As expected, our results showed that spat survival was higher
at lower temperatures (17 °C) and at shorter emersion times ( 19 h).
Within limits, metabolism in poikilotherms varies with tempera-
ture. As a consequence, O, demand diminishes as temperature
decreases, increasing the resistance of the animal to prolonged
emersion. The problem arises when a desired range of temperature
has to be maintained during shipment. We demonstrated that 1()0
g L"' of blue ice may be effective in keeping the temperature
lower than the upper thermal limit of the species (29 °C) for more

than 48 h, even at ambient temperatures above 38 °C. However
this quantity of ice will probably kill the animals as temperature
falls beyond the lower thermal limit of the species. Using half this
quantity (50 g L^' ). the problem of low temperature can be elimi-
nated because temperature only decreases to 14.5 °C, a tempera-
ture within the tolerable thermal range of the species (Sicard et al.
1999). However with 50 g L"' of ice, shipment times cannot last
longer than 26 h at ambient temperatures of 38 °C because tem-
perature in the coolers may exceed the upper thermal limit of the
species. This problem can be solved by using a thicker cooler with
greater insulation capacity. Another problem to be solved is the
thermal shock (2.5 and 5.0 °C h"') given to the animals during the
first 4 h of shipment using 50 or 100 g L"' of blue ice. This could
be minimized by acclimating the animals to the minimum expected
temperature in the coolers and packing them at this temperature in
a temperature-controlled room. Further research is needed to de-
termine whether this procedure would improve survival. In Pe-
naeus japonicits. 100% survival was obtained in shipments for as
long as 17 h out of water by cooling the animals 5 h from 24 °C
to 14 °C before air exposure (Samet et al. 1996).

We have devised a successful alternative for shipping scallop
spat. The potential application of this method could be the ship-
ment of scallop and other bivalve broodstock, and the transporta-
tion of other high-valued species like shrimp, lobster, or abalone to
live markets that are currently under expansion throughout the
world. The fire hazard that may represent the use of pure oxygen
could be reduced, with proper handling and shipping procedures,
and by optimizing the amount of pure oxygen used. The optimum
O^-air mixture remains to be determined.

ACKNOWLEDGMENTS

We thank Mr. Miguel Robles Mungaray from CIBNOR for
providing the catarina scallop spat for this study. Mr. Ignacio
Leyva gave technical assistance during the experiments. Drs. In-
ocencio Higuera, Ramon Pacheco, and Francisco Vargas from
ClAD-Hermosillo provided all facilities for the analysis of data
and the writing of the manu.script. Dr. Ellis Glazier edited this
English-language manuscript.

TABLE 2.

Hypothetical O, concentrations in the bags, at the end of the air-only and pure-O, catarina scallop {Argopecten ventricosiis) spat (3,5 mm

shell height and 3.7 mg dry tissue weight) shipping experiments.

Temperature

Emersion time

Standard O, uptake rate*

Total Oi consumed!

Final Oj air-

onlyt

Final O, pure-02§

(=C)

(h)

in water (mLO,"'h"')

on

emersion (mLOj)

(mLO,l

(mLO,)

17

19

0..";

10.2

1 1 .5.8

590

17

26

0.5

14.0

112.0

586

17

37

o..-;

19.9

106.1

580

22

19

1.1

20.7

10.5..^

579

22

26

Ll

28.4

97.6

572

22

37

I.I

40,3

85.7

560

28

19

2.4

48.3

77.7

552

28

26

2.4

65.9

60.1

534

28

37

2.4

93.2

32.8

507

Each bag contained 400 spat and 1.47 g dtw total biomass. Figures are assuming an independent O, uptake rate from available oxygen concentration.

* Data from Sicard et al. ( 1999)

1 109c of standard rate in water.

i Initial oxygen content = 2l9f of available bag volume.

§ Initial oxygen content = 100% of available bag volume = 600 mL.

770

Maeda-Marti'nez et al.

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Shumway. S. E. & T. M. Scott. 1983. The effects of anoxia and hydrogen
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Gonzalez. 1995. Relationship between the aerobic and anaerobic meta-
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puratus ( Lamarck )( Bivalvia) and Mytilus chilensis (HupeXBivalvia).
Comp. Biochem. Physiol. 1118:615-623.

Thompson, R. J., D. R. Livingstone & A. de Zwaan. 1980. Physiological
and biochemical aspects of the valve snap and valve closure responses
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Physiol. 137:97-104.

Vial, M. v., R. W. Simpfendorfer. D. A. Lopez, M. L. Gonzalez & K.
Oelckers. 1992. Metabolic responses of the intertidal mussel Perumyti-
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Biol. Ecol. 159:191:201.

Wang, W. X. & J. Widdows. 1993. Calorimetric studies on the energy
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Wells, R. M. G. 1980. Invertebrate respiration. The Institute of Biology's
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Joiinuil of Shellfish Reseiinh. Vol. 19. No. 2. 771-778, 2000.

SEASONAL VARIATIONS IN CONDITION, REPRODUCTIVE ACTIVITY, AND BIOCHEMICAL

COMPOSITION OF THE PACIFIC OYSTER, CRASSOSTREA GIGAS (THUNBERG), IN

SUSPENDED CULTURE IN TWO COASTAL BAYS OF KOREA

CHANG-KEUN KANG,* MI SEON PARK, FIL-YONG LEE,
WOO-JEUNG CHOI, AND WON-CHAN LEE

National Fisheries Researcli am! Development Institute,
Shining, Kijang-Gim.
Piisan 619-900. Korea

ABSTRACT Seasonal variations in condition index (CI), reproductive activity, and biochemical composition of the oysters, Cras-
sostrea gigas. in suspended culture in different nutritional conditions were compared between two bay systems (Jaran Bay and
Hansan-Koje Bay) of the southern coast of Korea from January 19% to September 1997. Differences in temperature and salinity were
not significant between stations, but chlorophyll a concentrations were significantly higher at Station Josan in Jaran Bay, an outer open
system, than at Station Osu in Hansan-Koje Bay, a semi-enclosed bay sy.stem with restricted food availability. CI and dry tissue weight
of a standard animal showed a similar seasonal cycle, with minimum values in late .summer and peaks in spring at both stations. In
the Josan oysters a rapid recovery in these components commenced in November 1996 with the simultaneous accumulation of reserves
(glycogen and protein) after the summer spawning. By contrast the Osu oysters recovered slowly 3 mo later in February 1997. The
values were also considerably higher in Josan oysters than in Osu oysters. At both stations ganielogenesis was initiated in late autumn
and the breeding period was extended over several months during the summer-early autumn period. Spawning intensity during summer
was, however, stronger in the Josan oysters than in the Osu ones. Food availability seemed to be a major factor in determining gonad
proliferation and thereby CI, when gametogenesis was initiated. Apparently, the high accumulation of glycogen and protein was
ob.served in the Josan oysters so that the absolute values for the standard animal were signitlcantly higher at Station Josan than at
Station Osu. These results indicate that difference in physiological states of the oysters cultivated in the two bay systems are strongly
related to site-dependent variation in the storage-utilization cycle of energy reserves (particularly glycogen), depending on food
availability. Our findings also suggest that it is necessary to readjust the cultivated density of oysters to procure enough wild seeds and
condition of oysters in Hansan-Koje Bay, taking carrying capacity of the bay into consideration.

KEY WORDS:

ability

Pacific oyster, Crassostrea gigas. condition, reproduction, biochemical composition, suspended-culture, food avail-

INTRODUCTION

Seasonal variations in condition and gametogenisis of marine
bivalves are strongly related to the energy storage-utilization cycle
and environmental factors such as water temperature and food
availability (Giese 1969, Gabbott 1975, 1983, Bayne 1976). Bayne
(1976) postulated that the cycles of energy storage and gamete
production can be overlapped temporally ("opportunistic" species:
Tellina tenuis, Abra alba, and Cerastoderma edule) or separated
clearly ("conservative" species: Mytilus edulis. Macoma balthica.
and Pecten maximus). Recent studies suggested that even within a
single species there might be interannual or local differences in the
cycles of energy storage and reproduction due to environmental
conditions, in particular nutritional condition (Bayne and Worrall
1980. Newell et al. 1982, Rodhouse et al. 1984, Bricelj et al. 1987,
Harvey and Vincent 1989, Navarro et al. 1989). These cycles in
cultivated bivalves can be different from their wild counterparts
(Rodhouse et al. 1984) and among populations of a species from
different locations (Brown and Hartwick 1988, Almeida et al.
1997, Okumu^ and Stirling 1998). In general, the suspended-
culture method of bivalves contributes to high tissue-growth rate
due to the better environmental conditions (i.e. food availability;
Rodhouse et al. 1984, Pazos et al. 1997).

Traditional culture methods for the Pacific oyster, Crassostrea
gigas, in the intertidal beds of Korea have been replaced with a
suspended-culture method since 1969. Intensive suspended-oyster

*Corresponding author: Chang-Keun Kang. National Fisheries Research
and Development Institute. Shirang. Kijang-Gun. Pusan 619-900. Korea.

culture has been developed in the semi-enclosed coastal bays on
the south coast of Korea. Owing to the development of oyster
culture using ropes suspended from long lines, oyster production in
Korea increased abruptly and was maximized up to 288,000 tons
in 1987. However, over a recent decade the oyster production was
unstable and slowly decreased. Such a recent decrease of oyster
production is considered to result largely from local declines of
growth rate due to the intensive culture (Yoo et al. 1980) and local
shortages in supply of healthy seed oysters (Park et al. 1999).

In some cases of suspended oyster-culturing grounds in the
southern coastal bays of Korea, condition of the oysters has been
lowered from year to year. Thus the culture period required to
produce a marketable product has been prolonged. At the begin-
ning of the development in Hansan-Koje Bay, seeded ropes were
suspended in early and mid-summer and then the cultivated oysters
were harvested after an approximate 9-mo growth. Recently, this
growth period for harvesting is extended to 16 mo, depending on
locations. This variability may be explained by the trophic capacity
of the bays in relation to the density of cultivated oysters and the
availability of food (Heral 1993). Deslous-Paoli and Heral (1988)
showed that seasonal variations in the condition and biochemical
composition of the cultivated bivalves could be affected by culti-
vated density at the same area. The densities overstocked within a
bay system may also affect the reproductive activity of the oysters.
Park et al. ( 1999) suggested that an overstocked bay, Hansan-Koje
Bay (Korea), is unfavorable for the collection of .seed oysters and
this phenomenon results from extremely low production of larvae
due to a prolonged pre-spawning stage and a low prevalence of
spawning oysters.

771

772

Kang et al.

The high reproductive activity of the oysters is still very im-
portant to collect enough seed for the oyster culture of Korea. The
condition, which is controlled by both the cycles of energy storage
and gametogenesis (Gabott 1975). determines the marketability of
comiTiercially exploited bivalve species. Information on the con-
dition and the cycles of gametogenisis and energy storage of oys-
ters in suspended culture is therefore valuable because of biologi-
cal and commercial interests.

This study investigated the seasonal variation in condition in-
dex (CI), reproductive activity, and biochemical composition of
suspended-cultivated oysters in two bay systems of the southern
coast of Korea. The objectives of the study were to compare physi-
ological states of the oysters cultivated in different environmental
(in particular, nutritional) conditions and to examine the site-
dependent variation in the role of storage or reserve materials
relative to condition and gametogenesis.

MATERIALS AND METHODS

Study Areas

This study was carried out in two bay systems on the southern
coast of Korea (Fig. 1 ). Two locations were chosen for this ex-
periment. One was at Osu in Hansan-Koje Bay with a total area of
56 km-^. This bay is a semi-enclosed system and the cultivated
oysters were suspended in the inner site of the bay. The other
station was at Josan in Jaran Bay, which is open to oceanic envi-
rontnents. Both of the sites have long been used as farming
grounds for the Pacific oyster, Crassoetrea gigas with very high
cultivated densities compared to other locations.

Oyster Preparation and Biometric Measurements

Oysters (shell length = 7 cm) collected from Koje Bay were
cultured on ropes suspended from a long line. Initial density was
about 500 individuals per rope. A total of 30 ropes at each station
were suspended from 1 m below the water surface and there was
a 3-m distance between ropes. Sixty oysters were randomly taken
from depth of 1 to 5 m below the water surface at monthly inter-

Figure I. i.oiution of the study urea. Hliick tetraj'ons iiutieale long-
line culturing grounds and arrows represent the .sampling stations.

vals from January 1996 to September 1997. To minimize compo-
sitional variations resulting from size class differences, oysters of
similar size were sampled. Samples were rapidly transported to the
laboratory and placed in filtered seawater at in situ temperature for
24 h to evacuate their pseudofaeces and gut contents.

Thirty individuals were cleaned to remove any attached epi-
fauna and adhering sediments, and whole weight was determined
for each individual. Shell length, width, and height were measured
to the nearest 0.1 mm using vernier calipers. Oysters were then
dissected carefully and wet tissue weight was determined after the
separated tissues were superficially dried with absorbent tissue
paper. The tissues were then frozen and stored at -80 °C until they
were analyzed. Shell valves were rinsed with distilled water and
weighed after drying in a furnace at 50 °C for 48 h. Dry tissue
weight was determined after freeze-drying for 48 h.

Reproductive Activity

Thirty individuals from each sample were used for microscopic
examination of histological smears. A transverse cut was made
across the body of the oyster and a 3-mm-thick section was fixed
in Bouin's solution. It was then routinely processed for histology
and 5-|jLm paraftm-imbedded sections were stained with iron he-
matoxylin-eosin. The stage of gonadal development was classified
and scored on a 0 to 4 scale according to Mann (1979). The
arithmetic means of the individual scores of the whole sample was
recorded as the Gonadal Maturity Index (GMI) for each sampling
date (see details in Dinamani 1987).

Biochemical Measurements

The dry tissue of 30 individuals was pooled and homogenized.
The use of pooled tissue from many individuals to determine av-
erage biochemical composition may provide useful information
because marine invertebrates in the field are often highly variable
in biochemical composition (Giese 1967). Ash content was ob-
tained by igniting a subsample (30-80 mg) of homogenized tissue
at 450 °C for 48 h in a muffle furnace. Protein was determined by
the colorimetric method of Lowry et al. (1951) after extraction
with normal sodium hydroxyde. Extraction for total lipid was per-
formed in a mixture of chloroform and methanol (Bligh and Dyer
1959) and lipid content was determined using the method of Marsh
and Weinstein (1966). Carbohydrate and glycogen were extracted
in 15% trichloroacetic acid and precipitated with 999c ethanol.
They were analyzed using the phenol-sulfuric acid method as de-
scribed by Dubois et al. (1956).

Standard Animal and Condition Index

To present absolute values for biochemical composition, the
composition of a standard animal of 22.496 g in dry shell weight
was calculated for each sampling date. Allometric equations of
log,, I dry tissue weight against log,,, dry shell weight for each
population at each sampling dale was determined by linear regres-
sion analysis. The results of the biochemical analysis were then
expressed in milligrams per standard animal All regressions were
statistically significant (/' < 0.001) except for June 1996 at Osu
station. A similar method was introduced for the clam Ta/u'.t de-
iiissiitus L. and T. pliilippinanini by Beninger and Lucas (1984)
and for the oyster C. gigas by Ruiz et al. (1992). CI was calculated
Irom the dry weights of tissue and shell according to the formula
CI = dry tissue weight (mg)/dry shell weight (g) (Walne 1976,
Urmvn and Hartvvick 1988).

Seasonal Variations in Crassostrea gigas

11?.

Environmental Conditions

At each sampling of oyster, water temperature and salinity were
measured //; situ using a CTD meter (Seabird Electronics, Inc.).
Duplicate water samples for measurement of suspended particulate
matter (SPM) and phytoplankton biomass (chlorophyll a concen-
tration) were collected at the water depth of 1 and 5 m with a .^-L
van Dorn water sampler. The water was passed through a 250- [j,m
mesh net to remove zooplankton and large particles. Water
samples (1-3L) for SPM determination were filtered through a
pre-weighed Whatman GF/C glass-fiber filter. The filters were
washed with 0.97f ammonium formate, dried at 80 °C. and then
reweighed. Chlorophyll a concentration was determined on ac-
etone extracts using the fluorometric method as modified by Par-
sons et al. (1984) with a 10 AU Fluorometer (Turner Designs).

Statistical Treatment

To test the difference between the two stocks of oysters in
mean values for each biochemical variable during the sampling
period, the paired comparison design was applied to the Wilcox-
on"s signed-ranks test (Sokal and Rohlf 1981 ). The Kendall's rank
correlation coefficients, t, were calculated to test the strength of
association among environmental parameters and oyster compo-
nents.

RESULTS

Environmental Conditions

Monthly mean water temperatures at the two stations are given
in Figure 2a. The water temperatures showed very similar seasonal
cycle, with maxima of approximately 26.5 °C in summer and
minima of approximately 6.5 °C in winter. Differences in tem-
perature and salinity were not significant between stations. Salinity
maxima of approximately 34 psu were recorded during late winter-
early spring in both years at the two stations (Fig. 2b). Salinity
minima were observed in summer in both years. During June
through August 1996, the values at Station Osu were lower than
approxiinately 32 psu at station Josan and a minimum salinity of
30 psu was recorded in Station Osu in July 1996.

SPM concentrations varied from 3.2 to 30.2 mg L"' and ex-
tremely high concentrations more than 20 mg L ' were observed
in February 1996 and August 1997 at both stations (Fig. 2c). No
correlations between SPM and chlorophyll a concentrations were

found. Chlorophyll a concentrations showed several peaks
throughout the year, but they showed maxima in spring (Fig. 2d).
The mean of experimental period was 1.43 ± 0.79 (SD) p.g L"' at
Station Osu and 2.48 ± 1.23 (j.g L'' at Station Josan. The differ-
ence in chlorophyll a concentrations between stations was statis-
tically significant (Wilcoxon's signed-ranks test. Osu mean =
4.63, Josan mean = 7.43; 0.001 < P < 0.01).

Biometry and CI

Biometric measurements showed that there was no growth in
shell length of oysters during the sampling period. Mean shell
lengths ranged between 68.9 and 95.6 mm at Station Osu. and
between 70.7 and 83.5 mm at Station Josan. Mean fresh and dry
tissue weights showed an apparent sea.sonal variation with maxima
in spring. Mean dry shell weight ranged between 15.620 and
30.861 g at Station Osu, and between 13.247 and 32.772 g at
Station Josan. The mean dry shell weight over the sampling period
at the two stations was 22.496 g.

The seasonal variations of CI were very clear at both stations
(Fig. 3). The maxima in CI were in April to May in both years and
were followed by an abrupt decline between June and August. At
Station Josan. a subsequent rapid increase in CI values was ob-
served in November 1996. However, at Station Osu, the minimum
value of 43 in August 1996 remained constant during the autumn-
winter period and a slow recovery occurred in February 1997, 3
mo after the recovery in November 1996 at Station Josan. In both
years the CI maxima were much higher at Station Josan than at
Station Osu. The maximum CI values at Station Josan were 104
and 133 in 1996 and 1997, respectively, whereas the values at
Station Osu did not exceed 90.

J I I I I I I L

JFMAMJ JASONDJFMAMJ JAS
199C 1997

Month

JFMAMJ JASONDJFMAMJ JAS
1996 1997

Month

Figure. 2. Seasonal variations in temperature (a), salinity (b), SPM (c),
and chlorophyll a at Stations Osu (open circle) and Josan (black circle)
from January 1996 to September 1997.

J FMAMJ JASONDJ FMAMJ JAS
1996 1997

Month

Figure. 3. Seasonal variation in CI at Stations Osu (a) and Josan (b)
during the experimental period.

774

Kang et al.

Reproductive Activity

The gametogenic cycles of the two cultured populations in
tenns of GMI are presented in Figure 4. Since differences between
male and female were not considered in the analysis of the bio-
chemical composition, GMI is here presented as the means of
pooled data from both sexes. There was good agreement in the
seasonal cycles of GMI between stocks. Gonadal tissue develop-
ment started in November and December and the GMI increased
progressively until June. The GMI maxima were recorded from
June to August when CI decreased sharply. Planktonic larvae of
oysters were observed throughout the summer from June to Sep-
tember in both 1996 and 1997. These results indicate that spawn-
ing activity continued during the summer period. The maxima
were then followed by a sharp decline due to spawning, which
ended in September.

Tissue Weight of a Standard Animal

Figure 5 shows .sea.sonal variations in dry tissue weight for a
standard animal (dry shell weight = 22.496 g). There were re-
markable seasonal variations in dry tissue weight in each stock and
the patterns were similar to those of CI, with peaks in April and
May, decreases during summer, and minima in early autumn. The
amplitude was greater at Station Josan. Therefore, with the excep-
tion of the time of minimum dry tissue weight in summer, standard
animal from Station Josan had significantly {P < 0.001 1 higher dry
tissue weight than that from Station Osu. At Station Josan, sub-
stantial increment just after the times of minima in dry tissue
weight was initiated in November 1996. However, at Station Osu,
no increase in dry tissue weight was found during late autumn-
winter 1996.

Biochemical Composition

Seasonal variations in mean percentage of almost all the bio-
chemical components showed clear seasonal trends and the pat-
terns of each component were similar between stations. Protein
percentages ranged from 40.5% to 66.8% of the dry tissue weight,
with maxima in summer when the dry tissue weights were minimal
at both stations. Lipid percentages were slightly higher in spring
than the rest of the year. The values tluctuated between 2.5% and
11.6% at both stations. In spite of significant differences in dry
tissue weight, no differences in the mean percentage compositions
of protein, lipid, and water showed between stations. Mean car-

te
o>

■5 4

C

o
C9

m

J F M A M
1996

J J A S O N D

Month

J F M A M
1997

J J A S

Fiuuri'. 4. .Seasoiuil >ariuti(in in ^loiiadul (levi'lopnu'iil, as Mann's go-
nadal maturity indi'v (1979) at Stations Osu (open harl and ,|(isan
(black bar).

400

J F M A M J J
1996

A S

ASONDJFMAMJJ
1997
Month

Figure. 5. Seasonal variation in dry tissue weight in standard animal
of 22.496 g in dry shell weight at Stations Osu (a) and Josan (b).
Vertical bars represent 95% confldence intervals.

bohydrate (also glycogen) percentage was however significantly
higher at Station Josan than at Station Osu (Wilcoxon's signed-
ranks test, Osu mean = 7.00, Josan man = 1 1.67: 0.001 < P <
0.01), with the values from 3.5% to 24.9% (average 12.0%) at
Station Osu and from 2.2% to 33.0% (average 16.8%) at Station
Josan. The levels were negatively correlated to the protein levels,
with maxima in the winter-spring season. Glycogen levels ac-
counted for most of total carbohydrate levels so that their seasonal
variations paralleled those of carbohydrate. Ash levels showed a
slight inverse relationship with carbohydrate levels, with minima
values of 1 1 .0% and 9.5% in May 1 996. and maxima of 23.0% and
17.0% in September 1996 at Stations Osu and Josan. respectively.
Mean ash percentage was higher at Station Osu than at Station
Josan (Wilcoxon's signed-ranks test, Osu mean = 11.06. Josan
mean = 5.50, P < 0.001). Water content ranged from 74.5% to
88.4% and from 78.3% to 87.2% of the fresh tissue at Stations Osu
and Josan, respectively.

The absolute values ol' biochemical components lor a standard
animal, calculated from the percentage composition and the dry
tissue weight (Fig. 5), are pre.sented in Figure 6 as milligrams per
standard animal. In addition, correlations between the environmen-
tal parameters and the oyster components observed during the
sampling period are summari/ed in Table I. GMI of oysters was
strongly correlated to temperature (0.01 < P < 0.001), but carbo-
hydrate (also glycogen) values of the standard animal were nega-
tively correlated to temperature at both stations. Kendall's rank
correlation matrices show that the accumulation and depletion
cycles of storage or reserve materials are different between sta-
tions. At station Osu both the mean CI and the standard animal dry
weight were significantly correlated to its protein and lipid values

Seasonal Variations in Crassostrea gigas

775

(A)Osu

(B) Josan

JFMAMJJASONOJFMAMJJAS
1996 1997

Month

JFMAMJJASONOJFMAMJJAS
1996 1997

Month

Figure. 6. Seasonal variation in weights of biochemical components in
standard animal of 22.4% g in dry shell weight at Stations Osu (A) and
Josan (B). P, protein; L, lipid; CHO, carbohydrate, G, glycogen. Ver-
tical bars represent 95% confidence intervals.

(P < 0.001 ). weakly correlated to the carbohydrate values, but not
to the glycogen values. Protein values were significantly correlated
to lipid levels (P < 0.001). but not to carbohydrate and glycogen
values. However, at Station Josan. both the mean CI and the stan-
dard animal dry weight were significantly correlated to almost all
biochemical components. Protein values were significantly corre-
lated to lipid values (P < 0.001) and also, though weakly, to
carbohydrate and glycogen levels (0.03 > P > 0.01). The differ-
ences between stations are attributed to the lack of accumulation of
carbohydrate (largely glycogen) from the autumn period of 1996 to
the end of the study at Station Osu (Fig. 6. c and d). Significant
differences in mean values for protein and carbohydrate contents
of the standard animal between stations were obvious from Wil-
coxon's signed-ranks test (Osu mean = 4.25 and 4.33. Josan mean
= 12.59 and 12.1 1, respectively; 0.01 <P < 0.001 for protein and
P < 0.001 for carbohydrate).

DISCUSSION

There were no differences in temperature, salinity, and SPM
concentration between the two stations observed. However, annual
mean chlorophyll a concentration was significantly higher at Sta-
tion Josan in Jaran Bay than at Station Osu in Hansan-Koje Bay.
The latter bay has been characterized by low chlorophyll a con-
centration and low primary production of phytoplankton compared
to other oyster-culturing grounds in the southern coastal bays of
Korea (Lee et al. 1991. Choi et al. 1997). It is well known that
water movement can determine the amount of food supply avail-
able to suspension feeders. Water exchange rates in Hansan-Koje
Bay are around 10% and 5% of the whole water volume of the bay
during the spring tide and the neap tide, respectively (Yoo et al.
1980). Therefore, it is unlikely that the food available to the oys-
ters is transported from the outer part. The suspended oyster-
culturing grounds have been developed densely with a total area of
1 1 km- in the bay. This area corresponds to 23% of total water
surface area of the bay. In relatively shallow areas with long resi-
dence time of water like Hansan-Koje Bay. filtration by a dense
population of suspension feeders can make a significant impact on
the phytoplankton biomass (Yoo et al. 1980, Cloern 1982, Officer

et al. 1982, Loo and Rosenberg 1989). In addition, at a low flow
rate of the bay, filtration rates of the oysters might be reduced by
filtered water being recirculated (Riisgard 1977). On the other
hand. Jaran Bay is characterized as a more or less eutrophic en-
vironment (Choi et al. 1997). Tidal current is relatively stronger
because the bay is exposed directly to the open ocean.

CI showed a similar seasonal cycle, with minimum values in
late summer and peaks in spring at both stations. However. CI
values were considerably higher at the Station Josan than at Station
Osu. Part of this difference in CI values was most likely due to
difference in food availability (largely chlorophyll a). Of major
interest in the seasonal variation of CI of the oysters from these
stations was the difference in the time of initiation of CI recovery
after the summer spawning between stations. After the summer
spawning, CI of Station Josan oysters commenced a rapid recovery
in November. However, those of Station Osu showed only a slow
recovery in February 1997, 3 mo later than at Station Josan. Simi-
lar variation was recorded in the dry tissue weight of standard
animal. These variations have important implications for cultiva-
tion and harvesting strategy because there is the greatest demand
for oysters between December and January in Korea. Our results
may imply that oysters from Station Josan are possible to market
during this period, but those from Station Osu are not suitable for
marketing at the same time.

The reproductive cycles of the oysters were similar between
stations. Gametogenisis was initiated at 10 °C at Station Osu and
15 °C at Station Josan in late autumn. The temperature range at
which initiation of gametogenesis and spawning in C. gigas occurs
is well summarized by Ruiz et al. (1992, Table IV of that work).
In the study areas from May to October, the water temperature
exceeds 18 °C to 20 °C, which is suggested as a minimum tem-
perature required to induce spawning (Mann 1979). The cycles of
gametogenisis and CI (also dry tissue weight of standard animal)
indicate that the breeding period of C. gigas from these areas is
extended over several months during the summer-early autumn
period, similarly to the case of New Zealand (Dinamani 1987). The
precise timing and intensity of spawning is difficult to determine
from a monthly sampling strategy. However, although a similar
seasonal cycle in gametogenisis between stations was recorded in
this study, the amplitude of seasonal fluctuation of CI was much
greater in Josan oysters than in Osu ones. Park et al. (1999)
showed that from the greater fluctuations of various CIs in Station
Josan. spawning intensities are stronger in the Josan oysters than in
the Osu ones. They also concluded that based on the measurements
of every 10 days for 4 mo (June to September 1997). both abun-
dance and lipid content of D-shaped larvae are much greater at
Station Josan than at Station Osu. and thus the spawners are physi-
ologically more healthy at the former station. Such a difference
between stations is probably a major factor in explaining the rea-
son why seeds of oysters are not settled and collected on ropes in
Hansan-Koje Bay.

The reproductive strategy of C. gigas can be considered an
adaptation to ambient environmental factors such as mainly tem-
perature and nutritional conditions (Lubet 1976. Ruiz et al. 1992).
For bivalve populations that experienced a similar thermal regime,
the availability of food in the environment and thereby the levels
of nutrient reserves within the animals directly support gonadal
growth and reproductive cycle (Giese 1969, Gabbott 1975, 1983,
Bayne 1976. Bayne and Worrall 1980. Newell et al. 1982, Rod-
house et al. 1984, Bricelj et al. 1987). Therefore, local variations
in SI, dry tissue weights, and spawning activities recorded in this

776

Kang et al.

TABLE 1.

Kendall's rank correlation coefficient matrix for temperature (T), salinity (S), chlorophyll a (CHL), Gonad Maturity Index (GMI),
Condition Index (CI), and dry tissue weight (DTW), protein (P), lipid (L), carbohydrate (CHO), glycogen (GLY), and ash content of a

standard animal.

T

S

CHL

GMI

CI

DTW

P

L

CHO

GLY

Ash

Station Osu

T

-0.67***

-0.33*

-0.50**

-0.19

-0.17

-0.11

-0.06

-0.41**

-0.34*

-0.43**

S

-0.53***

0.32

-0.38*

0.24

0.35*

0.18

0.21

0.43**

0.36*

0.44**

CHL

-0.16

0.59

0.30

0.35*

0.31

0.25

0.31

0.18

0.02

0.40*

GMI

0.43**

-0.21

-0.01

0.30

0.13

0.21

0.29

-0.07

-0.15

-0.25

CI

-0.43**

054***

0.05

-0.01

0.81***

0.69***

0.75***

0.51**

0.26

0.39*

DTW

-0.32*

0.49**

0.06

-0.02

0.91***

0.76***

0.72***

0.38*

0.20

0.57***

P

-0.31

0.36*

0.10

0.09

0.73***

0.79***

0.66***

0.26

0.06

0.46**

L

-0.33*

0.53***

-0.14

0.06

0.64***

0.65***

0.56***

0.41**

0.19

0.37*

CHO

-0.60***

0.83***

-0.19

-0.19

0.55***

0.51**

0.32*

0.64***

0.76***

0.42**

GLY

-0.59***

0.80***

-0.18

-0.15

0.56***

0.53***

0.34*

0.60***

0.96***

0.37*

Ash

-0.57***

0.39*

-0.04

-0.16

0.70***

Stati

0.72***
on Josan

0.71***

0.47**

0.40*

0.40*

*0.05 < P<0.()1: *»

0.01 < P<

0.001; **i

P< 0.001.

study can be expected to result from differences in accumulation
and utilization of storage or reserve materials.

The seasonal cycles in dry tissue weight of standard animal
reflected the reproductive cycle with the weights showing nia.xima
prior to the summer spawning and abrupt declines during spawn-
ing. The difference in the strength of seasonal weight fluctuations
between stations (Fig. 5), along with that in CI fluctuations, indi-
cates that the intensity of the spawning is different between stocks.
Although there was a general tendency that the seasonal variations
in the absolute values of biochemical components paralleled those
of dry tissue weights (Fig. 6), the seasonal patterns of carbohydrate
and glycogen contents differed distinctly between stations. In
Josan oysters, a rapid recovery of glycogen values began in No-
vember 1996 after the abrupt declines during spawning in summer
(June to September 1996) and peaked in April 1997. However, the
accumulation of carbohydrate and glycogen reserves in Osu oys-
ters (although there was a small peak in March 1997; Fig. 6) was
not observed from the autumn period of 1996 to the end of the
study.

The accumulation of glycogen reserves appears to be related to
the time of maximum phytoplankton biomass (Ansell and Trevail-
lion 1967, Ansell 1972). Glycogen reserves have been considered
to be the main energy reserves both for the formation of gametes
of marine bivalves, especially under conditions of nutrient stress
and also for the maintenance during nutritional stress (Beninger
and Lucas 1984 and refs. therein). Thus the lack in the accumu-
lation of glycogen al Station Osu presumably resulted from insuf-
ficient food availability within the bay and this might allow Osu
oysters to lead to low gamete proliferation. Over the study period
the absolute carbohydrate and glycogen values were considerably
higher in Josan oysters than in Osu ones. Deslous-Paoli and Heral
(1988) reported a similar pattern in ('. .i;/,i,'«.v from Marcnnes-
Oleron Bay (France). These authors reported that the levels of
glycogen were less than 5% of dry tissue weight in the oysters
overstocked with high cultivated density, whereas the levels were
more than 10% of dry tissue weight in oyster ponds with low
density of oysters in the same area. They also showed that a
deficiency of food due to an overstocking can disturb the physi-
ology, particularly the processes of gametogencsis and spawning,
inducing the failure of spat settlement.

Glycogen is transformed into lipid for the formation of gametes
(Gabbott 1976, Lubet 1976). Lipid reserves are lost in spawning of
adult female bivalves (Gabbott 1983). The variation in the absolute
values of lipid in the oysters from this study supports these hy-
potheses, with maxima prior to spawning. Since protein constitutes
the major organic component of bivalve oocytes (Holland 1978),
protein maxima prior to spawning are reasonable. Protein also
serves as an energy reserve during gametogencsis (Mann and
Glomb 1978, Adachi 1979. Barber and Blake 1981) and during
energy imbalance (Gabbott and Bayne 1973. Beninger and Lucas
1984). However, since gonadal development is an energy-
demanding process and the oysters in this study had only a very
short gonadal resting stage, it was difficult to assess the role of
each biochemical component as maintenance energy during the
energy imbalance period. Furthermore, the filtration and ingestion
rates decrease above 20 °C (Le Gall and Raillard 1988). whereas
oxygen consumption rates increase (Bougrier et al. 1995). There
may be therefore a synchrony in the energy-required timing for
maintenance and spawning during summer. Riley (1976) found
that from a controlled starvation experiment of C. nigiis. lipid and
protein were the main energy reserves. Whyte et al. (1990) con-
cluded that protein contributed more than carbohydrate to main-
tenance energy in oysters under conditions of extended food de-
privation, even when carbohydrate was apparently available in
sufficient quantity. The seasonal variation patterns in the absolute
values of lipid and protein were similar between stations, but the
protein value was significantly greater in Josan oysters than in Ous
oysters. A difference in the accumulation liming of these reserves
after spawning was found similar lo llial in the dry tissue weight
(Figs. 6 and 4).

Apparently, histological examination showed thai gametogen-
csis of the oysters observed was initiated simultaneously with the
accumulation (Station Josan) and with a minimum level (Station
Osu) of reserve materials in late autumn-early winter. Then gamete
development continued during the period of increase in reserve
materials throughout the spring and maximum levels in almost all
the biochemical components occurred al the moment of ripeness in
late spring, followed by a subsequent rapid decrease during spawn-
ing (June through September). This type of bivalve may be con-
sidered to he an opportunistic species (see definition in "Introduc-

Seasonal Variations in Crassostrea gigas

111

tion") that have a direct dependence on food availability in its
ambient environment. This finding is inconsistent with the results
of Ruiz et al. (1992) that for C. gigas in suspended culture in El
Grove (Galicia, Spain), the glycogen stored is used in the game-
togenesis and the protein and the lipid are utilized in winter when
available food is scarce. This discrepancy will have to be explained
by various endogenous and exogenous parameters.

In conclusion, the intensity of gamete proliferation and the
condition of the oysters in suspended culture in Korean waters
seem to be largely determined by the nutritional conditions in
ambient environments. Food availability is considerably restricted
in the semi-enclosed bay system with the high density of oy.sters.
In such a restricted nutritional condition, the accumulation of re-
serve materials, particularly glycogen and protein, is expected to
be insufficient to meet the energy required for increasing gonadal
development and tissue weight. Higher seasonal fluctuation in CI

and dry tissue weight of the oysters cultivated in the outer open
system suggests that they experience a superior nutritional condi-
tion, with much greater contents in reserve materials. As a result,
our findings indicate that the outer open system is a more suitable
site for the suspended culture to procure enough wild seeds and
condition of oysters. These results also conclude that for the en-
closed bay system like Hansan-Koje Bay. the cultivated density of
oysters must be readjusted, taking carrying capacity of the bay into
consideration (Heral 1993).

ACKNOWLEDGMENTS

The authors wish to thank shellfish farmers in Tongyoung and
Koje Island for providing their facilities cooperating with us dur-
ing the present study. Thanks are also given to anonymous referees
for their critical comments and suggestions on the manuscript.

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THE TRANSMISSION OF MICROSATELLITE ALLELES IN AUSTRALIAN AND NORTH
AMERICAN STOCKS OF THE PACIFIC OYSTER (CRASSOSTREA GIGAS): SELECTION AND

NULL ALLELES

DANIEL J. MCGOLDRICK,* ' ^ DENNIS HEDGECOCK,'
LOUISE J. ENGLISH,'^^ PUTTHARAT BAOPRASERTKUL,t ' ^
AND ROBERT D. WARD' ^

^CSIRO Marine Research. G.P.O. Box 15.^8. Hohart.

Tasmania, Australia 7001
'Cooperative Research Centre for Aquaculture, P.O. Bo.x 123,

Broadway NSW. Australia 2007
' University of California, Davis, Bodega Marine Lxdnnatory.

Bodega Bay. California 94923
'* Department of Aquaculture. University of Tasmania. P.O. Box 1215,

Launceston. Tasmania 7215, Australia

ABSTRACT Variation, transmission, and selection at 24 microsatellite loci are studied in five experimental families of the Pacific
oyster (Crassostrea gigas). Two families are from naturalized North American stocks, and three come from Australian stock. As
expected, there are multiple alleles at these loci and their segregating variation is reduced to four alleles or less in full sib progeny
groups. Two to 21 loci were tested per family. Eight of the 24 loci have only codominant alleles, but 16 loci also have non-amplifying
or null alleles. Of the 172 (43 x 4) parental sequences that were progeny tested, 30 (17%) were null alleles. Null alleles segregate in
both Australian and North American stocks and their presence is heterogeneous among crosses. Overall null allele frequency in North
American crosses was estimated to be 1 \% (eight of the 72 alleles progeny tested), just significantly less than the 22% (22 of the 100
alleles progeny tested) in the Australian stocks [P = 0.04). After accounting for nulls in genetic hypotheses, selection in the form of
significant deviations from Mendelian expectations is observed in 16 of 43 progeny tests (37%). There is no systematic association
between null alleles and selection, but analysis of dominance by sequential G-tests reveals non-additive kinds of zygotic selection. This
has also been recorded in two other oyster species and the blue mussel. It appears that null alleles at microsalellites and selection near
genetic markers are expected phenomena when studying transmission of genetic markers in bivalve molluscs. The implications of these
results for breeding, aquaculture, and population genetics are discus.sed.

KEY WORDS: Pacific oyster. SSLP, genetic marker

INTRODUCTION

The aquaculture production of bivalves (including oysters,
mussels, scallops, and clams) in 1997 was valued at soiDe 8 billion
dollars world-wide, and constituted some 7 million metric tons of
food (FAO 1999). Production of Pacific oysters {Cnis.KDstri'a gi-
gas) in the same year was nearly 3 million tons; it is the most
widespread, cultivated invertebrate on earth (Shatkin et al. 1997,
FAO 1999). A small, but growing fraction of the global production
of Pacific oysters is based on hatchery stocks. For example, all
Australian production comes from hatchery crosses followed by
nursery rearing and then grow-out. Selective breeding programs
for the Pacific oyster in Australia (Ward et al. 2000) and the
U.S.A. (Hedgecock et al. 1997 ) aim to produce broodstock with
improved domestication qualities. To assist in these programs, we
have begun to apply a suite of microsatellite loci. These, in asso-
ciation with other genetic markers including allozymes and AFLPs
are used in linkage mapping, trait mapping, pedigree analysis, and
marker-assisted selection.

Previous work on inbred families of the Pacific oy.ster revealed
substantial segregation ratio distortion at allozyme loci (Foultz
1986a, McGoldrick 1997). In addition, a heterozygosity growth

*Corresponding author. Present addre.ss: Texas A&M University, Horti-
culture/Foresl .Science Department, College Station, TX 77843-2145.

tPresent address: National Aquaculture Genetics Research Institute. Tum-
bon Klong 5 Amphure Klong Luang, Pathumthani, Thailand 121.

rate correlation and heterozygosity deficiency is observed at the
population level for bivalves in general (see Gaffney 1994) and
was first observed in the American oyster Crassostrea virginica
(Singh and Zouros 1978). There is a suggestion from the study of
inbred lines in Pacific oysters that particular allozyme alleles
might be linked to genetic regulatory backgrounds that segregate
in families and influence the ratio distortion for particular alleles at
many loci simultaneously (McGoldrick and Hedgecock 1997).
Here we examine whether distortion is occurring at microsatellite
loci in inbred and outbred crosses, and the nature of the distortion
(e.g. dominant, overdominant, or underdominant) when it occurs.
In oysters, detailed studies of microsatellite transmission are
few. Naciri et al. (1995) describe inheritance patterns for three loci
in the European flat oyster, Ostrea edidis. Fewer than 20 progeny
were examined for each of two single-pair crosses; parental geno-
types were inferred. Two loci gave progeny ratios that accorded
with Mendelian expectations. The third locus gave two homozy-
gote classes, but no heterozygotes in one family, and a large het-
erozygote excess in the other family. A cross between a heterozy-
gote for the two amplified alleles and a null homozygote could
explain the first result. For the second, the authors suggest that
lethal alleles hitchhiking with amplified alleles could be respon-
sible. A second flat-oyster paper (Bieme et al. 1998) examined
four microsatellite loci in about 80 larvae and post-larvae from two
full-sib crosses. Null alleles were not reported, but deviations from
Mendelian expectations were recorded in about one-half of the
cases.

779

780

McGOLDRICK ET AL.

Null alleles are not uncommon in single locus. PCR-based
typing in neutral DNA. Nulls frequently reflect changes in one of
the two PCR priming sites that prevent a primer from binding
efficiently, thus blocking amplification during the polymerase
chain reaction. Examples where mutations are known to produce
null alleles include a single-basepair transversion (Egglestontott et
al. 1997), a 1-bp insertion (Band and Ron 1997), a 4-bp deletion
(Jones et al. 1998), and an 8-bp deletion (Callen et al. 1993). More
commonly, the existence of null alleles is inferred at the population
level when observed heterozygote frequencies are less than those
expected at Hardy-Weinberg equilibrium (e.g. Foltz 1986b). In
such circumstances the frequency of the null allele may be esti-
mated as that which minimizes deviations from equilibrium. How-
ever, we use breeding data to provide more formal proof of the
existence of a null allele. Null alleles, once recognized, are man-
ageable in family studies, but somewhat less .so in population
studies (see Callen et al. 1993). Once identified, null alleles can be
treated as recessive alleles, while amplified microsatellite alleles
are scored as codominant alleles. The frequency of null alleles and
the question of whether they are homogeneous across stocks and
loci is an important issue for population genetic analysis, in de-
termining exclusion probabilities in pedigree analysis, and in
marker-assisted selection.

In this study we report the results of segregation tests of 24
microsatellite loci in five crosses of Pacific oysters. Null alleles are
present at many loci, but even when these are allowed for, devia-
tions from Mendelian proportions are frequently observed. The
forms of selection in other published studies of bivalves are also
investigated and the implications of these observations are dis-
cussed.

MATERIALS AND METHODS

Microsatellite Loci

Seventeen of the 24 informative microsatellite loci
(iicclCgl. . .ucdCg28) were developed at University of California.
Davis (McGoldrick 1997). Seven other informative microsatellite
loci (cmrCgl. . .cmrCgIS!) were developed at CSIRO Marine Re-
search. Hobart. Primer .sequences and available GenBank acces-
sion numbers are reported (Table 1 ).

Genomic Amplification

Genomic DNA template for North American samples was pre-
pared for amplification utilizing a small biopsy of adult mantle
tissue and a standard phenol chloroform method (Ausubel et al.
1994) and was typed at the University of California at Davis
Bodega Marine laboratory. Loci were amplified in 96-well sample
plates. After optimization of a sample of five loci (after Cobb and
Clarkson 1994). a 5-|jiL consensus reaction cocktail was estab-
lished (2 mM MgCU. 300 p.M dNTP mix with a 1:100 dilution
[v/v] of Dupont Renaissance"*' tetramethylrhodamine-6dUTP in
10 (xM stock dNTP. 5 pM of primer. 50 ng template, and 0.04 units
Taq polymerase from Promega Corporation [Madison, Wl]). A
primer set was not accepted unless it could generate scoreable
phenotypes with the consensus cocktail. Samples were amplified
in a Bio-Oven III thermocycler (Biotherm Corporation, Fairfax,
VA) with an initial denaturation at 92 ° for 2 min followed by 35
cycles of 92 ° for 30 sec. 55 ° for 30 sec. and 72 ° for 30 sec.
Following amplification. 4 (il of formamide loading dye (10 mL
formamide. 100 |xL 0.5M EDTA. and 2 mg bromphenol blue) was
added to each well and the samples were denatured at 92 ° prior to

TABLE 1
Primer sequences flanking Paciflc oyster microsatellites.

Locus

Clone

GENBANK

5'-Forward (labeled)

S'-Reverse

ucdCgOl

AE27

—

iicdCgOl

AM86

—

ucilCgOJ

AS88

—

ucilCg04

BR09

AFn.'51170

«(</C,i;a5

BS55

-

MdCg06

BV59

AF()51I72

iicilCxOS

CE48

AF051175

M-dCgO'J

CFOX

-

ucdCglO

CI75

-

ucdCgll

CK90

-

ucdCali

CU03

AF()51179

ucdCgl4

BY56

AF05I174

ucdCgIS

S72

-

iicdCglH

AP46

-

ludCgll

Byi2

-

MdCi'22

B.SIl

-

iicdCf;2S

CQ72

AF05I178

cntrCfi()2

Cl.^1

AF201461

nnrCiiO-^

F().^-4

AF20I462

cmrCfiOf)

HI 05

AF20I465

cmrCg6l

-

AF204062

cmrCfil41

-

AF204060

cmrCxl4J

-

AF204061

cmrCgl5l

-

-

CAAGCTTAAAAAAGCAAGTTTACG

TTGCAGGAAGCAAGAGATGA

GTTTGAACCCATGCAGAGGT

ATAATAATTAAAGGGGTTAAGGGG

GGGGTCTGTGATATCGGAGA

AAGCAACTATCAGTTTTTGGTAGC

CTTCTCACTTCACACACTCATCC

TTAAACTTGTGTAAAGCATTTGG

TGCACCAATTTGAGATGTGA

TTCAAAACGACATAGTCCACA

TGTGTAAATCAACAAAAGAAATGC

GGTGAAGGAAAACACAAAACA

TGATGCAGTAAGATTTCATTTCA

TCCATGTTTACTGCTACTTTTGG

GCCCTCTAAATTAAAATCTCTCT

CCCCAACTCAAACAGACGTT

TGTTTAATGATGTGTACCGCG

AGGAGATCATATCATAAGGAGACAGAG

CTTTGCCTGTG ATA AC AGTACGTATCG

ATTGTTTGCCGATACTGAGAGA

GATTGGTrGAAAAAATCACACG

ACCATTTGCACCTTTCCAAC

CTTGCCATATTOCCATGTGT

TGCTTCATTGITTGTTGTATGG

TGCGGTGCTATTATGAACCA

CTTGTTAACTGCCGGTGAGG

CAGAACTTTTGAGAGAGAGAGAGA

GTGGTAGCAATTGTGTCCTATG

GGTTCTACGCACAGTGCTGA

AATGAGCTGACAGTTCATAGGC

TTTAAACTTGTGTAAAGCATCTGG

CGTTCATCGATTTTCGCAAT

ACTGAGTTTGAAAATGTCACCG

ATCTGAGCTTGCATGGGAAC

GATCAATAATTTTCATGCCAGA

TTAGCTGCCGCTCAAGTTTT

ACAGGTAACCCCCTCCACTC

AAATGCTGTGCAGAGAAGCC

CCGCCATAGGTTTGAAAATT

TAGTCAGACGTTCCTAACTCTTCG

ATCAAATTGGCTGTATTTACAGTG

ATGTTTTACATTCTTACAGGTCATTCA

TTAAATrrCATTGACAATTATGGTCCCA

CTGACTGAAACTGCrrrGTTGA

TAACAGCAGCGCTACCATGC

TGACACTTGAAGCCTTGCAC

CTTTTACATGGAATTGTCACAGG

CATACACAAATTGCACTTATAGCA

The Transmission of Microsatellite Alleles

781

electrophoresis. A total of 4 |xL of dye-reaction mix was electro-
pliorcsed for 1.5 h at 60 watts on an 8% acrylamide (19:1. aery-
lamide:bis-acrylamide) gel containing 8 M urea and Ix TBE and
visualized using an FMBIO digital scanner (Hitachi. San Francis-
co. CA). Alleles were not sized.

Australian samples were examined at CSIRO Marine Research
in Hobart. Tasmania. Australia. Extractions of whole body tissues
were performed from 198-day-old juveniles and muscle tissue
from 395-day-old adults. Loci were amplified in 96-well trays in a
Perkin Elmer 9600 thermocycler using 5'- HEX, FAM-. or TET-
labeled primers (PE Applied Biosystems. Foster City, CA.). A
consensus reaction chemistry was again established in the absence
of fluorescent dNTP. Reaction cocktails consisted of 2 mM
MgCK. 200 (xM dNTP. I pM of primer. -10 ng template, and 0.02
units Tiuj polymerase (Promega Corporation). Samples were sized
and genotyped using an ABI377 sequencer with Genotyper soft-
ware (PE Biosystems).

Microsatellite loci with smeary, faint, or complex electropho-
retic banding patterns are not reported here. Two contaminating
individuals, one in family 97-2. and one in family 931L2 were
removed from analysis according to criteria described previously
(McGoldrick and Hedgecock 1997).

North American Oysters

Two inbred lines derived from selfed hermaphrodites (89-6 and
89-7) were crossed on June 29. 1993 to generate four genotypic
populations: 6x6.6x7. 7x6. and 7x7 (Hedgecock et al. 1995).
Approximately 1 year later (340 days post-fertilization), a hybrid
Fn population was made by sib-mating a male and female from a
7 X 6 F, hybrid line. Two hundred progeny were labeled in No-
vember (203 days post-fertilization) with a numbered bee tag at-
tached with cyano-acrylate adhesive and set out on a lease owned
by Hog Island Oyster Co. (Tomales Bay. CA). After about 1 year,
labeled animals were brought back to Bodega Marine laboratory
and held for DNA analysis. Twenty-one animals died prior to
DNA extraction.

In another experiment, two unrelated oysters were crossed to
generate an Fl, and two Fl sibs were mated in 1993 to generate
line 93IL2. The 72 progeny examined were thus 25% inbred.
Hatchery and grow-out protocols are described in Breese and Mal-
ouf (1974),

Australian Oysters

Six Australian lines derived from pair-crosses of unrelated
commercial broodstock were created on January 8. 1997. Animals
were grown in 140-L containers, settled, and stocked to an up-
welling nursery .system according to commercial practice. On May
8. 1997, animals were transferred to Duck Bay (Smithton. Tasma-
nia) where they were grown at low density in sectionalized seed
trays. Family 97-2 was examined for 21 microsatellite loci. Forty-
one oysters were .sampled at day 198 post-fertilization and typed
for 18 microsatellite loci (see Table 5). Ten of these loci plus three
other loci were examined in a supplemental sample of 3 1 older
adult animals taken on day 359. Four loci were typed in families
97-1 and 97-6 (see Table 6). from which samples of 39 and 41
juveniles were taken on days 198 and 359, respectively.

Tests of Mendelian Segregation

Mendelian segregation is tested with G-tests (Sokal and Rohlf
1995). Initially, segregation ratios are assumed to be derived from

neutral, Mendelian, codominant. and observable alleles. Second-
arily, null alleles are allowed to modify hypothesis testing. When
multiple hypotheses exist within a family, then all consistent hy-
potheses are considered within the family. For the purposes of
segregation tests. C-statistics are calculated, ranked in order of
decreasing probability, and the test with most likely ratio is taken
to be the correct one for the purposes of tabulation.

To uncover whether distorted segregation ratios indicate ga-
metic incompatibility, ineiotic drive, or viability selection, we use
partition G-tests for gametic and zygotic selection (Pham et al.
1990. Lorieux 1995). First, tests of the prior Mendelian hypothesis
are made. If this test is significant, estimates of allele frequencies
are made outside of the prior hypothesis and the genotypic pro-
portions are tested given these allele frequencies. Residual signifi-
cance indicates zygotic selection. Thirteen cross types are recog-
nized with various levels of informativeness. The informativeness
of G-tests for 13 unique types of segregation at a single locus
allowing for null alleles is shown in Table 2. Type 7 (or di-hybrid)
crosses are tested for 1 :2 ratio of the most frequent homozygote
class relative to the heterozygote (a test of deleterious recessive
gene action linked to the less frequent homozygote). In addition, a
1 : 1 ratio of the two homozygote classes is also tested to indicate
heterozygote advantage or disadvantage, again using G-tests. Col-
lectively, the.se tests are used to propose the simplest form of
selection that could produce the observed ratio.

For the purposes of tabulation we adopt a notation for present-
ing selection tests in tables. Allelic selection against parental al-
leles is underscored in the appropriate parent and resulting geno-
types e.g "A/B X C/D" in the parents indicates selection against the
transmitted "A" allele and "A/C. AID, BIC, BID" in the progeny
indicates that selection against the "A" allele results in deficiency
in progeny classes containing the "A" allele. Zygotic selection on
genotypes is indicated by set bracketing of specific genotypes in
progeny tests, e.g., "{AIB\". When zygotic and allelic selection
overlap in the same genotype, zygotic selection is indicated by set
brackets and alleles are underscored for the purposes of tabulation,
e.g, "AIB X CID" in the parental fields and "AlC. [AID], BIC. BID"
in the progeny indicates selection against the "A" allele plus fur-
ther residual zygotic selection against the "[A/D]" genotype.
When useful in tabulation, genotypic classes with undistinguish-
able parental alleles are indicated with parentheses with any alleles
requiring distinction due to selection contained within as before,
e.g. "A/O. X B/A" in the parental fields and "A/B, (A,0). B/0" in
the progeny indicates selection associated with the "O" null allele
but not the hidden "A " allele. When two null alleles are present
that are distinguished by their transmission, the second null is
indicated " O' " (see Tables 3-6 and Appendix 1).

RESULTS

There are 43 informative tests of Mendelian segregation plus
four tests to confirm fixed transmission of homozygous alleles.
The number of loci tested, presence of nulls, and forms of selection
(after accounting for nulls) is presented in two North American
and three Australian families.

Family I (7 x 6 - Hybrid Progeny from North America)

Eight loci were tested (Table 3). The significance level for
rejecting Mendelian segregation is therefore set at 1 - ( I - 0.05)"*
= 0.0064. A null allele is postulated in the female parent for

782

McGOLDRICK ET AL.

TABLE 2
Types of crosses when null alleles are segregating in a population.

Genotypes

C

oniponents

for tests of distortion

Parent 1

X Parent 2

Type

Ho

G,.„„„,;

^Pareitt2

G

Game tie

('ZySOlic

AO

OO

0

1:1

J

J

AO

AO

1

3:1

J

AB

OO

2

J

J

AB

AA

3

J

J

AB

CC

4

J

J

AA

BO

5

J

J

AA

BC

6

J

J

AB

AB

7

J

J

AB

AO

g

2:1:1

Uf

(J)

J

U)

AO

BO

9

1:1:1:1

J

J

J

J

AB

CO

10

1:1:1:1

J

J

J

y

AB

AC

11

1:1:1:1

J

J

J

y

AB

CD

12

1:1:1:1

J

J

J

y

° Partial information indicated hy parentheses.

ucdCgOl, giving the dam the phenotype M and the genotype O/M.
This permitted the appearance of F phenotypes (O/F genotypes) in
the progeny; the sire having the S/F genotype. Progeny numbers
then accorded with Mendelian expectations given the adjusted sig-
nificance level.

Four of the eight loci do not deviate significantly from Men-
delian expectations. Of the four that do deviate, three UicdCg04.
ucdCgOS, and ucdCglO) show a heterozygote deficiency due to
zygotic and apparent underdominant selection. One locus
(ucdCgU) shows a deficit of one homozygous class and an excess
of the other homozygous class, apparently due to a deleterious effect
linked to the F allele transmitted by one of the parents (see Table 7).

Family 2 (931L2 - 25% Inbred Progeny from North America)

Ten loci were tested (Table 4). The significance level for re-
jecting Mendelian segregation is therefore set at I - ( I - 0.05)'""
= 0.0051. Null alleles are postulated for five loci. One locus
(iicdCg09) has one parent with a F phenotype and one with a S
phenotype, but gives S, F, S/F, and null phenotypes in offspring; in
this instance the parents are presumed to be O/S and O/F geno-
types. In two other loci {iicdCglO and iicdCgl4). a null allele is
postulated for either the male or female parent to permit the ob-
served progeny phenotypes. Locus ucdCgl5 is ostensibly a cross

between an F phenotype sire and a null homozygote dam, but it
yielded F, S, F/S, and null phenotypes. It is presumed that the sire
has an O/F genotype and the dam has an O/S genotype, but that the
S allele failed to detectably amplify in the female parent. The fifth
locus was iicdCglH. This cross has an S sire and an F/VF dam, and
the segregation yielded four phenotype classes in the progeny:
SA'F, F, S/F. and VF. We postulate that the sire genotype is S/O
and the dam genotype F/VF.

Six of the ten loci do not deviate significantly from Mendelian
expectations. Of the four that do, ucdCg09 shows a deficiency of
the null homozygote genotype, iicdCgI4 and ucdCgll show de-
viations for all three genotypes, and iudCg28 shows a deficiency
of genotypes containing the VF allele and is explained if a delete-
rious effect is associated with the VF allele.

Family 3 (97-2 - A Non-Inbred Pair-Cross from Australia)

Twenty-one loci were tested (Table 5). The significance level
for rejecting Mendelian segregation is therefore set at 1 - (1 -
0.05)"-' = n.()()24. Null alleles are postulated for 12 loci. Seven
loci have both parents designated heterozygous for a null allele and
an amplified allele, for one locus one parent is designated as a null
homozygote, and for four loci either the dam or sire is designated
a null/amplified allele heterozygote.

TABLE 3
Segregation tests of microsatellite transmission in crossbred North American cross 7 x 6A.

Locus

Sire >

Dam

Type

Gcnotypic classes

in progeny

Ratio

H„

G

P

7 X 6A:

mclCgOl

S/F

O/M

II)

O/F

:M/F

■M/S :0/S

27:28:46:42

:l:l:l

7.88

0.0485

iiclCnlU

F/S

F/S

7

F/F

:F/S

:S/S

27:X4:.^6

:2:1

4..TO

0. 1 1 65

ualCgt)4

I-/S

F/S

7

F/F

IF/SI

:S/S

43:37:35

:2:1

\5.n

0.0004

ucilCgOS

F/S

F/S

7

F/F

.F/S

:S/S

24:68:38

:::!

}.41

0. 1768

iialCaOf)

F/S

s/s

7

F/S

:S/S

69:77

:l

0.44

()..5078

ucdCi-OS

I-/S

F/S

7

F/F

■iF/SI

.S/S

3.5:44:48

:2:l

14.22

0.0008

ucJCmIO

F/S

F/S

7

F/F

■ IF/SI

.S/S

58:55:39

:2:l

15..50

0.0004

iutlCgl3

F/S

F/S

7

F/F

:F/S

:S/S

19:60:49

:2:l

14.20

0.0008

().(K)64.

The Transmission of Microsatellite Alleles

783

TABLE 4.
Segregation tests of microsatellite transmission in full-sib mating 93IL-2.

Locus

Sire

Dam

Type

Genotypic classes in progeny

Ratio

93IL-2. Most likely Inpotheses

ucdCgOl

F/S

F/S

7

F/F

ucclCg02

S/S

F/S

Jl

F/S

ucdCi;04

S/S

F/S

3

F/S

uciiCg09

F/O

"O'/S

9

F/O'

iiCilCglO

F/S

O/S

8

F/O

ticdCvl3

F/S

F/S

7

F/F

iudCgl4

'•s/o

F/S'

S

F/O

ucdCgIS

F/O

■ovisi

9

F/O'

ucdCgJl

F/S

F/S

7

F/F

ucdCglS

S/O

F/VF

9

S/VF

:F/S
:S/S
:S/S
:F/S
:F/S
:F/S
:F/S
:F/S
:F/S
-0/VF

:S/S

■S/O
:S/-
:S/S

:S'/(S. O)
:S/0
:S/S
■.F/O

:0/0'

.0/0'
:S/F

::22:17
1:28

|;22

■:2L25:6

i:22:19

;:29:11

1:7:20

:l8:20:L'i
9:23:32
L^:5:26:16

2:1

1

1

1:1

1:1

LI

1.83

0.04005

0.49

0.4835

1.24

0.2663

13.76

0.0033

7.41

0.0246

1.71

0.4260

13.54

0.0012

0.75

0.8625

18.81

0.0001

15.96

0.0012

" A second segregating null allele is indicated by O' in the dam.
to amplify in the female parent, see text. /", ^ = 0.0051.

'A second segregating slow allele is indicated by S' in the dam. ""The slow allele failed

TABLE 5.
Segregation tests of microsatellite transmission in Australian pair cross 97-2.

Locus

Sire X Dam

Type

Genotypic classes

in progeny

Ratio

H„ C

P

ucJCg02

172/201

172/172

3

172/172

: 172/201

15:18 1

1

0.27

0.6013

ucJCg03

]\yi5(,

143/156

12

113/143

:l 13/156

:I4.VI56

: 156/ 156

3:13:10:4 1

1

1 : 1 9.53

0.0230

ucdCg04

I39/LS9

159/159

-

159/159

32 1

-

-

ucdCgM

1 56-7182

07166

9

156/0'

:0'/182

: 156/166

:166/I82

2:24:6:29 1

1

1:1 .39.72

<0.000l

ucdCgOS

249/332'

07272

9

249/0'

:249/272

:272/332

:332/0'

0:0:15:17 1

1

1:1 44.49

<0.000l

ucdCg09

0/2I4

07164

9

O/O'

:0/l64

:0'/214

:164/214

4:11:4:13 1

1

1:1 8,54

0.0361

ucdCglO

197/222''

153/175

12

153/197

:15.V222

:175/197

: 1175/2221

16:14:28:5 1

1

1:1 17.95

0.0004

NcdCgi:

0/93

0/97

9

O/O

:0/93

:0/97

;93/97

6:13:2:7 1

1

1:1 9.23

0.0263

tadCgl3

148/184

0/148

8

(O, i48)/l48

:0/l84

: 148/1 84

41:3:7 2

1

1 21.87

<0.0001

ucdCgl4

150/180

126/133

12

126/150

:126/180

: 133/1 50

: 133/ 180

20:6:13:8 1

1

1:1 9.69

0.0214

iicdCglS

160/171

O/160'

8

(O. 160')/160

:I60'/171

:0/l71

3:10:8 2

1

1 12.11

0.0023

ludCglS

O/106

104/106'

8

(O. I06')/106

:O/104

:104/106

17:4:10 2

1

1 2.95

0.2291

iudCg2l

134/144

O/O

2

0/134

:0/144

9:15 1

1

1.52

0.2182

ucdCg22

229/254

229/254

7

229/229

:229/254

:254/254

4:21:7 1

T

1 4.01

0.1349

ciiirCg02

265/265

265/283

3

265/265

:265/283

23:16 1

1

1.26

0.2610

cmrCgO.<

437/470

437/470

7

437/437

:437/470

:470/470

8:18:10 1

->

1 0.22

0.8946

cmrC^06

0/136

0/136

1

0/0

:-/l36

22:41 1

3

3.07

0.0797

cmrC^6l

216/216

216/224

3

216/216

:2 16/224

25:13 1

1

3.86

0.0496

cmrCfiNl

0/1S6

186/204

8

186/204

:186/-

:O/204

13:20:7 1

1

1 1 .83

0.4009

clnrCgN.^

0/1 50

07145

9

0/0'

:0/l45

:O'/150

:145/150

2:11:9:18 1

1

1:1 14.92

0.0019

cnjrCgI5I

270/274

270/278

11

270/270

:270/274

:270/27

:274/278

13:11:5:11 1

1

1:1 4.08

0.2526

■■ The 156 allele is weakly amplifying and can be below detection, see text. "The 222 allele is also weakly amplified, see text. '^The 332 allele is again weakly amplified, see
lexl. P , is 0.0026.

TABLE 6.
Segregation tests of four microsatellites in Australian pair crosses 97-1 and 97-6.

Locus

Sire X

Dam

Type

Genotypic classes i

a progeny

Ratio

H„

G

P

97-1

cnirCg61

216/216

216/216

-

216/216

39

1

-

-

cmrCgNI

174/204

O/204'

8

O/174:i204'.O)/204

174/204'

24:10:5

1:2:1

23.20

<0.0000

cmrCgl4J

0/I5Q

150/155

8

0/155 : (0. 155)/150

150/155

14:12:13

1:2:1

5.96

0.0509

cmrCglSI

260/274

0/278

10

O/260 : 0/274

260/278: 274/278

10:12:7:9

1:1:1:1

1.38

0.7093

97-6

cmrCg6I

216/216

216/216

-

216/216

39

1

-

-

cmrCgNI

0/196

196'/ 178

8

(O. 196)/ 196' : 196/178

0/178

23:6:10

2:1:1

2.27

0.3208

cmrCgl43

146/155

150/160

12

146/150 : I46'/I60

150/155:155/160

7:4:18:11

1:1:1:1

10.93

0.0121

cmrCglSI

2SI/2SI

281/281

-

281/281

37

1

-

-

P^, = 0.016,

0.025.

784

MCGOLDRICK ET AL.

TABLE 7.
Analysis of selection at 12 microsatellite loci in 5 Paciflc oyster families.

Family

Type

(jr„iai

f-rolal

('Parenll

"Parenl2

^Gametic

Gzvgonr

P Parent 1

'Pareiill

p

Gametic

p

ZvfiolU-

Form

Action

7x6
iicdCg04

1

15.77

0.0004

1.11

14.65

0.2912

0.0001

Z

u.d..s

iicclCfiOH

1

14.22

0.0008

-

-

2.67

11.55

-

-

0.1025

0.0007

Z

u.d..s

iicdCglO

1

15.50

0.0004

-

-

4.76

10.74

-

-

0.0291

0.0010

Z

u.d.,s

ludCgli

1

14.20

0.0008

-

-

14.19

0.01

-

-

0.0002

0.9278

G

d.p

93IL2

ucdCg09

9

13.76

0.0033

1.14

6.31

7.45

6.31

0.2848

0.0120

0.0063

0.0120

G

d,f

ucdCgl4

8

13.54

0.0012

-

-

2.79

10.74

-

-

0.0946

0.0010

G

d,ni

iicdCgll

7

18.81

0.0001

-

-

16.91

1.91

-

-

<0.0001

0.1673

G

d.p

ucdCgIS

9

15.96

0.0012

-

9.87

9.87

6.09

-

0.0017

0.0017

0.0136

G

d.f

97-2

ucdCg06

9

39.72

<0.0001

37.16

1 .33

38.49

1.23

<0.0001

0.2483

<0.000l

0.2669

G

m

iicdCglO

12

17.95

0.0004

10.20

0.14

10.34

7.61

0.0014

0.7054

0.0013

0.0058

G,

Z

m.s

iicdCgl.i

8

21.87

<0.0001

-

-

20.22

1.65

-

-

<0.0001

0.1996

G

m

ludCglS

8

12.11

0.0023

-

-

11.89

0.22

-

-

0.0006

0.6370

G

m

iicdCgNS
97-1

cinrCgNI

9

14.92

0.0019

5.01

8.40

13.40

1.52

0.0253

0.0038

0.0003

0.2177

G

f

8

23.20

<0.0001

_

_

9.66

13.54

0.0019

_

0.0019

0.0002

G.

Z

m.s

97-6

cmrCgI43

12

10.93

0.0121

8.40

2.53

10.92

0.01

0.0038

0.1119

0.0009

0.927!

G

m

G, allelic selection; Z, zygotic viability selection; u.d.. underdominance; d. dominance; p. allelic .selection by one of the parents, but unresolved; m. male
allelic selection; f, female allelic selection; s. specific selection pattern.

Particular mention must be made of three loci: ucdCgOb.
ucdCgOS, and ucdCglO. Locus iicdCg06 is a four-allele system,
with the sire being 156/182 and the dam 0/166. Four progeny
classes are expected. 156/0, 156/166, 0/IS2. and 166/182. This
locus was examined in both young and old progeny and the 156
allele is weakly amplifying. In the first set of tests, of the juveniles,
the 156 allele is detected twice. However, in the second set of
(older) progeny, the 156 allele is not detected, although it is as-
sumed to be present in progeny having the 166 or no amplification
phenolype. Overall, there is significant distortion (P < 0.0001). It
appears that this 156 allele is under strong selection in progeny due
to a linked deleterious recessive gene or a deleterious interaction
with factors transmitted by the dam.

For locus ucdCg08, the sire shows a single strong band at 249
bp and the dam shows a single strong band at 272 bp. However, the
249 allele is not detected, not even weakly, in any progeny. Prog-
eny show either a 272-bp phenotype (n = 1.5), a 332-bp phenotype
(/; = 10; an allele that amplifies weakly), or no amplification
product (/; = 7). This locus is multiplexed with locus iiidCgN.
which in this family presents four alleles where all individuals
showed appropriate genotypes (no non-amplified individuals) and
where the sire is consistent with the progeny genotypes given the
dam. The progeny for iicdCg08 are consistent with the following
explanation: the dam is a 272/0 hetero/.ygote and the sire is a
249/332 hetero/.ygote. The absence of the 249 allele in progeny
suggests that the 249 allele might mark a lethal interaction when
combined with factors in the dam's genetic background (however.
see "Discussion"). The 332 allele at iicdCgOS does not amplify
strongly using these PCR conditions (perhaps due to its larger si/e
or a mutation in the priming site) and .so is not reliably scored in
the progeny; even though we can score the sire as a 249/332. we
score the borderline 332 allele as a null in progeny. The segrega-
tion at this locus tits expected Mcndelian ratios when we score the

dam's 272 allele as if it were a dominant segregation with the
pooled class 272/- and O/O. This will give two consistent pheno-
typic classes. 272. and null, in a 1:1 ratio — very close to the ratio
observed.

Locus IicdCglO is a four-allele system, with the sire being
197/222 and the dam 153/175. Four progeny classes are expected,
153/197, 153/222, 175/197. and 175/222. This locus was examined
in both young and old progeny. In the first set of tests, of the spat,
the 222-bp allele is below detection by the ABI system. However,
in the second set of (older) progeny, the 222 allele is detected,
although more weakly than the smaller alleles. Furthermore, while
the segregation is close to expected in the spat, scoring the 222 as
a null allele (;; = 32, P = 0.0451), it is aberrant in the older
individuals (n = 31. P = 0.0003), with overall significant distor-
tion (P = 0.0004). This is due to a relative lack of the genotypes
with weak 222 allele and a significant relative deficiency of the
heterozygote 175/222 (Tables 5 and 7). We note that these four
alleles are separated step-wise in si/e by about 20 to 2.5 bp — very
close to the size of the forward PCR primer (Table I ). Whether
changes in PCR kinetics (such as extension efficiency or priming
errors) generated the \ariable detection of the 222 allele or whether
there is an age-dependent effect was not resolved. Regardless of
the amplification intensity of the 222 allele, it also appears that it
is under .selection in this family due to association with a delete-
rious gene, plus an interaction with the chromosomal segment
marked by the 175 allele.

Fifteen of the 21 loci do not deviate significantly from Men-
delian expectations. Of the six that deviate, three are borderline
deviations from the adjusted significance levels. The three loci
showing strong deviations were ucdCgUS and ucdCglO (discussed
above) and iicdCgl3. For locus iicdCf;l3. 80.4'/; of progeny have
the l4iS-bp phenotype instead of the 50'/r expected, apparently due
to a linked deleterious effect associated with the IS4-bp allele.

The Transmission of Microsatellite Alleles

785

Family 4 (97-1 - A Non-Inbred Pair-Cross from Australia)

Four loci were tested (Table 6). One or other parent is hetero-
zygous for a null allele and amplified allele in three instances.
Three of the four loci accord with Mendelian expectations. The
deviant locus. cmrCgMl. gives progeny ratios that are quite dif-
ferent from those expected, even though all expected phenotypes
are observed.

Family 5 (97-6 - A Non-Inbred Pair-Cross from Australia)

The same four loci were tested as in family 97-1 (Table 6). One
parent is heterozygous for a null allele and amplified allele. Three
of the four loci accord with Mendelian expectations and the fourth
is marginally significant following corrections for multiple tests.

DISCUSSION

Null Alleles

A total of 47 tests of Mendelian transmission were carried out.
43 of which were segregating for more than one allele. Null alleles,
including alleles such as iicdCglO^'' (see Table 51 with borderline
amplification, were postulated in almost one-half of the tests (22 of
47 or 47%). It is possible that some borderline null alleles might
amplify more intensely under less stringent PCR conditions (e.g.
addition of more primer and lowering of annealing temperatures),
but we have pooled these borderline nulls into the same category
as completely null alleles. The completely undetectable null alleles
might result from a more severe mutation (e.g. a small deletion in
the priming site rather than an extended length of the microsatellite
allele). Given that 43 informative segregation tests were carried
out. 43 X 4 = 172 parental alleles were examined. Overall, 30 of
the 172 parental alleles (17%) were null or non-amplifying alleles.
One allele did not. for unknown reasons, amplify in a female
parent (iicilCglS'' : Table 4). Null allele counts were slightly stock-
dependent, being more common in Australian stocks (22 of 100
parental alleles. 22%) than North American stocks (8 of 72. 11%).
however the difference is only marginally significant (P = 0.04).
More crosses have to be examined to comment meaningfully on
null allele incidence at individual loci, although there is a sugges-
tion that null alleles might be more common at some loci than
others. For example, ucdCg04 was examined in three crosses (two
North American and one Australian) with no evidence for null
alleles, while at the other extreme. ucdCg09 was examined in two
crosses (one North American and one Australian) and in both cases
the apparently homozygous parents were heterozygous for ampli-
fied and null alleles.

The presence of null alleles is often presumed to result from the
primer design process or PCR artifacts such as paralogous loci, but
progeny tests reveal that null alleles are inherited and result from
mutations in the template. Multiplex PCR allows us to rule out
non-specific inhibitors of PCR or poor template preparations as
explanations of null alleles. Sequence specific inhibitors cannot be
ruled out. The presence of null alleles in multiplex PCR reactions
in one family and not another rules out systematic mismatches of
the primers and implicates either template variation at the priming
site or perhaps large insertion events that prevent extension during
the thermal cycling. The high frequency of null alleles is not solely
a property of any one genomic library or microsatellite primer set
used. To date, multiple independent libraries have produced prim-
ers with null alleles in Pacific oysters (McGoldrick 1997. Magou-
las et al. 1998. English unpublished). Multiple laboratories have

observed null alleles in oysters, some running other types of PCR
markers. For example, Hu and Foltz (1996), while examining
scDNA markers in American oysters, found three in.stances of
"abberant genotypes" in families that can be explained by null
alleles.

Since null alleles occur in one pedigree and not others, primer
design is rather "hit and miss." Redesigning primers is feasible if
a few loci (perhaps five) are to be typed for population genetic
work or for a limited number of pedigrees, but becomes much
more difficult for a bivalve genome mapping study requiring about
100 markers as anchor loci and with complete codominant expres-
sion for all alleles. There is no a priori mechanism to guarantee
that nulls will not appear at the redesigned primers in a new pedi-
gree or in some proportion of priming sites in a natural population.
Nevertheless, it is quite clear from these breeding studies that null
alleles are frequent at microsatellite loci in Pacific oysters and
perhaps all PCR-based markers in bivalves that amplify neutral
DNA.

Null alleles at allozyme loci have been reported in oysters. In
the American oyster (C. virginica) null alleles were reported at
Mpi and LMp-2 after testing progeny from five crosses (Foltz
1986b). The importance of these relatively rare observations at
allozyme loci in influencing general deficiencies in heterozygosity
has been down-weighted relative to larval viability selection (e.g.
Mallet et al. 1985, Gardner 1992). Overall, heterozygote deficien-
cies for microsatellite loci in Tasmanian and Japanese samples
Pacific oysters (English unpublished. McGoldrick and Huvet un-
published) appear to be considerably larger than those observed for
allozyme loci (English et al. 2000). This is most likely related to
the higher prevalence of null alleles for microsatellites than for
allozymes, and suggests there is a greater level of segregating
variation in untranscribed DNA than in transcribed DNA. Se-
quence evolution at priming sites can be explained by additional
mutational mechanisms, perhaps involving recombination, that act
in addition to neutral point mutations (e.g. through deletions and
insertions) and with longer persistence in neutral DNA. Alterna-
tively, population level phenomena such as hybridization and in-
trogression of diverged chromosomal segments (Hirase 1930, Imai
and Sakai 1960, Thomson 1959, also reviewed by Gardner 1997)
might also be important. Hybridization and hypotheses about dif-
ferences between transcribed and untranscribed DNA have some
published basis and the relative contribution of each remains un-
resolved at this time.

Null Alleles in Gene Mapping

Nulls can be accommodated in gene mapping. Given any mi-
crosatellite segregation, the most complete classification would be
parents "A/B x C/D" crossed to give progeny classes "{A/C. A/D,
B/C. B/D]" (type 12 in Table 2). Should one of these alleles be a
null allele, the segregation would be scored in the form "A/C, A/0.
B/C. B/0" (e.g. type 10). Note that the change in score from
observed allele D to unobserved allele O does not change the
underlaying counts in these progeny categories, the segregation
tests, degrees of freedom, or the mapping results. Therefore,
changing the priming sites or conditions to favor amplification of
the D allele does not produce any statistical gain in mapping.
Further, should the allele C in the second parent be indistinguish-
able from the A or B allele in the other parent (reducing the cross
type from "A/B x C/O" to "A/S x B/O"; type 5). the segregation
would be of the form -A/B. B/B. B/O. A/O". This would be equal

786

McGOLDRICK ET AL.

in information content to the standard segregation of "A/B x B/B".
Here we simply pool the classes "A-" and "B-" recognizing the
"A/0" as a legitimate genotypic class and achieve equal power to
the "A/B X B/B" case. Similarly, should both alleles in one parent
be null and the other parent heterozygous amplified/null (type 2),
the results are again equal to an " A/B x B/B" segregation. More-
over, should one parent be heterozygous for a null and the other
homozygous for nulls (type 0), we again have equal power to the
"A/B X B/B' segregation if template reactions are controlled with
multiplex PCR so that the "O/O" genotype can be .scored reliably.
If both parents are heterozygous for nulls and indistinguishable
amplified alleles "A/0 x A/O" (or type 1), we would have some
mapping information (equivalent to a 3:1 segregation), but would
not have expected any in the case of "A/A x A/A ". Lastly, we note
that if both parents are heterozygous for nulls and distinguishable
amplified alleles (type 9), we have four recognizable classes and
complete classification, e.g. "A/0 x B/0" gives "A/S. /K/O. B/O.
0/0. "

These facts suggest that at worst we get a 1 : 1 ratio, and in large
studies, decreasing gain for effort in resolving null issues by re-
ducing PCR stringency or redesigning primers for highly variable
markers. Therefore, should a mapping project with 50 to 100 mi-
crosatellites plan for this level of power (e.g. using dominant
AFLP markers and microsatellites together), there is little problem
with null alleles in mapping. Certainly, the great fecundity of
bivalves can accommodate the 398 progeny needed to detect loose
(30 cM) linkages in the assortment of two dominant loci (see
Allard (1956]) for a detailed analysis of the numbers of progeny
needed for linkage mapping with dominant markers). Indeed, or-
ganisms with large family sizes (e.g. most invertebrates and
plants), even if they have null alleles, have obvious natural advan-
tages that make them desirable for mapping with microsatellites.

Selection Associated with Microsatellite Markers

About one-third of microsatellite segregation ratios showed
significant ratio distortion (16 of 43 or 37.27r) when null alleles
are accounted for. This proportion comes after significance adjust-
ments are made within each of the five crosses for multiple tests of
segregation. There is no significant association between segrega-
tion distortion and the presence of null alleles ( 12 tests showed no
distortion and null alleles. 15 showed no distortion and no null
allele, eight showed distortion and null alleles, and eight showed
distortion and no null allele, P = 0.724).

.Segregation distortion at the level of genetic markers is clearly
quite common in Pacific oysters. Allo/yme analysis of the progeny
of sibs from selfed hermaphrodite Pacific oysters also revealed
significant .segregation distortion (McGoldrick and Hedgecock
1997). again affecting about one-third of tests (16 out of 51 or
liWc). Significant distortion has also been recorded in the Hal
oyster. Oslica ediilis. again in about one-third of tests of micro-
satellite segregation (Naciri et al. 1995. Bierne el al. 199S). Many
other published studies, most of which arc based on ullo/ymc data
(Appendix I ). have also reported segregation distortion in bi-
valves. Thus the distortion does not appear to be associated with
the use of microsatellite loci, but is a general bivalve phenomenon.
In our studies, strong zygotic viability selection cannot be luled out.

Selection in Bivalves

There is much to he gaineti by investigation ol the lorms of
selection and markers associated with segregation distortion in
other bivalves (Appendix 1). Selection is often associated with
single alleles (also sometimes referred to as "gametic" whereby

selection can be explained by simple deleterious recessive genes
linked to an observed allele). For simple segregation ratios of type
0 through 6 (Table 2). apparent .selection on specific genotypes has
been reported in the genus Cmssostrea 19 times (C viri^inica 16
times and C. gigas three times). No data were found for flat oysters
(genus Ostrea), but in Mytilidae (represented by Mytitus edidis),
distortion has been reported five times for these .seven types. The
simpler types of segregation (type 0-6) cannot ever reveal any
underlying non-additive properties to the selection observed at the
level of these markers, so the context within which the observation
is made can lead to a biased conclusion which implicates only
linked deleterious recessives. In fact, deleterious recessive action
alone would grossly under-represent gene action at genetic mark-
ers in bivalves. This can be observed when types of crosses and
markers are used that have more informative segregation (e.g.,
types 7-12).

For more informative segregation ratios, selection on specific
genotypes apparently occurred in all four taxa (C gigas. C. vir-
ginica, O. edulis, and M. edidis). There were 14 such segregation
distortion events in the American oyster (C. virginica). one case
outside this study in the Pacific oyster (C. giga.s), 18 cases in
mussels (M. ediilis). and three cases in flat oysters (O. edidis:
Appendix 1 ). No attempt is made to correct for experimental effort
in these cases, but by taking these events case by case and when
the information is available, underdominant selection patterns of-
ten appear (with a lower fitness for the heterozygote). This implies
there are associated negative interactions between chromosomal
segments containing the observed locus. For example, in the di-
hybrid crosses with distortion (type 7 crosses), five of seven events
(71%) included some form of underdominance and this occurred in
all four bivalve taxa (Appendix 1 ). The present study concurs. For
example, the North American family 7x6 has three of four cases
(75%) apparently underdominant using the microsatellite markers
(Tables 3 and 7). These results are intriguing when combined with
the empirical observation at locus iicdCgOH concerning the 249-bp
allele (Table 5). The absence of the 249 allele in progeny suggests
that the 249 allele might mark a lethal interaction when combined
with factors in the dams genetic background. An alternative hy-
pothesis is that the sire's 249 allele is a paralogous amplification
product not belonging to iicdCgOS. but this would not explain why
the 249-bp fragment was not observed in any progeny nor why it
had a microsatellite stutter pattern. In addition, if there was some
sort of template contamination in the sire that produced the 249
allele, then all the other loci in this family should have had spu-
rious alleles observed in the sire, but no unexplained alleles were
observed at other loci. While the segregation of the 249 allele
might be an unexplained artifact, it is also consistent with an
inleraclion hypothesis.

This underdominance al the family level is potentially very
serious because gene frequencies become naturally unstable with
major population genetic and evolutionary implications in terms of
frequency dependent selection. Underdominance in segments (in-
cluding all sufficiently linked genes) can lead to fixation of intro-
gresscd gene segments in metapopulations or randomly drifting
small populations of bivalves should one or the other segment
attain and maintain a critical frequency threshold (e.g. Crow 1986).
This is particularly relevant in light of low estimated effective
sizes in hatchery stocks (Hedgecock and Sly 1990). Further, it is
possible to cause extinction by means of gene replacement if an
inlrogressed segment confers susceptibility to sonic periodic envi-
ronmental trigger such as a disease outbreak or en\ironmental
shifi. The potential cost of this type of event could be the value of

The Transmission of Microsatellite Alleles

787

the entire culture industry if a commercial broodstock becomes
affected and is not managed with this contingency in mind. In
evolutionary terms, bivalve populations might remain well adapted
and genetically stable for long periods of time, but might unex-
pectedly and rapidly approach extinction in response to a recurrent
environmental trigger. Further research into the prevalence of un-
derdominance in bivalves is needed and awaits more defined link-
age mapping especially within the context of stock importation and
gene frequency modification during selective breeding.

ACKNOWLEDGMENTS

We thank the Aquaculture CRC. the Fisheries Research and
Development Corporation, the USDA Western Regional Aquacul-
ture Consortium, and the National Research Initiative Competitive
Grants Program (92-37206-8003 and 95-37206-2318) for funding
this work. Will Borgeson and Greg Maguire for help producing the
family lines. Bronwyn Innes for genetic typing, and Jawahar Patil,
Nick Elliott, Peter Rothlisberg, and Vivienne Mawson for com-
ments on the manuscript.

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788

MCGOLDRICK ET AL.

APPENDIX 1.
Reported occurrences of segregation distortion for bivalve Mollusc families.

Species

Cross

Locus

Parent I x Parent 2

gl

g-'

g3

g-i

Ratio H„ G

P

Type 3 AB X AA

C. virginica

Cross 1

Ap-I

lOO/IOH'

108/108

I08'/108

108/100

18:44 1

1 11.25

0.0008^

C. virfiinica

Cross 1

Gpi

100/100

78/58

100/78

100/58

18.44 1

1 1 1 .25

0.0008'

C. viriiinica

Cross 2

6PCDH"''

mm

100/100

100/100'

100/72

318:157 1

1 55.67

0.0000'

C. virginicd

Cross 4

Pgm-2

J00'AH2

100/100

lOO'/lOO

100/82

38:9 1

1 19.25

0.0000'

C. virginica

Cross 5

Pgm-2

IO0/>Q

100/100

100/100

100/82

412:299 1

1 1 S.04

0.0000'

C. lirginica

Cross 7

Lap-I

loom

100/100

100/100

100/93

209:170 i

1 4.02

0.0450'

C. virginicu

Cross 7

Uip-2

100/100

100783

100/100'

100/83

109:266 1

1 67.80

0.0000'

C. virginica

Cross 10

Cot-2

7H/10I)'

100/100

78/100

100/100'

25:8 1

1 9.19

0.0024'

C. virf>imfa

Cross 10

Lap -2

JOO/SJ

100/100

100/100

100/83

25:8 1

1 9.19

0.0024'

C. yirf^inica

Cross 10

Gpi

e/c

c/c

e/c

c/c

32:14 1

1 7.24

0.0071''

C. viriiinica

Cross 10

Gpi

e/c

c/c

e/c

c/c

61:36 1

1 6.52

0.0107''

C. virginica

Cross 4

CV-/95"-""

B/B

A/B

(A/Aj

A/B

B/B

5:3:24 0:

: 1 0.00

0.0000''

C. gigas

92-97-5

Pgm

A/B

A/A

A/A

A/B

9:24 1

1 7.07

0.0078'

C. gigas

92-97-5

Uip-2

A/C

C/C

A/C

C/C

9:24 1

1 7.07

0.0078'

C. gigas

92-97-5

Dap-2

A/A

A/B

A/A

A/B

21:6 1

1 8.83

0.0030'

M. eiiulis

FAM II

Esl-D

100/121

100/100

100/100

121/100

85:26 1

1 33.03

0.0000'

M. editlis

FAM II

Pgd

}/4

3/3

3/3

4/3

109:62 1

1 13.09

0.0003-

M. ediilis

64 x5

Pgi

100/100

88/100'

100/100'

88/100

96:126 1

1 4.07

0.0437"

M. edtilis

29 X 10

Lap

100/100

1007105

100/100'

100/105

15:34 1

1 6.63

0.0100"

M. edttiis

64 X 101

Lap

1 00/1 OS

100/100

100/105

100/100

78:131 1

1 4.80

0.0285"

Type 4ABxCC

C. virginica

Cross 7

Gpi

II2/7S

100/100

112/100

100/78

220:162 1

1 8.84

0.0029'

C. virginica

Cross 8

Gpi

100/100

112/78

112/100

100/78

113:75 1

1 7.73

0.0054'

M. edulis

64 X 101

Pgi

100/100

90/96

96/100

90/100

107:148 1

1 6.62

0.0101"

Type 6 AA X BC

C. virginica

Cross 4

Gol-2

100/100

78/279

78/100

100/279

14:31 1

1 6.58

0.010.3'

C. virginica

Cross 5

Gni-2

100/100

78/279

78/100

100/279

291:411 1

1 20.61

0.0000'

C. virginica

Cross 5

Uip-2

115/115

100/00

115/00

115/100

413:335 1

1 8.15

0.0043'

C. virginica

Cross 5

Gpi

100/100

78/58

100/78

100/58

314:440 1

1 21.15

0.0000'

Type 7 AB X AB

C. virginica

Cross 3

Mpi

91/100

91/100

91/91

191/100/

100/100

55:88:70 1:

M S.26

0.0160'

C. virginica

Cross 10

Gpi

e/c

e/c

le/el

e/c

Ic/cl

14:68:11 1:

2:1 21.02

0.0000''

C. gigas

92-97-5

Acan-l

A/C

A/C

A/A

lA/CI

C/C

21:5:1 1:

1:1 33.92

0.0000'

M. edulis

FAM I

EsiD

100/121

100/121

100.100

1121/1001

121/121

73:90:22 1:

!:1 29.01

0.0000-'

M. edulis

64 X 5

Lap

100/105

100/105

105/105

1100/105/

100/100

43:75:102 1:

!: 1 47.38

0.0000"

M. edulis

64 X 5

Lap

100/105

100/105

105/105

1100/105/

100/100

43:75:102 1:

!:l 47.38

O.O(HX)"

O. edulis

CI

Oedu.JI2'"

224/230

224/230

224/224

1224/230/

230/230

17:9:52 1:

M 70.95

o.oooo*

O. edulis

C2

Oedu.B(f"

9S/I01

98/101

98/98

98/101

1101/101/

25:50:5 1:

!:l 19.61

0.000 1''

Type 8 AB X AO

C. virginica

Cross 2

(yPGDH

100/72

I007O

100/-

IOO'/72

10/72/

318:157:0 2:

: 1 0.02

0.8967'

C. virginica

Cross 4

Cl'-/W"-""

B/0

A/B

lA/OI

/A/B/

B/-

5:3:24 1:

:2 8.88

0.0118'

Type II ABx

AC

C. virginica

Cross 2

Gol-2

78/100

787279

78/78'

/JVJIOO

78/279

100/279

81:107:101:17: 1:1

: : 1 38,05

0.01X10'

C. virginica

Cross 3

Coi-2

78/ 100

7£/279

78nK_

1787100/

78/279

100/279

51:22:66:69 1:1

1 : 1 30.68

0.0000'

C. virginica

Cross 3

Gpi

100/78

78758

ioonK_

100/58

78/7£

78/5S

27:72:48:73 1:1

1 : 1 28.63

0.0000'

C. virginica

Cross 4

Pgm-2"''

100782

lOO/O

100/-

100/82

IO/82/

38:9 2

1 4.68

0.0.30.5'

C. virginica

Cross 8

Ap-I

116/108

1 167100

1116/116'!

II 6' /1 08

116/100

1108/100/

37:57:58:33 1:1

1:1 II .29

0.0102'

C virginica

Cross 10

iMp-l

100/82

ioir/93

100/100'

100/93

100'/82

93/82

11:0:16:12 1:1

1:1 23.49

0.()(HH)'

M. edulis

FAM I

Pgm-2

100/92

100/80

100/100

100/92

100/80

92/80

46:30:35:23 1:1

1:1 8.32

0.0398'

M. edulis

FAM I

Hey

100/157

100/28

100/28

157/28

100/100

157/100

73:13:69:23 1:1

1:1 70.44

O.IKHKV

M. edulis

FAM II

Imp

94/96

94'AJ8

94/94'

96/94'

98/94

98A)6

27:50:32:49 1:1

1:1 10.67

0.01.16'

M. edulis

32 X 10

Pgi

93/100

88/93

93' /1 00

88/100

93/93'

88/93

76:29:16:8 1:1

1:1 79.40

0.0000"

M. edulis

45 X 39

Pgi

91/100

93/100'

100/100'

93/100

91/100'

91/93

63:101:59:86 1:1

1:1 1511

0(1017"

M. edtdis

45 X 42

Pgnt

100/103

9,VI03'

103/103'

100/103'

98/103

98/100

13:35:14:23 1:1

1:1 14.11

0.0028"

M. edulis

29 X 10

Pgm

97/100

I00'/I07

100/107

97/107

imioo'

97/100'

36:71:55:74 1:1

1:1 16.52

0.0009"

M. edulis

32 X 10

Pgm

94/107

100/107'

107/107'

100/ 107

94/107'

94/100

47:34:29:19 1:1

1:1 12.73

0.0053"

M. edulis

M X 5

I'gtii

97/ im

I00'/I04

100/104

97/104

100/100'

97/100'

47:32:67:50 1:1

1:1 12.76

0.0052"

M. edulis

M X 101

Pgm

97/100

9r/l04

Km/ 104

97/104

97'/l00

97/97'

30:39:74:67 1:1

1:1 26.72

0.000"

O. edulis

CI

Oedu.TS'"

106/124

I24'/I28

124/124'

124/128

106/124'

106/128

17:12:32:25 1:1

1:1 1 1 III

0,01 :7''

Type 12 ABx

CI)

C. virginica

Cross 1

Uip-2

115/108

100/00

115/00

115/100

108/00

108/100

15:8:24:7 1:1

1:1 13.21

0.0042'

C. virginica

Cross 2

Gpi

112/100

78/58 112/78 112/58

100/78

100/58

65:109:109:194

1:1

1:1 70.74

0.0(X)0'

C. virginica

Cross 4

Gpi

106/100

78/58

106/78

106/58

100/78

l(H)/58

9:21:4:14 1:1

1:1 13.85

0.0031'

C. virginica

Cross 8

Adk

110/92

IO0AI6

1 10/100

110/96

lim/92

96/92

38:57:28:36 1:1

1:1 10.91

0.0122'

' BL-auiiuinl 1983. "Hvilsom and Thciscn 1984. 'Foil/ 1986. ''Hu cI al. 1993. 'Hu and Foil/, 1995. 'McGoldrick 1997. 'Bicme 1998.

Journal of Shellfish Research. Vol. 19. No. 2. 789-797. 2000.

MICROGEOGRAPHIC DIFFERENCES IN GROWTH, MORTALITY, AND BIOCHEMICAL
COMPOSITION OF CULTURED PACIFIC OYSTERS {CRASSOSTREA GIG AS) FROM

SAN QUINTIN BAY, MEXICO

ZAUL GARCIA-ESQUIVEL,* MARCO A. GONZALEZ-GOMEZ,
DAHEN L. GOMEZ-TOGO, MANUEL S. GALINDO-BECT, AND
MARTIN HERNANDEZ-AYON

Institiito de Investigaciones Oceanologicas. Universidad Aiitonoma de Baja
California. Apdo. Postal 453, Km. 107 Carretera Tijuana-Ensenada.
Ensenada. B.C.. Mexico

ABSTRACT Changes in shell height, tissue dry weight (TDW). mortality, and biochemical composition of Crassoslrea gigas were
evaluated at two commercial sites in San Quentin Bay (SQB), Mexico, during the first 9 mo posl-settlement. Shell growth rates were
1 .5x higher at the mouth (0.36 mm d"' ) than the head of SQB (0.23 mm d~' ). Tissue dry weight was also 7- to 8-fold higher in oysters
from the mouth (2.1-2.16 g TDW oyster"') than the head of SQB (0.23-0.33 g TDW oyster"' I at the end of the 9-mo experiment.
Market size (9 cm) was reached after 9 mo at the mouth and at an estimated age of 13 mo at the head of the hay. Highest mortality
occurred within the first month post-settlement i509c-60'7c) and reached approximately 63% to 87% throughout the whole study period.
Proteins (48%-64%) and lipids (2%-9%) were the inost abundant biochemical components during early spat development. When
oysters reached a size between 48 and 55 mm (shell height) in the fall, glycogen ( l%-22%) replaced lipids as the main energy depot.
The changeover occurred earlier in oysters at the mouth than in oysters from the head of the bay. It is suggested that between-site
differences in growth and biochemical composition in oysters are the result of longer immersion/feeding period experienced by oysters
at the mouth of SQB.

KEY WORDS: Oyster. Crassoslrea gigas. San Quentin, biochemical content, growth, condition index

INTRODUCTION

San Quentin Bay (SQB) is a shallow coastal lagoon (2 ni mean
depth) located between 30° 24' and 30° 30'N and 1 15° 57' and
1 16° 01 'W. on the northwest Pacific coast of Mexico. Its produc-
tivity (0.24-0.94 g C m' d~') and hydrodynamics are strongly
tide-dependent and influenced by alongshore upwelling systems,
which maintain a high nutrient supply to the lagoon during the
spring months (Alvarez-Borrego and Alvarez-Borrego 1982,
Millan-Nunez et al. 1982). The residence time of the water in the
bay varies from hours near the mouth to days at the head of SQB
(Juarez-Villarreal 1982). Therefore, any changes in the available
food, either in quantity or quality, or any microgeographic differ-
ences in environmental conditions may have implications for the
successful culture of suspension-feeders.

Hatchery production of commercially important marine bi-
valves, such as oysters, is well established and provides the basis
for economically viable industries on both coasts of North
America. The Pacific oyster. Crassostrea gigas. is a species intro-
duced from Japan and it is the mainstay of the oyster industry in
the Pacific coast. This industry relies largely on hatchery produc-
tion of larval stages in American hatcheries and remote setting of
pediveligers around the world. C. gigas was introduced into SQB
(Ensenada. Mexico) in 1975. and its acceptance in the local market
prompted the adaptation of mass production techniques in the
laboratory and the field by local farmers (Islas-Olivares 1975).
While larval cultures proved to be uneconomical, oyster produc-
tion via remote setting increased from approximately 100 to 2.000
tons live weight year"' over the last 20 years (J.C. Gardufio pers.
comm.). This production was based on remote setting of pedive-
liger larvae on oyster or scallop shells, early spat rearing in sub-

*Corresponding author.

tidal rafts, and suspended juvenile-adult grow-out in intertidal
structures called "racks."

The substrate with newly settled oyster spat is typically placed
inside wide-mesh bags and suspended from floating rafts along the
channels of SQB. At the end of the rearing period, a series of 7
mother shells are connected through a polypropylene rope (overall
length 1.2 m) and suspended in intertidal culture "racks" made of
wood or PVC plastic frames (Polanco et al. 1988). The oysters
remain in these units (approximately 6.2 x 2 x 1.2. length x width
X height) until they reach the harvesting size of approximately 9 to
10 cm (Polanco et al. 1988). The culture process is extensive, since
most operations involve a substantial amount of human labor and
minimal manipulation once the oysters are transferred to the water
column. The efficiency of these commercial units remain anec-
dotal, largely because the lack of coordination between aquacul-
turists and researchers, and the budget constraints to travel to and
from SQB. a pristine site located approximately 250 km from
academic centers. A previous study (Acosta-Ruiz 1985) reported
oyster growth rates on experimental systems different from those
commercially utilized. Therefore, the present study was aimed at
assessing the growth, mortality, and biochemical changes of the
Pacific oyster. C gigas during two production cycles at two com-
mercial sites of SQB with differing residence water times.

MATERIALS AND METHODS

Setting and Early Rearing

Two batches of oyster larvae were sent overnight inside insu-
lated boxes in the form of a wet "paste" wrapped with nylon cloth
and paper towels. Ice packs were also placed inside the boxes in
order to maintain a cold atmosphere (approximately 5 °C) during
transport. Full details for the storing and shipping conditions of
competent (ready-to-set) larvae are given elsewhere (Jones and
Jones 1983). Both oyster batches came from the Whiskey Creek

789

790

Garci'a-Esquivel et al.

TABLE 1.

Morphometry and settlement success of two oyster (Crassostrea gigas) batches in San Quintin Bay. Spring and Summer = larvae were set in

April and July of 1995, respectively. Mean iX = ±95% confidence interval) or median values (Md, was 25% and 75% percentile range) are

shown for each variable. Mann-Whitney test was used for comparison of medians. Comparisons of means were carried out with a

two-sample Student test, ns = Non-significant difference, ** = Significant differences at a = 0.01, NS.

Variable

Spring batch

Summer batch

Shell height (jj.m)
Dry weight (]xg)
Ash-free dry weight (fjLg)
Shell width (fjim)
Setting

No. shells per tank
No. larvae per milliliter
Setting time
No. spat per shell

Md = 321.8 (316.8-326.7)
Md = 6.6 (6.1-7.0)
X = 2.20 (±0.06)
X = 297.6 (±2.7)

63,000

1.2

59 h

X = 46.5 (±7.4)

Md = 236.7 (316.8-326.7); ns
Md = 5.5 (5.4-5.7): **
X = 2.14 (±0.04); ns
X = 290.2 (±2.7); **

30.000

0.6

48 h

X = 29.9 (±4.5)

Oyster Farm (Oregon) on April 20 (Spring experiment) and July
20, 1995 (Summer experiment). Upon arrival to the Institute de
Investigaciones Oceanologicas in Ensenada (I.I.O.), triplicate sub-
samples of 30 to 100 larvae were individually measured under a
microscope and weighed in terms of dry and ash-free dry weight
(see below). The rest of the larval paste was packed again as
described above, and transported by land (approximately 4 h) to
SQB where oyster setting took place. Overall shipping time from
Oregon to SQB was approximately 36 h.

Larval setting was fully carried out by local fisherman. Brietly,
about 400 half-oyster shells were packed in individual bags made
of nylon rope (diagonal mesh 7 cm). Bags with shell substrate were
deployed in cylindrical fiberglass tanks of 8,500 L (diameter =
300 cm, height = 120 cm). The total amount of bags with shell
substrate per .setting tank was different for the (Spring) and (Sum-
mer) batches (Table I ), but the ratio of larvaeisubstrate was about
the same in both cases, e.g. 170:1 (swimming larvae:shell). The.se
numbers are about twice as much the ratio typically used by oyster
growers in America (Jones and Jones 1983), but no attempt was
made to modify local practices. Setting took place over 60 h
(Spring) or 48 h (Summer) under closed conditions and aeration
was provided through air stones. At the end of the setting period
the bags with substrate were transferred to subtidal floating rafts
located along the channels of SQB (Fig. 1 ). where they remained
until ready for grow-out, in inlertidal racks.

Mortality

Prior to transferring the newly settled spat to the rearing chan-
nels, a total of 21 (Spring) and 33 (Summer) shells were removed
from the bags and labeled. The number of spat attached to the inner
side of the shell was counted with the aid of magnifying glasses
and recorded. The same shells were recovered at the end of the
rearing period in order to count the nuttiber of spat present in their
internal and external sides. Percentage of mortality during the
rearing phase (floating rafts) was based on the number of spat set
on the internal side of the shell. From this point on both sides of
the shell were used for mortality estimates.

At the end of the rearing period, the shells with spat were strung
on sections (1.2 m length) of polypropylene rope. The entire as-
sembly is locally known as "sarta." and consists of 7 equally
spaced shells per rope. Ten sartas with shells were labeled and
suspended in intertidal culture racks located at the head (Fig. 1)
and near the mouth of SQB (5 sartas per site. 1 s;ir-ta per rack). All

experimental sartas were intermingled with the rest of those de-
ployed by local fishermen (total 1 10 sartas per rack) in order to
avoid any density-dependent bias throughout the experiments. In
the Spring experiment, monthly/bimonthly mortality was assessed
"in silu" by counting the total number of live and dead oysters in
each sarta. Evaluations for the Summer experiment were carried
out at the beginning of the experiment (post-settlement), the end of
the rearing phase (or beginning of grow-out phase), and the end of
the experiment. Empty shells (open valves and no tissue) were
recorded as dead organisms in all cases. At the end of the experi-
ment, all 10 sartas were taken to the laboratory where the oysters
were carefully detached from their substrate and counted. The
percentage of cumulative mortality (%M) was calculated by using
the following equation:

9'fM = [l -(N,/No)]x 100

where N,, = initial number of live oysters per sarta and N, =
number of live oysters at time t.

Growth and Biochemical Changes

At the same sites selected for mortality evaluations (Fig. I ).
120 sartas were labeled and suspended in 10 commercial culture

Figure I. Rearing and grow-out experimental sites In SQB. C. gigas
was reared subtldally al the channel (C'll) during the first month of
post-larval life. Further grow-out was carried nut at the head (H) and
near (he nionlh (M) of the left of SQB.

Growth. Mortality, and Composition of C. g/gas

791

racks. A total of 5 racks per site and 1 2 sarias per rack were used
for this experiment. Between 3 and 10 sartas (1 per culture rack)
were monthly/bimonthly collected from each site, taken to the
laboratory inside coolers, and placed in a cold room (4 C) until
processing the next day.

Handling of larvae, sarta's manufacturing, and grow-out opera-
tions were carried out by local fishermen. Labeling, distribution of
experimental sartas, sampling, and processing were carried out by
us in order to avoid any bias in estimating the actual efficiency of
the production units.

Sample Processing

Setting larvae were placed in pre-combusted aluminum pans
and rinsed with a small amount (approximately 1 niL) of isotonic
ammonium formate (2.75%, w/v) to eliminate salts. The solution
was quickly removed from the pans with the aid of a Pasteur pipet
and tissue paper. Pans with larvae were oven-dried at 60 °C for 24
h. weighed, and combusted at 450 "C for 24 h. The ash-free dry
weight of oyster spat was obtained by the difference between dry
and ash weights.

The sartas with oysters were processed in the laboratory as
follows: oysters were removed from the substrate with the aid of a
flat-head screwdriver and hammer. The shell substrate had to be
carefully broken in order to separate intact oysters located on the
inner side of the substrate or hidden between other experimental
oysters, and therefore the use of a screwdriver was preferred in-
stead of a knife. Fifteen intact oysters per sarta were randomly
selected and measured with digital calipers (±0.01 mm) on their
longest dimension and weighed (live weight). The shell and soft
tissue of each individual were then separated in order to estimate
condition index. The .shell was dried at room temperature for 24 h
and weighed, while the whole tissues were oven-dried ( 105 "C, 24
h) and weighed. The remaining oysters were frozen at -20 °C and
freeze-dried for later biochemical analysis.

Condition index (CI) was calculated on a gravimetric basis
using the following equation (Crosby and Gale. 1990):

CI = (tissue dry weight (g) x 1.000]/

[capacity of the inner side of the shell]

where the capacity of the inner side of the shell (g) is the difference
between the weight of whole (live) oyster and the dry weight of the
shell.

Tissue weight of small (<3 cm) oyster spat was determined by
carefully separating their shells with the aid of dissecting forceps
under microscope, followed by complete tissue/fluid recovery with
Pasteur pipettes, transfer to aluminum pans, and oven-drying at
105 °C for 24 h. No attempt was made to estimate the condition
index of these small oysters, due to the impossibility of separating
intact organisms from the shell substrate.

Weighing of larvae/early oyster spat was done on a Perkin-
Elmer AD2Z electrobalance (±0.001 mg) while juvenile/adult
stages were weighed on a Mettler balance (±0.1 mg). Absolute
growth rates (AGR, |jLm d~') were computed from linear regres-
sions from the slope obtained between shell height and age of the
oysters.

Biochemical analyses were performed on Spring oysters only.
Six to 35 oysters per sarta (depending on size) were dissected, and
their adductor muscle and remaining tissues (viscera, gills, and
mantle) were pooled into two separate fractions. Three replicates
(sartas) were processed on each sampling date, but pooled sartas

were used when needed for small sizes. Muscle and remains were
freeze-dried, pulverized with the aid of a coffee grinder, and stored
(-70 °C) inside tightly closed jars containing dessicant. These
samples were later used for biochemical analyses in 1997. Total
tissue nitrogen (N) was quantified with the micro Kjheldahl
method (AOAC 1990) and proteins were estimated by multiplying
N X 5.8 (Gnaiger, 1983). Tissue glycogen was precipitated with
ethanol, converted to glucose by acid hydrolysis, and quantified
enzymatically (Pfleiderer 1983). Lipids were extracted with etha-
nol :chloroform (2:1 and 1:2, Bligh and Dyer 1959) and quantified
gravimetrically.

Environmental Variables

One thermograph (Ryan Instruments, mod. TempMentor) was
deployed at the mouth of SQB on July 10, 1995. Another termo-
graph (I.I.O.-UABC, mod. TDS-85) was also deployed at the head
of SQB on the .same date and continuous (hourly) temperature
recordings were obtained through November 14, 1995.

Monthly evaluations of total particulate matter (TPM) were
carried out for each location between July 1995 and April 1996.
Triplicate water samples were collected in plastic bottles (250 mL)
at 65 cm above bottom with the aid of manual vacuum pumps
(approximately 25 cm Hg, vacuum). A nitex screen (30-|xm mesh)
was placed at the entrance of the tubing in order to remove larger
particles. The samples were transported to the university inside
coolers (approximately 2°C^ °C) with a total elapsed time be-
tween collection and processing of less than 8 h. Water samples
were filtered through pre-washed and pre-combusted glass fiber
filters (GF/F). A final rinse with isotonic ammonium formate
(2.75%) was done in order to eliminate salts. The filters were dried
at 90 °C for 20 h, weighed, combusted at 450 °C for 2 h, and
re-weighed. TPM was obtained directly from the dry weight and
the organic fraction (POM) was obtained by the difference be-
tween the ash and dry weights.

Statistics

A multifactorial two-way ANOVA (2x2x5) was used to test
the effects of the seeding season (Spring and Summer) and culture
site (mouth and head) on the mean oKserved mortality of oysters at
the end of the experiment, with 5 replicates (sartas) per site. Ab-
solute growth rates of oysters cultured on different sites and seed-
ing seasons were compared with a test of multiple comparisons of
slopes (a posteriori Tukey"s test). The same test was used to
compare tissue growth, previous transformation of raw data (mg
dry weight) to Ln ( 1 + weight in grams). A two-sample parametric
(Student f) or non-parametric test (Mann-Whitney) was used at
each experimental site to test the effect of seeding season (Summer
versus Spring) on the oysters' condition index. Time effects were
not statistically tested because the effect of this factor was obvious
in all ca.ses. A two-way ANOVA test was independently used to
compare the effects of time (age) and tissue type (muscle and
viscera) on the relative (percentage) protein, lipid, and glycogen
content in oysters from the mouth and head of SQB.

With the exception of the comparisons of slopes (Zar 1984). the
rest of the statistical tests were carried out on a personal computer
with the software SigmaStat for Windows, version 2.0 (Jandel Sci-
entific, Chicago, IL). Assumptions of normality and homoscedas-
ticity were tested with Kolmogorov-Smimov and Bartlett tests
prior to analysis. Non-parametric methods were used when any of
these conditions were violated.

792

Garci'a-Esquivel et al.

RESULTS

Larval Condition

The initial shell height (median, 322 and 327 |j,m) and organic
content (2.1 and 2.2 (jig) of pediveliger larvae were not .signifi-
cantly different in the Spring and Summer batches, yet Spring
larvae exhibited significantly heavier and wider shells than Sum-
mer larvae (Table 1 ). Spring larvae also showed higher settlement
success (46.5 ± 7.4 spat shell"', mean and SE) than Summer
oysters (29.9 ± 4.5 spat shelP').

Growth, Condition Index, and Mortality

A decoupling was observed between shell and tissue growth.
Shell growth rate was linear throughout the study period (Fig. 2a),
whereas tissue accretion was exponential in oysters from the
mouth of the bay, with a lag period during the first (Spring) or
second (Summer) month of post-larval development (Fig. 2b). Tis-
sue growth was linear in oysters from the head of SQB, but the rate
of accretion was significantly lower than the rate of shell growth
(Fig. 2, a and b).

Sartas located at the mouth of the lagoon were rapidly covered
with sticking sponges, green algae, and bryozoans during the sum-
mer months, while those at the head of SQB were essentially free
of epibionts. Despite this condition, shell growth rates were higher
at the mouth (0.34-0.37 mm d"' ) than the head of SQB (0.20-0.23
mm d"'), irrespective of seeding .season (Fig. 2a). No significant
differences in shell growth rate were observed between spring- and

summer-seeded oysters cultured within the same location (Tukey
test for multiple comparisons of slopes, mouth q = 1.71,P>0.05:
head q = 1.61, P > 0.05). Market size (9-cm shell length) was
reached after 9 mo post-settlement at the mouth and at an esti-
mated age of 13 mo at the head of the lagoon.

Tissue growth was also higher in oysters from the mouth than
the head of SQB (Fig. 2b). At the end of the experiments, TDW
was 7- to 8-fold higher for oysters from the mouth (2.10-2.16 g)
than the head of SQB (0.23-0.33 g). Juvenile oy.sters smaller than
15 mm (Spring) or 30 mm (Summer) exhibited a high (>70) CI, but
these values dropped to a minimum during the fall and winter
months (Fig. 3a). Condition index increased again in oysters from
the mouth, but not from the head of SQB, towards the end of the
winter (Fig. 3a). Overall, oysters from the mouth consistently ex-
hibited higher CI than those at the head (Fig. 3a). Spring oysters
from the mouth also exhibited significantly lower CI than Summer
oysters ( Mann-Whitney T = 958. /), = 31, «, = 45: P = 0.013).
However, no significant differences were detected in the mean CI
between Spring and Summer oysters from the head of SQB (Stu-
dent r test, t = 0.400, n, = 35, «, = 47; P = 0.690).

Most of the C. gigas mortality (54%-68%) took place at the
channels within the first month post-settlement. Mortality was neg-
ligible (<5%) once the oysters were transferred to the intertidal
racks, except in Spring oysters located at the mouth of SQB. This
batch exhibited the greatest cumulative mortality during the first 2
mo post-settlement (Fig. 3b). A two-way ANOVA test carried out
at the end of the experiment showed significant site (F = 3.35; d.f.
1. 31 =; P = 0.012), but no significant batch (seeding season)

A

Channel

^ (a)

.S

-

o

Mouth

/-L ' '.

f"

64 -

n

Head

Y

Oo'

h

/^

— n

^

p-

X

■

^/

^9

.n- • • •

.n

•5

T)

32 -

/rr^

■ a-

. n-

,c

A

O^

W

0 -

A^

A

o •
. o
. o

1

1 1

1 1

1 1

1

1 1

1

C

73

c
o
O

100 -

A
O

Channel
Mouth

K ^'^ \

.

□

Head

80 -

(

-

\ X^°

60 -
An

\J^ nT * o ^ 5

1 1 1

1 1 1 1 1 1 1 1 1

100

Time Post-settlement (months)

Hjiure 2. Tcmpiiral ihanncs of slull hcifihl (ul and tissue dry weight
(b) in C. Rig,as cuUnred at two silos ( month and head I of .SQB. Pre-
liminary rearinf; ((irsl month post-settlement) was carried out at chan-
nels. Data shown for oysters set in .\pril (continuous line) and .luly
(dotted linel of 1995. Vertical bar = SK.

Time Post-settlement (months)

KiRure .V Temporal changes of condition index (a) and percentage of
niortalily (h) e\hil>ited b> ('. ^i^aa cultured at two sites (mouth and
head) of .SQB. Preliminary rearing (first month post -settlement) was
carried out at the channels. Data shown for oy.sters set in April (con-
tinuous linel and ,lul> (dotted line) of 1995. \ertical bar = SE.

Growth. Mortality, and Composition of C. gigas

793

effects on the oyster's cumulative mortality (F = 7.16, d.f. = 1.
31: P = 0.077). A highly significant interaction between both
factors was also detected (F = 21.30; d.f. = 1. 31: P < 0.0001 ).
Overall mortality from setting through harvesting (9-citi shell
height) ranged from 687r to 769r (889f. Spring batch from mouth
site).

Biochemical Patterns

The amount of proteins, lipids, or glycogen was consistently
higher in oysters from the mouth than the head of SQB (Fig. 4).
Lipid was the main energy depot during early C. i;ii;a.s develop-
ment, but glycogen was e.xponentially accumulated during the fall.
Glycogen replaced lipids as the main energy depot when oysters
reached a size of approximately 50 mm after 5 mo (mouth) or 7 mo
(head) post-settlement (Fig. 4. a-d). When expressed on a percent-
age basis, lipid and glycogen content were also lower in oyster
tissues from the head of SQB, and the lipid/glycogen storage pat-
tern was the same as previously described (Fig. 5). Glycogen was
accumulated during the early fall, changing from approximately
1% to 21% (mouth) or from 0.3% to 7% (head) during the period
from September to November (Fig. 5). During this period, per-
centage lipid remained relatively constant (4%-5%) in oyster from
both sites (Fig. 5). A two-way ANOVA test showed that the per-
centage of protein did not significantly change with increasing age
in oysters from the mouth, yet all three energy substrates (protein,
lipid, and glycogen) were significantly affected by tissue type and
its interaction with the oyster's age (Table 2). Muscle showed
lower glycogen content than tissue remains in oysters from the
mouth, but no clear differences were detected in lipids (Fig. 5. a
and c). Oyster age. tissue type, and their interaction also affected
the relative amount of proteins in organisms located at the head of
SQB (Table 2). yet no significant age or interaction effects were
detected in lipid content (Table 2). The relative amount of glyco-
gen in oysters from the head was only affected by age (Table 2).
with higher values observed at the end of November when glyco-
gen concentration in oyster tissues was highest (Fig. 5. b and d).

MOUTH

HEAD

D J F so

Time Post-settlement (months)

Figure 4. Temporal changes in the absolute amount of protein, lipid,
and carbohydrate in the adductor muscle (a and b) and remaining
tissues (c and dl of C. gigas cultured at two sites (mouth and headl of
SQB. Data shown from the fourth month of age through the end of the
experiment for oysters set on April of 1995. Arrows indicate the timing
when glycogen replaced lipids as the main energy depot. Vertical bar
= SE.

0
68

o4^1

Protein

Lipid

Glycogen

-^^

SONDJF so

Time Post-settlement (months)

Figure 5. Weight-specific content of proteins, lipids, and carbohy-
drates in the adductor muscle (a and b) and remaining tissues (c and
d) of C. gigas cultured at two sites (mouth and head) of SQB, Data
shown from the fourth month of age through the end of the experiment
for oysters set on April of 1995. Vertical bar = SE.

Environmental Variables

A failure of the thermograph deployed at the mouth of SQB
prevented the collection of temperature records at this site after
mid-September. However, based on the available data from the
previous months, it was observed that the water temperature was
consistently colder at the mouth than at the head of SQB (Fig. 6).
The temperature followed a tidal rhythm, with stronger effects
observed at the mouth of the lagoon. Summer temperature gradu-
ally increased at the mouth from 19.3 °C (July) to a peak of 24 °C
in September and from 19.5 °C to 24.8 °C at the head of SQB.
Daily temperature differences within sites ranged from 1.7 °C to
2.8 °C (mouth) and from 1.7 °C to 2.0 °C (head). It is noteworthy
that between-site temperature differences (approximately 1.2 °C)
were similar to or lower than the daily variability recorded within
sites (Fig. 6).

A trend of increasing TPM was observed at both study sites
towards the winter months, with values ranging from approxi-
mately 3 mg (June) to 10 mg TPM r' (February), except in
September where an abnormally high TPM value (approximately
32 mg r') was recorded (Fig. 7a). No statistical differences in the
mean TPM concentration were found between sites (Mann-
Whitney. T = 86.0, P = 0.162). POM remained relatively con-
stant ( 1 .2-2.3 mg r' ) throughout the experiment at both locations,
except at the end of August (approximately 0.2 mg T') and the end
of September, where POM reached up to 6 mg I"' (Fig. 7a).

Estimations of aerial exposure time based on the tidal height at
each one of the sites indicated that oysters from the head of SQB
consistently experienced longer (23%-26%) immersion time than
those at the mouth. In addition, higher aerial exposure was ob-
served in the winter months (Fia. 7b).

DISCUSSION

Growth

One of the most conspicuous observations of the present study
was the decoupling between shell and tissue growth during early
spat-juvenile development. Shell growth was linear and tissue

794

Garci'a-Esquivel et al.

TABLE 2.

Two-way ANOVA tests for the effects of oyster age and type of tissue on the relative (percentage) content of proteins, lipids, and glycogen in
cultured Crassostrea gigas from two sites (mouth and head) of San Quintin Bay. DF = Degrees of freedom; P = probability of rejecting the

null hypothesis.

Source of
variation

Protein

Lipid

Glycogen

DF

F

P

F

P

F

P

Mouth

Age

3

0.41

0.751

26.37

<0.001

70.41

<0.()01

Tissue

1

7.25

0.016

194.68

<0.001

15.81

0.001

Age X Tissue

3

2.18

0.131
Head

20.12

<0.001

5.03

0.001

Age

3

9.56

<0.00)

0.47

0.711

12.05

<0.001

Tissue

I

19.57

<0.001

140.68

<0.001

1.09

0.312

Age X Tissue

3

9.39

<().()()1

2.14

0.135

1.82

0.184

growth was exponential, with a substantial lag period in oysters
from the mouth of SQB. Alternatively, shell and tissue growth
were linear in oysters from the head of SQB, but the latter was
accrued at a much lower rate than the shell (Fig. 2). Despite the
observed decoupling, tissue content was still a major component of
the total body weight of oyster post-larvae, as shown by the high
condition indices found at these sizes (<4 mm), when compared to
juvenile-adult stages (Fig. 3a). The rapid shell growth exhibited by
C. gi^as during early development may have an adaptive signifi-
cance for predator avoidance (Garcia-Esquivel and Bricelj 1993
and refs, therein), yet the major implication of the observed de-
coupling may lie on the fact that tissue mass was sensitive to
developmental changes and seasonal (temperature) stressors. Thus
high values of CI were associated with a relatively high proportion

MOUTH

25 -

,

i

p

20 -

u

0

f\

s

(T)

15 -

V

i

Q.

1

1 1

1

E

0)

t-

Time (months)

Figure 6. Continuous records of sea« ater lempiralure al the head and
mouth of SQB during the period of .July through November of 1995.

of tissue body mass, typical of early post-larval stages. Garcia-
Esquivel (2000) has also shown that shell height of laboratory-
reared C. gigas spat (0.32-5 mm size range) scaled allometrically
with a mass (tissue dry weight) exponent of 2.75. thus contlrming
that tissue content was a major component of early oyster stages.
On the other hand, low CIs coincided with the highest (Spring
batch) and lowest (Spring and Summer batches) seasonal tempera-
tures reported for SQB (this study, Alvarez-Borrego and Alvarez-
Borrego 1982). It follows that CI values below 60 were indicative
of poor physiological condition (high water content) of the oysters
in the field, whereas values above 80, typically found during the
spring and the fall, reflected a good physiological condition.

Oysters from the mouth reached the market size of 9 cm after
8 or 9 m post-settlemenl. while those from the head of SQB
reached the same size after approximately 13 lo 14 mo. These

36

24 -

o

O 12

(a)

organic fraction
I///I mouth
iV^^ head

fe

170

o

E 125

0)

E
i" 80

0)

m 35

mouth

O

— I—
M

J J A

Time (months)

Figure 7. femporal changes in the amount of particulate matter In the
surface seawaler (a) and mean aerial exposure time experienced by C.
gigtis al the head and mouth of SQB. The total amount of particles
(hatched bars) and organic particles (shadow bars) are shown for each
culture site. Vertical bar = SE.

M

Growth. Mortality, and Composition of C. cigas

795

growth rales are higher than those recently reported in the litera-
ture. Thus r. aifiiis needed approximately 20 mo to increase their
shell height from 1 to 7 cm in tray cultures located at Bahia de la
Paz. near the entrance of the Gulf of California (Arizpe 1996).
while tray-cultured oysters from the coast of Portugal increased
their size from 2 to 8 cm within a period of 14 mo (Almeida et al.
1997). The striking similarities between the growth curve exhib-
ited by the Spring and Summer batches of C. gigas within a culture
site suggests that seasonal changes of environmental variables
were not limiting the growth of this species. However, site-specific
differences in growth rates appeared to be associated with micro-
geographic differences within SQB. since the oyster spat had a
common handling history from setting through rearing phases.

Mortality

Spring larvae had heavier shells than the Summer ones. These
differences were not reflected in the magnitude of oyster mortali-
ties during the rearing or grow-out phase at SQB. since both
batches experienced heavy losses within the first month post-
settlement and followed the same pattern thereafter, with clear site
effects. The greatest spat mortality was observed within the first
month post-settlement at the channels at a size smaller than 6 mm
(Figs. 2a and 3b). Although the exact timing of the mass mortality
could not be re.solved from the approach utilized in this study,
detailed laboratory studies have shown that most of the post-larval
mortality (up to 509^) of C. gigas takes place within the first week
post-settlement (Garcia-Esquivel 2000). These mortalities appear
to be associated with the amount of pre-metamorphic energy re-
serves and the degree of their utilization during metamorphosis
(Gallager et. al. 1986, Haws et. al. 1993, Garcia-Esquivel 2000),
but the lack of biochemical data in these stages prevented any
conclusion from the present study.

Mortality during the grow-out phase (intertidal racks) was neg-
ligible, and overall losses during the whole production cycle are in
agreement with the SO'/r to 109c mortality previously reported in
raft-cultured oysters from SQB (Islas-Olivares 1975, Rainos-
Amezquita 1987). The presence of abundant epibionts at the mouth
of SQB during the summer suggests that those species could have
clogged the filtering apparatus of Spring oysters and were partially
responsible for the high post-rearing mortality found at the mouth
in this particular batch. Sartas deployed during the summer did not
have enough time to get colonized at high densities with these
opportunistic species. On the other hand, sartas located at the head
of SQB were intermittently exposed to the air. thus preventing any
significant fouling throughout the experiments. High mortality of
adult oysters (>9 cm) from the outer part (mouth) of SQB has
already been reported elsewhere (Terrazas-Gaxiola 1986. Caceres-
Martinez et al. 1998). The mortality at this site was partially at-
tributed to the biofouling of encrusting organisms, including the
sponge Hulichondiia sp.. the ascidian BoliyUoides sp.. and the
hydroid Tiirbiilaria crocea (Inclan-Rivadeneyra and Acosta-Ruiz
1989). Despite the potential influence of biofouling organisms and
predators on the cultured oysters at SQB. their specific role has not
been seriously addressed to date. Recent studies carried out by
Caceres-Martinez et al. ( 1998. 1999) have shown the presence of
worms (Polydora sp.) near the valve edges of C. gigas and around
the siphon area of the black clam (Chione fliictifraga) from SQB.
The authors did not find any statistical relationship between oyster
mortalities and the presence of Polydora sp.. yet the infestation
ranged between I and 6 wonns per oyster (Caceres-Martinez et al.

1998) and I and 48 worms per clam (Caceres-Martinez et al.
1999). Therefore, further studies are required in order to identify
the role of potential predators/epibionts on the observed oyster
mortality.

Biochemical Patterns

C. gigas followed the same biochemical pattern previously de-
scribed for the fiat European oyster O. cdiilis (Holland and Han-
nant 1974), with rapid rate of glycogen accumulation after meta-
morphosis and a shift from lipid to glycogen storage during the
fall. The magnitude and pattern of glycogen accumulation ap-
peared to be related with the preparation for C. gigas gametoge-
nesis in SQB. Gametogenesis would have been interrupted in the
winter when the oysters exhibited a low CI and were forced to
utilize endogenous glycogen, resulting in greater energy losses in
the oysters at the head (Fig. 4). Although none of the oysters from
this study exhibited ripe gonads during the 9-mo experiment, a
previous study showed that the reproductive cycle of C. gigas at
SQB starts with gametogenesis in April, followed by partial and
complete spawning throughout July and August (Paniagua-Chavez
and Acosta-Ruiz 1995). Accordingly. Mann ( 1979) found that first
maturation of this species was anticipated by a continuous increase
of carbohydrates in the tissues, up to a peak coincident with early
active and late active stages of gametogenesis (presence of fol-
licles, spermatogonia-oogonia. and spermatocytes-oocytes),
whereas a subsequent decrease of glycogen was matched by the
presence of ripe organisms. The author did not measure lipids
directly, but it can be suggested from the ratio of carbohydrate to
total organic carbon that the former was preferentially accumu-
lated only when this ratio was greater than 0.6 (Table 1 in Mann,
1979).

The fall-winter decrease in glycogen content in oyster tissues
was apparently due to a combination of low water temperature and
lower food availability during this period (see below), whereas the
slower growth and lower glycogen content chronically exhibited
by oysters from the head of SQB most likely resulted from a
combination of frequent reliance on this substrate and shorter feed-
ing periods during aerial exposure, rather than differences in the
amount of food available between sites. Throughout the year, oys-
ters located at the head consistently experienced approximately
23% to 26% longer immersion time than oysters from the mouth of
SQB (Fig, 7). It is known that C. gigas typically shows higher
digestive capacity with increasing immersion time, but also reach
smaller sizes than those continuously submerged (Moal et al.
1989). Furthermore, there is no evidence of compensatory mecha-
nisms for increasing feeding rates or absorption efficiency with
increasing aerial exposure in marine bivalves (Shick et al. 1988).
It follows that between-site differences in glycogen content most
likely resulted from a combination of a higher utilization of this
substrate during anaerobiosis and shorter feeding periods experi-
enced by oysters with longer aerial exposure. In the absence of
water, oysters tend to close their valves, are unable to capture food
particles, and solely rely on the anaerobic utilization of endog-
enous glycogen, free amino acids, and proteins to fulfill their en-
ergy needs. Bivalves typically depress their metabolism down to
approximately 2% to 9% under aerial exposure (Shick et al. 1989)
and consequently the cost of anaerobiosis is drastically reduced
under these conditions. Therefore, the most significant effect of
aerial exposure may not be the utilization of large amounts of
endogenous glycogen reserves during anaerobiosis, but the limited

796

Garci'a-Esquivel et al.

availability of ingested energy to the oysters. This in turn would
translate into lower energy available for growth and lower capacity
for glycogen storage in oysters subjected to increasingly longer
aerial exposure time. Such an explanation is consistent with the
findings reported by Moal et al. (1989) and the site-specific dif-
ferences observed in the present study, in terms of oyster growth
and tissue energy content.

Optimal ingestion rates of C. gigas are known to occur at 19 °C
(Bougrier et al. 1995). On the other hand, continuous temperature
recordings carried out at SQB during the experiment showed a
temperature maxima of approximately 26 °C in September, and
consistently higher values (mean difference of 1.2 °C) at the head.
Likewise, Alvarez-Borrego and Alvarez-Borrego (1982) recorded
mean annual temperatures of 17 °C at the mouth and 18 °C at the
head of SQB. with maximum of 23 °C to 25.3 °C in September and
minimum in November and December (12.9 °C to 13.3 °C). There-
fore, the temperature regime at SQB seemed appropriate for opti-
mal growth and completion of the storage-reproduction cycle in C.
gigas throughout the year, except in the winter where the lower
temperatures would induce a reduction in the physiological rates
(oxygen consumption and clearance rates) of the oysters. It is
noteworthy that the absolute amount of TPM steadily increased
towards the winter in both study sites, but the POM remained
constant (Fig. 7), thus suggesting that higher TPM concentration in
the fall/winter resulted, by dilution, in poorer food quality. No
clear differences in TPM or POM were found between sites; how-
ever, detailed time series previously carried out in the summer over
a 10-d period indicated that chlorophyll a concentration and pro-
ductivity was 3-fold higher at the mouth than the interior of SQB
(Millan-Nunez et al. 1982). The discrepancies between both stud-
ies may have resulted from the lack of temporal resolution during
discrete water samplings and the smaller size (<30 |jim) of particles
analyzed in this work. In any case, the actual effects of aerial
exposure and changes in environmental variables (food and tem-
perature) need to be partitioned in the future, with specific experi-
mental designs.

ACKNOWLEDGMENTS

This project was partially supported by grants awarded to
Z. G. E. by the Consejo Nacional de Ciencia y Tecnologia (ref.
CONACYT 4290T) and the Sistema de Investigacion del Mar de
Cortez (ref. SIM AC 980106020). We are very thankful to fisher-
men from Juan Cota S. de P. R.L., Ostiones Guerrero, and
Acuicola San Quentin for their logistic support in the field, and
providing the experimental organisms and space.

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Journal of Shellfish Research. Vol. 19. No. 2, 799-808. 2(X)0.

SURVIVAL AND FEEDING ACTIVITY OF OYSTER SPAT (OSTREA EDULIS L) AS A
FUNCTION OF TEMPERATURE AND SALINITY WITH IMPLICATIONS FOR CULTURE

POLICIES ON THE SWEDISH WEST COAST

EVA MARIE RODSTROM* AND PER R. JONSSON

TJcinio Marine Biological Laboratory

Gotehorg University

SE-452 96 Stromstad. Sweden

ABSTRACT This study evaluates the effects of temperature and salinity on feeding activity and survival rate for spat of the European
oyster. Osirea ediilis originating front the Swedish west coast. The main objective was to investigate local adaptations to hydrographic
conditions with low temperatures and salinities with consequences for culture policies. In laboratory experiments, mortality increased
at lower temperatures except at salinities below 1 8 7cc. Highest mortality was found in combinations of very low salinities and the high
temperature treatment (10 °C). Feeding activity, measured as fecal production, was significantly lower in 5 compared to 10 °C. Feeding
rate began to decline at 28 9cc and ceased at 16 7cc. The time to recover feeding activity depended upon the duration of low-salinity
exposure. Spat exposed to salinities below 16 %r did not regain their feeding activity when returned to full salinity, indicating
permanent damage. Increased exposure time also lead to higher mortality after the return to full salinity. Comparison between rapid
and gradual change of salinity indicates a potential for acclimation. In a model forced by field data on temperature and salinities,
survival trajectories were calculated for different water depths and for different seasons. The model predictions were tested in a field
experiment where the model could explain winter and spring monalities in terms of temperature and salinity. In the summer, however,
the model underestimates mortality, indicating the importance of such other sources of mortality as fouling and predation. To minimize
mortality, this study suggests that culture policies should include deployment below 6 m with initial transfer of spat to the sea at
midsummer.

KEY WORDS: Ostrea edulis. shellfish, survival, growth, temperature, salinity, aquaculture

INTRODUCTION

The European flat oyster, Ostrea edulis. has been used for
human food since ancient times, and extensive mariculture is docu-
mented from the days of the Roman Empire (Yonge 1960). Exca-
vated piles of oyster shells dating from the bronze age (500-1.500
BCE) bear evidence of a significant fishery on natural stocks of O.
edulis in Scandinavian waters (Dannevig 1953, Yonge 1960).
However, in present times, Scandinavia represents the northern
margin of the geographic distribution of O. edulis, and climatic
changes have probably greatly influenced its local abundance
through time. The Swedish west coast, influenced by the Baltic
Sea and several river discharges, may be a particularly adverse
region with the combination of low salinities and temperatures,
which is characteristic of the eastern parts of the seas of Kattegat
and Skagerrak. Suboptimal hydrography has been suggested as an
explanation for infrequent and irregular recruitment of O. edulis
along the Swedish west coast (Sparck 1924, Ostergren 1925). Nev-
ertheless, the conditions for survival and growth of juvenile and
adult O. edulis are advantageous with high summer temperatures,
high seston concentration (Lannergren 1983, Rodstrom 1989) and
the apparent absence of the lethal parasite Bonamia ostreae
(Mortensen 1993). Because of the low and irregular natural re-
cruitment, successful exploitation of O. edulis along the Swedish
west coast will most likely be dependant on land-based spawning
and the production of spat for subsequent transfer to the sea. An
analysis of the economic prerequisites of a future mariculture de-
velopment in Sweden will require detailed knowledge about spat
survival and growth under local hydrographic conditions. Early
life-stages are usually particularly sensitive to suboptimal tempera-
tures and salinities (Kinne 1970. Kinne 1971, Shumway 1996,

*CorTesponding author. E-mail: Eva.Marie.Rodstrom@tmbl.gu..se

Walne 1979). The length and variability of these adverse periods
will vary with water depth and season. Especially during winter
and early spring oysters, will often experience both low tempera-
tures and low salinities.

The objective of the present .study was to study experimentally
the effect of salinity and temperature on survival and feeding ac-
tivity of Ostrea edulis spat. The experimental results were incor-
porated into a model driven by local field data on temperature and
salinity. The model is used to explore optimal culturing policies
with respect to the time of spat transfer and the depth of tray
deployment. Finally, the predictions of the model were tested in a
field study of spat survival.

MATERULS AND METHODS

Material

All experiments were carried out using oyster spat produced at
the Tjamo Marine Biological Laboratory (58°, 53' N, 11°. 8' E)
from controlled spawnings of locally collected adult Osirea edulis
L, 1758. Adult conditioning and the culturing of larvae and spat
were performed following the protocols described by Walne
(1979) and Wilson (1981). After 1 month of conditioning, the
broodstock oysters released larvae that were collected on a 90-(xm
screen. Larvae were then transferred to 34 %<. filtered seawater (0.2
|i.m) in 50-L barrels (2 larvae mL"') and fed the microflagellate
Isochrysis galbana (Parke) (clone T-iso) at 1*10'^ cells mL~'. The
filtered seawater was maintained at 20 °C, stirred by aeration, and
changed every second day. After about 12 days, the larvae devel-
oped into competent pediveligers and began to settle on PVC-
plates offered as settlement substrate. Larvae that successfully
completed metamorphosis were gently removed from the substrate
with a razor blade and placed in upwclling columns (diameter: 12
cm, height: 30 cm) with an air-lift drawing water through the

799

800

RODSTROM AND JONSSON

bottom, which was covered with a 200-|im screen. The up-welling
columns with spat were immersed in a 100-L tank with a constant
flow of seawater (20 °C, 34 %o). The spat were grown on a non-
limiting supply of /. galhcma (1*10^ cells mL" ' ) and were allowed
to grow for 6-8 weeks to a shell length of ca 7 mm before they
were used in the experiments (except for the field experiment,
where spat were 18 mm before transfer to the sea). All cultures of
/. galbana were grown in batch on f/2 media according to Guillard
(1983).

Survival and Fecal production as a Function of Temperature
and Salinity

The first series of laboratory experiments were designed to test
the combined effects of salinity and temperature on spat survival
and feeding activity. The range of salinities tested. 12-34 %f,
covers the range of local field salinities. The experiments were run
at 5 and 10 °C, where 5 °C represents a suboptimal temperature at
which the oysters are still feeding although at lower rates (Rod-
strom 1989). Befoe the experiments, all oyster spat were kept
individually in lOO-mL plastic beakers at 34 %r and 10 °C for at
least 10 days to check that the spat were alive and feeding. All spat
were fed before and during the experiments with Isochrysis gal-
bana at a concentration of 1*10'^ cells mL"'. Because field salini-
ties may change rapidly (Fig. la), the oyster spat did not get the
opportunity to acclimate to the new salinity before the experiment.
Field temperature varies more gradually (Fig. lb), and the spat

0 50 100 LSO 200 250 300 350 400

50

300

3.50

100 1.50 200 250

Day No.

HKiiro 1. .\nniiiil varialion in hydniuraphy in surface waters of
Tjarno Marine KinloKJcal l.ahoraliirv. (al .Salinity {'i,). (b) Tempera-
ture ( C). Data from 1989 at (l-m and lO-m depth.

were acclimated to the experimental temperatures by changing the
temperature gradually 0.5 °C per day.

The seawater in the experiments was pumped from 40-m depth
(34 %f ) and filtered through a sand filter to a 2.'i-m^ head tank. For
salinities lower than 34 '?f, seawater was diluted with deionized
and distilled water. The salinity was determined with a tempera-
ture-compensated refractometer (accuracy ±1 Voc). Each salinity
was mixed 24 h before the experiment and then gently aerated
before use. Isochrysis galbana was added and mixed in each sa-
linity just before the experiments. During water change, the spat
were temporarily drained, while the beaker was rinsed. Then, new
seawater with the appropriate salinity and fresh /. galbana was
added, and the spat were returned to the beaker. Spat were ran-
domly allocated to each treatment, and all beakers were randomly
distributed in space. The experiments were carried out in constant-
temperature rooms. Every second day, all beakers were examined;
an oyster spat was defined dead if it lay open and could no longer
close the shell valves when mechanically disturbed.

The effect of temperature and salinity on feeding activity was
tested by recording the fecal production, which was assumed to
give a time-integrated estimate of feeding activity. Only band-
shaped feces were recorded, which were assumed to represent
ingested material and not pseudo-feces. The relation between sus-
pension feeding and fecal production was examined in a separate
study of clearing rate (see below). The experimental beakers in the
survival experiment above were examined every second day, and
the amount of feces produced during 48 h was recorded semiquan-
titatively, where 0, 1, and 2 indicate absence of feces, low feces
production, and high feces production, respectively. The experi-
mental treatments lasted for 27 days in 10 °C and for 29 days in
5 °C. After the experiments, all spat were directly transferred
back to 34 %c and 10 °C, after which the monitoring of survival
and fecal production continued for 29 days in 10 °C and 39 days
in 5 °C. If no other information is given, 10 replicate spat for each
combination of salinity and temperature were used. Daily mortality
rates and 95 % confidence intervals were calculated from the slope
of the linear regression of the natural logarithm of the number of
surviving spat against time. Treatment effects of temperature, sa-
linity, and time on fecal production and interactions among factors
were tested with an analysis of variance (ANOVA). The linear
model used to describe the scores of fecal production (X) was:

X„u,n = )x + S, -H Tj + W, + ST,j + SW„ + TWj, + STW^^,

+ I(ST),„„ + WI(ST),„ij, + e„„y,„ (1)

where salinity (S), temperature (T), and week (W) are considered
as fixed factors, and individual spat (I) is a random factor nested
within the factors S and T. The assumption of homoscedasticity
was tested using Cochran's C statistic (Winer et al. 1991 ). In all
statistical tests, a type I enor rate (a) of 0.05 was used.

Survival and Fecal Production as a Function of Ia>w Salinities

A second experiment was run to in\cstigatc in more detail how
low salinities aflect survival and feeding activity. This experiment
was run in the same way as described above, and the salinities
tested were 20, 18, 16, 14, 12 %c and a control in 34 %f. However,
this time only one temperature ( 10 °C) was included. After 1 week
in 34 '('( and 10 C. the oyster spat were Iransfeired to the new
salinity directly without acclimation. Spat were exposed to one of
the low salinities for 27 days, after which they were transferred
back lo 34 '?< and studied for 40 more days. Survival and fecal

Survival and Feeding of Oyster Spat

801

production were recorded as described above. Daily mortality rate
was estimated with regression analysis.

Suniral and Fecal Production as a Function of Imw Temperatures

To investigate the effect of very low temperatures on survival
and fecal production, two experiments were performed. In the first
experiment, survival at the temperatures 4, 2, and 0 °C was stud-
ied at 34 %r and 22 %r. The oyster spat were initially acclimated to
10 °C and 34 %c. Oyster spat were transferred to the different
salinities before the experiment started, and the temperature was
gradually lowered to each treatment temperature (1 °C day""').
During this period, all spat were kept in the same container (one
for each salinity) until the treatment temperatures were reached,
when the spat were transferred individually to 100-mL plastic
beakers. Ten replicate spat for each factor combination were used.
The experiments were performed in temperature-controlled water
baths. Water was changed every second day and the spat fed
Isochiysis galbana (1*10*^ cells mL"'). The oyster spat were ex-
posed to the low temperatures for 30 days. Daily mortality rate was
estimated with regression analysis, as described above.

The second experiment examined the impact of low tempera-
tures on fecal production in 5, 4 and 3 °C at the salinities 34, 24,
and 20 '7c<. The oyster spat were individually placed in 100-mL
plastic beakers and were initially acclimated to 10 °C and 34 %o.
Before the experiment started, three replicate spat were transferred
to the different salinities, and the temperature was gradually low-
ered to each treatment temperature ( 1 °C day"'). Fecal production
during 48 h was recorded as described above and the score (X) is
described with the linear model:

X„k = |x -H Si -(- Tj -h STjj -I- e^,

k(ij)

(2)

analyzed with an ANOVA with temperature (T) and salinity (S) as
tlxed factors.

Fecal Production as a Function of Rapid or Gradual Change
in Salinities

This experiment was performed to test for an effect of the
time-scale of salinity changes on spat feeding activity, measured as
fecal production. Gradual acclimation was compared to direct
transfer of spat to suboptimal salinities. Also examined was the
question of whether previous acclimation to low salinities would
increase feeding performance when directly transferred to even
lower salinities compared to spat being acclimated at higher sa-
linities.

The oyster spat were gradually acclimated to lower salinities
with 1 or 2 %c per day. They were fed /sochr\'sis galbana (1*10'
cells mL~') every second day when the water was changed. All
spat were kept in the same container until the target salinity in the
specific treatment was reached. After that, spat were individually
transferred to 100-mL plastic beakers. Three replicate spat for each
factor combination were used. Before the actual recording of fecal
production started, the spat were allowed to acclimate to the treat-
ment salinity for 2 more days. For each experimental transfer,
three control individuals from a pool of spat kept at 34 %( were
treated identically and transferred to new beakers with 34 %c. The
temperature during the experiment was 14 °C. Fecal production
after 24 h was recorded semiquantitatively on a scale where 0, 1,
2, and 3 indicate absence of feces, low, medium, and high feces
production, respectively. The following protocols were used:

1. spat initially acclimated in 34 %o and directly transferred to
28, 24, 20, 16, and 12 %c, respectively:

2. spat initially acclimated in 24 %< and directly transferred to
34, 20, 16, and 12 %i. respectively;

3. spat initially acclimated in 20 %c and directly transferred to
34, 16, and 12 %c, respectively,

4. spat initially acclimated in 16 %<■ and directly transferred to
34 and 12 %r, respectively; and

5. spat initially acclimated in 12 Vci and directly transferred to
34 %c.

Survival and Feeding Activity as a Function of Exposure Time at
Low Salinity

Previous experiments suggested that recovery from low salini-
ties was affected by the duration of the exposure. To examine the
effect of exposure time on survival and feeding activity, oyster spat
were exposed to the suboptimal salinity 16 %c for 1, 2, 3, and 4
weeks, respectively. For each exposure period, 10 replicate spat
were used. Before the experiment, all oyster spat were kept in 34
9?r and fed hochrysis galhana (1*10' cells mL~') every second
day when the water was changed. The spat were then transferred
to 16 %c directly without any previous acclimation. After the dif-
ferent exposure times, the spat were transferred back to 34 %p and
followed for another 54, 47, 40, and 33 days, respectively. Survival
and fecal production were recorded during the experiment as de-
scribed above. Daily mortality rates during the recovery phase was
estimated by linear regression as described above, and the scores
of fecal production (scale: 0, 1 . 2) the first week of recovery were
tested with a one-factor ANOVA with incubation period as a fixed
factor.

Clearing Rate as a Function of Salinity

In the experiments described above, feeding activity was in-
ferred from fecal production. To test the assumed relationship
between suspension feeding and fecal production, the actual rate of
suspension feeding was studied and compared with measured fecal
production. The clearing rate of oyster spat was determined in 34,
28, 24, 20, 16, and 12 %v by measuring the disappearance of
suspended hochrysis galbana cells exposed to oyster spat. All spat
were initially kept in 34 %f . 1 4 °C, and fed /. galbana (1*1 0'' cells
mP'). The spat were kept in 100-mL plastic beakers during the
experiments. At each salinity, three replicate spat (one spat per
beaker) and two control beakers without spat were incubated for
24 h. One sample ( 1 mL) of the /. galbana concentration from each
beaker was collected at the start and after 24 h and fixed in Lugol
(acid iodine-iodide). The fixed samples were allowed to settle for
24 h in the wells of a multidish (Nunc, 3.5 mL). The wells were
then examined with an inverted microscope (Nikon Diavert, 200X)
and video-recorded images of /. galbana cells were counted with
an image analysis .software (IPLab. Signal Analytics, Inc.). The
volume cleared per unit time (F) of /. galbana by the oyster spat
was calculated from the decrease in concentration during the in-
cubation according to:

F = (ln(C/Co) - ln(E/Eo)] * V/t (3)

where C and E are the concentrations at the start (0) and after some
time (t) of the controls and the sput treatments, respectively, and V
is the volume of the experimental container. The estimated clear-
ing rates were finally compared to the scores of fecal production.

802

RODSTROM AND JONSSON

Model of Spat Survival and Feeding Activity as a Function of
Local Hydrography

The significance of the laboratory experiments on spat survival
as a function of salinity and temperature during local field condi-
tions was explored in a model simulation. The main objective was
to analyze the effects of the time at transfer of spat to the sea and
the depth of deployment. Data on temperature and salinity were
collected at the Tjiirnci Marine Biological Laboratory between
1981 and 1991 (unpublished data). One dataset consists of ap-
proximately daily recordings of surface (0.5 m) temperatures and
salinities. By combining this time series with a dataset consisting
of monthly recordings of vertical (0-30 m) temperature and salin-
ity profiles, a matrix of daily temperatures and salinities was es-
timated covering every meter extending from the surface to a depth
of 15 m. Based on regression analysis, the following relations were
used to estimate temperature (t) and salinity (s). at depth (z) from
surface recordings (t,,, Sj,):

t(z) = (-0.0008 1 2 * m"* -f 0.02 1 * m ' - 0. 1 73 * nr

-fO.474 * m-0.318) * z-l-T,, (4)

s(z) = (-0.04426 * s„ + 1.372) *■/. + s„

(5)

where m is the month (1 to 12). Validation of these relations
showed that error rarely exceeds 10 % for temperature and 5 % for
salinity.

From the results of the laboratory experiments on spat survival
as a function of temperature and salinity, a survival matrix was
constructed (Fig. 2). Daily survival rates for temperatures between
-I to 25 °C and salinities between 1 1 and 34 7oc were estimated by
inter- and extrapolation from the experimental results. No reduc-
tion in survival rates was assumed in salinities between 24-34 %c
when temperature is above 10 °C (Newkirk et al. 1995). The
experiments also revealed that exposure at low salinities (< 18 %<)
imposes a stress load resulting in delayed mortalities when re-
turned to higher salinities. This effect was included in the model as
an added mortality when returning to salinities s 19 %c according
to a loading function estimated from a curve fit to experimental
data (.see Fig. 4) as;

added daily mortality = 0.03 * ( I - exp(-0.005 * h")] (6)

-'^('■c,

Fi^uri' 2. .\ jiraphic rc|)ri'si'nl;ilicin (il'llii' nuilri\ of (hilly survival r;iU'
of Oslrea vduli\ spat as a riiniiion of li'Mipcratiirc (C) and salinity ('i< I
iisvd in the model siiiuilullons. Survival rates are inter- and extrapo-
lated from experlmenlai results.

where h increases by I for each day at salinities < 18 %r and
decreases by 1 at salinities a 19 %c (h > 0). Sensitivity analysis
showed that the added mortality had little effect on the simulation
results. The forcing from the teinperature and salinity matrices
generates a time trajectory of daily survival rates that was ex-
tracted for each depth, and the total survival over the time period
was calculated. Time trajectories for 120 days at three different
starting times were explored, April 1, June 1. and September I.
This was repeated for temperature and salinity data for the 9 years
between 1981 and 1989.

A less rigorous model was formulated to examine the relative
effect of local hydrography on feeding activity. Because feeding
activity was recorded as ranked fecal production, the objective was
only to compare relative feeding activities for different depths and
at different seasons. A matrix of feeding activity as a function of
temperature and salinity was constructed from the experimental
data. With the forcing from the temperature and salinity matrices
(1981-1989). a time trajectory of daily activity was extracted for
each depth between 0-15 m. and for the same three starting times
as for the mortality simulation. An attempt was also made to
combine feeding activity with food availability by multiplying
activity scores with local chlorophyll u measurements, available
in the model as a matrix of monthly vertical profiles calculated
from 5 years of field measurements (unpublished data from the
Tjamo Marine Biological Laboratory). The output from the simu-
lations of the feeding activity model is a cumulative sum of daily
activity times chlorophyll a concentration. Computer simulation of
the mortality and feeding activity models was performed in
MATLAB® 4.2 (MathWorks Inc) for the Apple Macintosh.

Field Experiment of Oyster Spat Survival and Growth

A field experiment was designed to test how much of the /;; siiii
survival rate could be explained by the mortality rates observed in
the laboratory experiments and predicted by the model described
above. The experiment was carried out adjacent to a blue mussel
culture outside Grebbestad about 30 km south of the Tjarnci Ma-
rine Biological Laboratory. A set of 148 oyster spat (18.3 ± 0.3
mm, mean ± SE, n = 148) were transferred to the sea in Novem-
ber 1988, and survival and growth was followed to November
1991. The spat were cultured in suspended, plastic trays (600 x
400 X 140 mm), stocked at a density of 350 spat m"- and deployed
at an average depth of 4.5 m. All spat used were hatched at the
laboratory using brood-stock oysters from local populations. The
spat were examined in the field for survival and shell length on
eight occasions, and at each sampling occasion, the spat were
relayed into clean trays. The presence of fouling organisms and
potential predators was also recorded.

RESULTS

Survival as a Function of Salinity and Teinperature

The mortality rate was highest in the lowest salinities where
exposure to 12 %<■ killed all spat within a week (Fig. 3). Salinities
lower than 18 '}',r are apparently suboplimal; only 40-50 'i of the
spat survived 16 ''/,, for 30 days. The effect of the temperature
reduction from 10 lo 5 °C was more complex where there was an
increase in mortality below 28 7r<. except for the lowest salinities
tested where the effect of temperature was reversed, and mortality
increased with temperature (Fig. 3). At very low temperatures (0-4
°C) mortalities were generally low (Table 1 ). There was almost no

Survival .and Feeding of Oyster Spat

803

0-
10

20 25

Salinity (%o)

30

35

Figure 3. Daily mortality rate (mean ± 95 % CI) of Ostrea edulis spat
as a function of salinity and temperature.

effect at 34 %< and a slight increase of mortality toward lower
temperatures at 22 7((. After exposure to low salinities, the spal
were transferred back to 34 %c and survival was recorded during
this recovery period. During the recovery period, mortality rate
was higher for the spat previously exposed to the low salinties 14
and 16 %c as compared to spat incubated in higher salinities. An
increased mortality during the recovery phase was evident also in
the 20 and 24 9t( treatments for spal incubated at 5 "C. Mortality
during recovery declined 1-2 weeks after transfer to 34 %c. A more
detailed study showed that future mortality after exposure to low
salinity (16 %c) is dependent on the exposure time (Fig. 4). Sur-
\i\al was initially high in all four exposure treatments, and a
difference in mortality rate was first evident after the spat were
transferred to 34 %o. Mortality dramatically increased after 2
weeks of exposure to 1 6 %r.

Feeding Activity as a Function of Salinity and Temperature

Feeding activity in different combinations of temperature and
salinity was inferred from studies of the fecal production. Fecal
production has the advantage of integrating feeding activity over
time and was also easily observed and quantified on an ordination
scale. The assumed relationship between what we considered as
fecal production and actual feeding rate was tested by measuring
the clearing rate of oysters feeding on hochrysis galhana. Figure
5 shows the relation between fecal production (in relative units)
and the clearing rate (mL h"' g"') as a function of salinity at 14 °C.

Feeding activity dropped dramatically after direct transfer of
oyster spat from 34 SJt to salinities lower than 28 %( (Fig. 6). Spat
in the 18. 20. and 24 '7cc treatments regained their feeding activity

TABLE 1.

Daily mortality rates (mean ±95% CI) as a function of low
temperatures at two salinities.

Salinity

Temperature

22 7rc

34 %,

OX
2°C
4=C

0.012

0.0074

0.0035

0
0.0()3()

0

0.03

16^., I week

Time of low salinity exposure

Figure 4. Daily mortality rate (mean ± 95 % CI) of Ostrea edulis spat
after exposure to low salinity tl6 Vtt} for periods of 1, 2, 3, and 4 weeks,
respectively. Temperature was 10 'C.

after some days; whereas, spat in the 12. 14, and 16 Vcc ceased to
feed. The time to regain feeding activity after transfer to 34 %c
increased at lower salinities, especially in the low temperature
treatment. Analysis of variance of the linear model (Eq. 1) shows
that there is a significant interaction between the effects of tem-
perature and salinity on feeding activity (Table 2). This is inter-
preted as a general decrease of feeding activity with lower salinity
and that the low temperature treatment (5 °C) further reduces
feeding but only at low salinities (Fig. 6). Note that feeding ac-
tivity at salinities <20 7(i is not considered in the statistical analysis
because of high mortality rates. Feeding activity at low tempera-
tures (3-5 °C) was significantly reduced (Table 3) and completely
ceased at 3 °C independent of salinity. The time to recover full
feeding activity when transferred back from low to high salinity
(34 9ic) increased at lower salinities and could take several weeks
(Fig. 6). Similar to the effect on survival, the recovery of feeding
activity after exposure to low salinities depended on the exposure
time (Fig. 7). After 1 week of exposure to 16 %c with no feeding
activity the spat quickly recovered when transferred to 34 %c. As
the time of exposure increased so did the recovery phase (Fj 3, =
6.9. P = 0.001).

Feeding Activity as a Function of Gradual Acclimation to
I^ew Salinities

This experiment was performed to investigate how feeding ac-
tivity responded to a gradual acclimation to low salinities com-

c
o

3
T3
O

D. 40

u -"■

80
60-

r

'~-

'00

' KN.

Ic

1

■so

£

•^.^

60

u

rA

^

40

m

c

,'0

n)

<U

0

U

34 %»

Salinity

Figure 5. Fecal production (Vr of maximum score) and clearing rate
(mL h ' g"', mean ±95 % CI) for Ostrea edulis spat in different
salinities at 14 C.

804

RODSTROM AND JONSSON

^

o

3

o

o

u

[I,

U lu 2U 30 40 50 faO 70 SO 90

0 10 20 30 40 50 (lO 70 SO 90

-*- 14 %«. 10°
-•-12%», 10°
-o- 12 %c. 5°

1U"C: L

" ^

34 '/(,, luwer salinities

back 10 34 '7t,.

hack to 10°

Fijiure 6. Time trajectories of fecal production ( % of maximum score)
of Oslrea ediilis spat in dilTerent salinities at 5 C and 10 C. Below the
panels are transfer protocols for the different treatments.

TABLE 2.

Analysis of variance of the effects of temperature and salinity on
oyster spat feeding activity, measured as fecal production.

Source

d.f. Mean Square

Tetiiperature

1

30.0

23.2

<0.0001

Salinity

3

25.9

20.1

<0.0001

Week

3

0.792

1.45

0.23

Iniiividual (Temp.. Salinity)

48

1.29

2.81

<0.0()01

Temp. * Salinity

3

3.92

3.03

0.038

Temp. * Week

3

0.518

0.948

0.42

Salinity * Week

9

1.49

2.73

0.0057

Temp. * Salinity * Week

9

0.891

1.63

0.11

Individual (Temp., Salinity)*

Week

144

0.546

1.19

0.12

Residual

224

0.460

The factor "Week" identifies variation among different weeks during the J
30-day experiment, and the factor "Individual" identifies variation among
the studied spat in each combination of temperature and salinity. I

Temperature levels: 5 and 10 °C; salinity levels: 20. 24. 28. and ^47t,.

Simulation of Spat Survival as a Function of Local Hydrography

The over-all goal of the present study was to elucidate the
effects of low temperatures and salinities in the field on survival '
and feeding activity of oyster spat. Based on the results from the ,
laboratory experiments, we wanted to extrapolate to local field
conditions to obtain information about expected mortalities as a
function of the depth of spat deployment and the sea.son of spat i
transfer to the sea. From hydrographic datasets on temperature and
salinity at the Tjarnii Marine Biological Laboratory, survival tra-
jectories were calculated using the mortality rates estimated in the
laboratory experiments. Results from simulations of the model for
9 ditferenl years of hydrographic data show that both the depth of
deployment and the time of spat transfer strongly affect average i
mortality rate. Mortality rate increases above a depth of ca 8 m. in
particular for spat transferred in April (Fig. 9). Mortality is gen- I
erally highest for spat transferred in April; whereas, it is very low
for transfers starting in .luly. Also note that the variability of mor-
tality among years is highest in the surface waters. i

Simulation of Spat Feeding Activity as a Function of j

iMcal Hydrography ■

Results from the simulation of the feeding activity model can
only be used to infer qualitative effects of depth and timing of spat
transfer to the sea. Activity is predicted to increase with depth with
more saline water (Fig. 10a). Over the 120-day period, cumulative

pared to a rapid change in salinity. Figure 8 shows the feeding
activity of oyster spat where each of the five panels represents a
salinity that was reached by gradual acclimation. The columns in
each panel reprcsenl the feeding activity for spal directly trans-
ferred to new salinities. The experiment shows that it was not
possible to acclimate the spat to 12 %r. However, spat gradually
acclimated to 16 %( performed better compared to spal directly
iranslerred from .34. 24. or 20 7,r. Note thai the feeding activity of
the control spat in .34 7<i declined over lime indicating a general
deterioration of spat quality. This should, however, not change the
conclusions about the effect of gradual acclimation.

TABLE 3.

Analysis of variance of the effects of low temperatures and low

salinities on oy.ster spat feedinn activity, meu.sured as

fecal production.

Source

d.f.

Mean Square

Temperature

")

7.0

14.5

0.0002

Salinity

1

1.0

2.08

0.15

Temp. * salinity

4

0.33

0.692

0.61

Residual

18

0.481

Temperature levels: 3. 4 and 5 "C; salinity levels: 20. 24. and 34 %t

Survival and Feeding of Oyster Spat

805

KXl-i

w-

^ ^

80-

f^

m-

c

o

60-

o

3
T1

50-

r>

Q,

4U-

.(u-

M

u.

:n-

1(1-

11 J

34%.

16 %o, 1 week
16 %t, 2 week
16 %o, 3 week
16%c, 4 week

V) 40

Day No.

,34^,

16'5, ,

34 7,0

,34'?„

16'!,

34 %,

1

|34'5,

16 7,, ,

34

">' 1

1 34<;,

161.

1

^A",,

Figure 7. Time trajectories of fecal production ( % of maximum score)
of Oslrea edulis spat during exposures to 16 %< salinity for 1, 2, 3, and
4 weeks including subsequent transfer back to 34 %c. Below the panel
are transfer protocols for the different treatments.

activity is greatest for spat starting in July and least for spat start-
ing in April. Because food availability varies both with depth and
season, the food uptake will depend both on activity and food
concentration. In a coarse attempt to account for food availability,
we ran a similar simulation but multiplying activity with a chlo-
rophyll a matrix compiled for the depths 0-15 m and for the
different seasons (monthly resolution). Inclusion of food availabil-
ity changes the depth profile, compared to the profile of activity,
mainly by reducing the advantage at greater depths (Fig. 10b).

Field Experiment of Oyster Spat Survival and Growth

The daily mortality rate and the shell growth rate of a cohort of
oyster spat transferred to the sea in suspended trays are shown in
Fig. 1 la. Also shown is a simulation of the mortality model based
on depth-specific data on temperature and salinity for the time
period covered in the field experiment (Fig. lib). Mortality of
oyster spat shows maxima in the summers of 1989 and 1990 with
rates not explained by the model. Mortality may have been caused
by a combination of the observed fouling by blue mussels, Mylihis
edulis L.. and the presence of predatory sea stars. Aslericis nibeiis
L.. At other times of the year, field mortalities are similar to what
is predicted by the model. As expected, shell growth rate shows a
time trajectory with maxima during the summer months and with
very low rates during the winter months.

DISCUSSION

Local Hydrography and the Choice of Experimental Conditions

Ostrea edulis disappears when going southwest from the Swed-
ish Skagerrak to the northern parts of the Kattegat (Korringa
1976). Most probably the distribution of O. edulis is limited by low
winter temperatures and occasional low salinities in this region.
Changes in the relative magnitude of river discharge, particularly
in the spring, the high-salinity Jutland current, and the brackish
Baltic current lead to large variations in salinity often on short time
scales. Reproduction of Ostrea edulis is negatively infiuenced by
the local variations in hydrography on the Swedish west coast.
Low summer temperatures are supposed to prevent gonad matu-

U%^ li'H. 24 It 20 .i<

Lj

20 %* 16 %c 12 %<- C 34 %- 54 %.

34^

241,

M-St

16%..

12%. CU%c

1

16%c

1

1

1

34%.

'

16%=

12%

C34%c

\l%t C34S.

Salinity

Figure 8. Fecal production (% of maximum score) of Ostrea edulis
spat after rapid and gradual transfer to lower salinities at 14 °C. Each
panel represents the salinity to which larvae were gradually accli-
mated. Each treatment within a panel represents a salinity to which
larvae were rapidly transferred. A control (C 34 %c) shows the fecal
production for larvae which remained at 34 %r. Treatments with no
recorded fecal production are indicated with 0.

ration and subsequent release of larvae (Sparck 1949, Sparck 1951,
Wilson and Simons 1985). According to local oyster fishers, suc-
cessful spawning and settlement only occur every 6 years. This
lack of continuity is the main reason for the recent interest in
land-based spawning to improve spat production. Given the po-
tential option to produce spat of Oslrea edulis in a land-based
hatchery, the present study is focused on the effect of hydrography
on spat mortality and feeding activity when transferred to the sea
for subsequent culturing.

The response to fluctuating temperature and salinity in marine
organisms often depends on the rate of change (Alderdice 1972,

0-|

•^^■^ y* '-"""^

2-

y_^ /■ m'"^

l_ , / ^^

4-

I , / /_

J . / y_^

?^-

f I — f^

J= 8-
Q 10-

i P — '

12-

L^

— •— April-July

14-

1 &

-»- July-Oci
— t— Sept-Dec

0.002 o.otu o.onfi 0,008

0.01

0.014

Mortality rate (day )

Figure 9. Model simulation of average daily mortality rate (mean ±
SD, n = 9) as a function of depth and the time at transfer to field
conditions. Three scenarios are simulated, transfer of spat from the
hatchery to field conditions in the beginning of April, July, and Sep-
tember, respectively. Time of exposure is 120 days.

806

RODSTROM AND JONSSON

A u-

— * —

Apri
July-
Sept

— 1 — r

V t

~\

2-

x. ' \

\

\^ \

\

4J

\ \

\

N^ \

\

?.'■

\_ r

\

\ \

^ \

■£ 8-
Q.

\ \

\

\

Q 10-

' / \

\

^ \

12-

"A

-liilv , \ \

"a

14-

Oct , \ 1

'yA

\

16-

-, — . — . — . — 1 — . — . — . — • — 1 — ' — 1 — -— 1 — r

40

B 0

2

4-1

D.

Q 10

12-

14

16

50 60 70 80 90

Feeding activity (relative units)

•—April-July
■•— July-Oct
Sept-Dec

100

40 50 60 70 80

Food uptake (relative units)

90

Figure 1(). (a) Simulated feeding activity (relative units; mean ± SD, n
= 9) as a function of depth (m) and the time at transfer to field con-
ditions, (b) Simulated food uptake (relative units; mean ± SI), 11 = 9) as
a function of depth (ml and the time of transfer to field conditions.
Three scenarios are simulated, transfer of spat from the hatchery to
field conditions in the beginning of April, July, and September, re-
spectively. Time of exposure is 120 days.

Kinne 1970. Shiimway 1W6. Theede and Lassig 1967). Daily
temperature changes along the Skagerrak coast are moderate and
rarely exceed 1 C day"' (Fig. lb). However, because the Skager-
rak coast receives an ever-changing contribution of currents from
the North Sea and the Baltic that are further mixed with river input,
the salinity may change by several parts per thousand per day (Fig.
la). The rate of temperature and salinity changes in the experi-
ments of spat mortality and feeding activity were selected to retlect
the rate of change ob.served in the field. Temperature was conse-
quently changed at a maximum of I °C day ' while spat were
directly transferred between different salinities. The direct trans-
fers between full salinity (.M 7i<) and the lowest salinities tested
were obviously more rapid than found in the field. The comparison
between direct transfer to low salinities and a more gradual change
(1-2 %( day"') indicates that oyster spat may maintain feeding
activity at lower salinities if the change is gradual (Fig. S).

Effects of Temperature and Salinity mi Mortality anil I'eedinjt Activity

Low salinities and tempcr;itures. in the range observed in the
field, clearly can increase mortality rate and reduce feeding activ-
ity. Daily mortality rates increased sharply below salinities of ca
209(1 depending on temperature (Fig. 3. Table 1 ). Feeding activity

NDJ FMAMJ J ASONDJ FMAMJ J ASON
1989 1990

0,008

5- 0.006-

NDJ FMAMJ J ASONDJ FMAMJ JASON
1989 1990

Figure 11. (a) Daily average mortality rate and growth of Oxfrea erfu/is
spat (mm day"') in suspended tray culture under field conditions,
1989-1991. (b) Model simulation of daily mortality rate for the time
trajectory of salinity and temperature at 5 m during 1989-1991.

declined below 28 %o and stopped below 20 %(. Lowering the
temperature from 10 to 5 °C further reduced feeding activity,
which ceased completely at .^ "C. independent of salinity (Fig. 6.
Table 3). Generally, mortality increased when the temperature was
reduced from 10 to 5 °C (Fig. 3). The exception was at very low
salinities when mortality increased dramatically in the 10 °C treat-
ment. It may be speculated that the higher metabolic activity at 10
°C (Child and Laing 199S. Shumway 1996) forced the oyster spat
to feed more often, resulting in more frequent exposure to the
lethal salinity. In contrast, at low temperatures the spat could re-
main closed for longer periods of time escaping the deleterious
effect of ambient low salinity. This is supported by the findings in
Hutchinson and Hawkins (1992) that the scope for growth declined
in combinations of high temperatures and low salinities. Based on
experimental comparisons (Fig. 8) between gradual and rapid
changes in salinity, average feeding rate will not depend strongly
on the rate of change (max. 3-4 %<) of field salinities (Fig. la).

Ijing-Tenn Effects of Low Salinity Exposure

The duration of the exposure to the suboptimal salinity 16 %i
affected subsequent mortality and feeding activity of spat when
transferred to full salinity (Figs. 4 and 7). When exposed for up to
2 weeks, spat rapidly resumed their level of feeding activity, and
no increased mortality could be detected. However, when exposure
exceeded 2 weeks, there was a lag period for up to 2 weeks before
the normal level of feeding activity was attained. A similar effect
of long exposure to 16 ''u was found for spat mortality that con-
lirnied to be high after transfer to full salinity. This pattern may be
explained assuming that spat can close their valves for a limited
time when encountering such adverse conditions as low salinities.
After some time, depending on physiological conditions, the spat

Sl'RVIVAL AND FEEDING OF O^ STER SpAT

807

are forced to \ enlilate and feed v\ ith risks of damage and mortality
(Nell and Paterson 1970).

Earlier observations from the Skagerrak coast by Woilebsck
(1903) and Dannevig (1953) showed extensive mortalities during
March to June when oysters were exposed to low salinities and low
temperatures. Similar observations have been reported recently by
local fishers on the Swedish west coast. Newkirk et al. (1995) al.so
observed that some areas in Nova Scotia, Canada, were unsuitable
for Osrreci eihili.'i because of suboptimal periods of low salinitv and
low temperature. Mortality during cold winter temperatures is.
however, apparently low. It may be speculated that the combina-
tion of increasing temperature and low salinity will cause high
spring mortality because of an increase in metabolic demand forc-
ing the oysters to feed frequently and expose tissues to deleterious
salinities. It is also possible that increased spring mortality is
caused by a previous cold stress period (Newkirk et al. 1995).

Fecal Production and Clearing Rate

Feeding activity was measured as fecal production, which was
quantified on an ordinal scale. The rationale is that fecal produc-
tion integrates feeding over time, is a direct measure of feeding
activity, and it is easy to measure. The correlation between esti-
mated clearing rates and observed fecal production was generally
good (Fig. 5). Estimated clearing rates for 12-mm spat feeding on
Isochiysis galhana were 8-20 mL h^' at 14 °C. This compares
reasonably with; for example, Walne (1972), who reported clear-
ing rates for IO-nim spat of 25 mL h"' at 21 °C.

How Much of Field Observations Are Explained by the
Mortality Model?

A critical question in the present study is whether the experi-
mental results of mortality and feeding activity can be extrapolated
to field conditions and, if so, whether conclusions could be drawn
about the expected significance of observed mortality and feeding
patterns. As an attempt to validate the experimentally estimated
mortality rates, we exposed spat to field conditions for 2 years and
monitored mortality and growth (Fig. 11a). Using a mortality
model based on the mortality experiments and field data of tem-
perature and salinity, we simulated mortality patterns expected for
the field experimental period (Fig lib). The field experiment
showed high mortalities in April and July for spat during their first
year. The model correctly predicts the mortality rates found in the
spring but strongly underestimates rates in the summer. The high
mortality in the field experiment during summer may have been
caused by a combination of intense biofouling or predation. In
conclusion, temperature and salinity may explain mortality rates in
winter and spring, but biological interactions become more impor-
tant during summer and autumn. It can also be seen that spat suffer
less mortality during their second year. This may be explained by
the usually increasing tolerance against suboptimal temperatures
and salinities with age (Walne 1979), possibly mediated by a
greater capacity to remain closed and tolerate starvation. A further
explanation could be size-dependent predation.

The growth rates found in the field clo.sely follow predictions
from the laboratory experiments on feeding activity as a function
of temperature and salinity. Most of the growth occurs between
May to November (Fig. 1 la). April and May, when food is abun-
dant, give poor growth possibly caused by the combination of low
salinities and relatively low temperatures. This view is also sup-
ported by the model simulation of feeding activity during this
period (Fig. 10a).

Recommendations for Management of Cultures

To explore the significance of observed mortality and feeding
patterns for the management of spat transfer to the sea and sub-
sequent culture, we simulated different management policies. By
using the model of mortality and feeding activity based on labo-
ratory experiments and field data on hydrography, we simulated
transfer of spat at three different seasons and at 16 different water
depths (Figs. 9, 10a). The simulations show that mortality is high
during spring above a depth of 6 m. The variability among years
also increases sharply in the surface waters. We recommend that
spat be kept below 6 ni during winter and spring to reduce mor-
tality. Of course, a flexible policy would be to take advantage of
the year-to-year variability in hydrography by continually adjust-
ing culture depth to prevailing conditions. However, considering
the rapid changes in hydrography, this approach would probably
be prohibitively expensive. Around July and through summer, the
spat mortality caused by temperature and salinty is at its minimum
throughout the water-column. Feeding activity may also be at its
maximum during this period. Because food uptake is expected,
from field data on chlorophyll a. to be high regardless of depth
(Fig. 10b), an option exists to escape the heavy fouling at the
surface during this period (unpublished data) by deploying the spat
below 6 m. In the autumn, mortality increases slightly but is still
almost independent of depth. Because food uptake is expected to
decline with depth (Fig. 10b), we recommend that spat be kept
above 6 m during this period. Finally, because local hydrography
may differ on small spatial scales because of the extensive archi-
pelago and local land run-off. the selection of culture locality may
also be important.

Populations of Ostrea edidis on the Swedish west coast expe-
rience extreme hydrographic conditions. This may have selected
for local tolerance to low temperatures and salinities. Future work
should test for the existence of local genetic populations and com-
pare geographical differences in tolerance. This knowledge will be
particularly important for management policies of introductions of
O. cdiilis from areas with less extreme hydrography.

ACKNOWLEDGMENTS

We thank Olle Danielsson and Mats Ulmestrand who helped
with the field exposure of oyster spat, Jan Karlsson who provided
the broodstock of oysters, and Carl Andre, Kent Berntsson and
Rutger Rosenberg for valuable comments. Financial support to
E.M.R. for the study was provided by the Swedish Agriculture
Research Council and the Colliander, Adlerbertska, and Anna Ahr-
enberg Foundations.

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RODSTROM AND JONSSON

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Joiinuil of Shellfish Reseiirch. Vol. 19. No. 2, 809-814. 2000.

AN IN SITU STUDY ON THE SURVIVAL AND GROWTH OF CRASSOSTREA VIRGINICA
JUVENILES IN BON SECOUR BAY, ALABAMA

IMAD G. SAOUD,' DAVID B. ROUSE,' RICHARD K. WALLACE,'
JOHN E. SUPAN,- AND SCOTT RIKARD'

Department of Fisheries and Allied Aquacultures, Auburn University
Auburn. Alabama 36849

'Louisiana Sea Grant. Louisiana State University,
Baton Rouge, Louisiana 70803

ABSTRACT Experimenlal plots were established at a relic oyster reef on the eastern side of Mobile Bay, Alabama between July 1998
and November 1999 to determine whether elevated beds might improve oyster survival and growth. Oysters (Crassostrea virginica)
were spawned in a hatchery and the spat were allowed to settle on small oyster shell fragments and on whole oyster shell. Two-
month-old juveniles (15-18 mm) were deployed in polyethylene oyster bags on bottom and on underwater shell pads 20 cm and 40
cm above bottom. Oysters on whole shells were deployed outside bags in order to evaluate predation. Remote sensing data loggers were
deployed near bottom and 40 cm above bottom to measure temperature, salinity, and oxygen concentration. Growth (increase in height),
survival, and condition of oysters in bags at the three experimental depths were compared. Temperature and salinity varied between
1 1 .8 °C-32.8 °C and 4.4 ppt-29.7 ppt. respectively. Periodic anoxic events, which lasted from a few hours to 6 days, were documented.
Oysters at the three experimental levels grew to approximately S5 mm during the first year. Total mortality was observed at all three
levels during the second summer when oxygen levels dropped to 0 mg L"' for five consecutive days while water temperature was 28
°C. If the 17 months monitored during this study are typical, restoration of Fish River Reef will be difficult due to periodic anoxic
events.

KEY WORDS: oyster, oxygen, hypoxia, anoxia

INTRODUCTION

Bon Secour Bay is an embayment located in the southeastern
segment of Mobile Bay, Alabama (Fig. 1). Although, some areas
of Mobile Bay support a viable oyster fishery, Bon Secour Bay has
no commercially harvestable oyster reefs today. Ritter ( 1895) sug-
gested that overfishing depleted this part of the bay of harvestable
oysters. Various reasons have been proposed to explain why viable
self-sustaining oyster populations have not returned to the region.
Explanations have ranged from the subsidence of the Gulf Coast
tidal region to deforestation of the surrounding drainage basin
causing an increase in freshwater and silt input (Mackin 1951).
Others have suggested an increase in the number of oyster drills.
Stramonita haema.stonui (Eckmayer 1979). Eckmayer (1983) re-
ported a near total mortality of oyster spat in Bon Secour Bay in
1979 due to a freshet. Cake and Eckmayer (1982) summarized
reasons for poor production as reduced spatfall, periodic oxygen
depletions and hurricanes. Oxygen depletions in August 1967 and
July 1978 have also been blamed (Eckmayer 1979).

A peculiar phenomenon that occurs sporadically on the eastern
side of Mobile Bay is the "jubilee." During jubilees, considerable
numbers of demersal fish and crustaceans are driven toward the
eastern shores of the Bay for several minutes up to a few hours
(May 197.3). Jubilees are wind driven events that result in low
oxygen water masses moving eastward along the bottom, forcing
benthic animals in front of them. They typically occur in the sum-
mer under specific water and wind conditions (Loesch 1960, May
19731. Information necessary to predict where and when jubilees
might happen, how long they last, and the height of the anoxic
layer above the bay bottom is lacking. Moreover, the periodicity of
anoxic conditions in Bon Secour Bay, and any impacts on oyster
survival is not known.

The goal of the present study was to determine the feasibility of
restoring a relic oyster reef at the Fish River site by the Stale of
Alabama. We measured water parameters (oxygen concentration,

temperature, salinity) continuously for several months. We tested
for correlations between in situ water parameters and juvenile
oyster survival and growth. Hatchery produced juvenile oysters
were deployed at the experimental site at various depths and the
null hypothesis that oyster survival and growth on 20 cm and 40
cm raised platforms was equivalent to survival and growth of
oysters deployed on the bottom was tested.

MATERIALS AND METHODS

Research Site and Protocols

Eastern oysters, Crassostrea virginica. were strip-spawned and
the larvae reared at the Louisiana State University oyster hatchery
at Grand Isle, Louisiana. Larvae were divided into two batches;
one was allowed to settle on crushed oyster shell and one on whole
oyster shell. Juvenile Crassostrea virginica were reared for ap-
proximately 2 mo in a flow-through system. Ten underwater plat-
forms were constructed at the Fish River Reef (ERR) (Fig. I) in
Bon Secour Bay, using 4-mm thick plastic sheets, either 20 cm or
40 cm wide, and 360 cm long. The plastic sheets were rolled
lengthwise and the ends connected to form cylinders with a diam-
eter of 1 13 cm and a crossectional area of 1 m". Plastic mesh was
tied across the bottom of each cylinder. The cylinders were then
placed next to each other on bottom at the study site (2.8 m deep)
and filled with oyster shell so that platform heights were 20 cm and
40 cm above bottom. Shell material was also dispersed on the
bottom around each platform. Oysters were deployed on bottom
and on the platforms at the two heights on July 30, 1998 and on
August 15, 1999.

Remote continuous-monitoring devices (RCMD; Minisonde
and Datasonde 3 by Hydrolab) were deployed close to the plat-
forms from July 30. 1998 through February 2000, at 3 cm and 40
cm above bottom in order to monitor oxygen concentration, tem-
perature, and salinity in the vicinity of the deployed oysters. No

809

810

Saoud et al.

City of Mobile

Gulf of Mexico

SCAL£ 1 :4e0000

Figure 1. Mobile Bay. Point "A" is the approximate location of Fish
River Reef.

RCMDs were deployed from November 1998 till February 1999,
and only one was deployed at 20 cm above bottom during Febru-
ary and Marcfi 1999. RCMDs were exchanged and calibrated twice
weekly. A water column profile of temperature, salinity and dis-
solved oxygen was taken using a YSI 35 hand-held instrument
whenever the RCMDs were exchanged. Wind speed and direction
data were obtained from the Alabama Weather Information Ser-
vice.

First Deployment

Two-month-old oysters produced in the hatchery and retained
on a I2..'i-mm mesh were deployed on July 30. 1998. .Six poly-
ethylene oyster bags (12..'i-mm mesh) were filled with approxi-
mately 500 oyster juveniles ( 15.2 mm ± 0.44 SE) and deployed on
the platforms. Two bags were deployed on bottom, two were de-
ployed on the 20 cm high platforms and two on the 40-cm high
platforms. The oysters were then sampled monthly between Au-
gust and November and bimonthly thereafter. During sampling,
approximately .10 oysters from each treatment (height above bot-
tom) were placed in a marked container and returned to the lab.
The oyster bags were brushed and shaken vigorously under water
in oriler to remove deposited sill. The presence or absence of
liHiling organisms and predators was documented. Growth was
estimated as the difterencc between the average height (hinge to

opposite margin) of oy.sters in a sample and the average height of
oysters in the preceding sample. On June 8, 1999, the oyster bags
were brought to the surface, counted, and live oysters from the two
bags at each height were combined into a 25-mm mesh bag and
redeployed to the same platforms. Percent survival was estimated
from the ratio of live to dead oysters. Hemolymph was taken from
ten oysters and checked for Dermo, Perkinsus marinus, using the
fluid thioglycollate method described by Ray (1952). Intensity of
P. marinus infection was asses.sed according to Mackin (1962).

Exposed shell strings were used to control for mortality from
predation. Oysters that were allowed to settle on whole oyster shell
were counted and a hole was drilled in each oyster-shell substra-
tum. Five shells were attached together by passing a string through
the holes and tying a knot on either side. Ten shell strings were
then deployed at each depth and held in place by inserting brass
clips into holes drilled in the sides of the platforms. A shell string
from each treatment was removed and transported to the lab at the
same time samples were taken from the oyster bags. On June 30,
1999, the oyster shell attached to strings were found to have wild
C. virginkci juveniles settled on them and were replaced with new
shell strings carrying known numbers of hatchery spawned juve-
niles on July 6.

On June 8. 1999, the height of oysters froin the bottom treat-
ment and from the 40-cm platform was measured, and the oysters
were cleaned, weighed, and their condition index determined ac-
cording to methods suggested by Crosby and Gale (1990).

Second Deployment

A second batch of 2-month-old oysters were sieved through a
12.5-mm mesh and the retained oysters were deployed on the
platforms on August 6, 1999. Three 12.5-mm mesh oyster bags
were filled with 300 juveniles (17.99 mm ± 0.36 SE) each. One
bag was deployed at each of the three experimental levels at the
study site. During sampling, bags were lifted onto a boat and live
and dead oysters were separated and counted. A random sample of
25 live oysters was taken back to the lab for additional measure-
tnents. In the lab, oysters were cleaned of all epibionts. blotted dry,
weighed, and their height measured.

Average heights and weights of oysters at each depth were
compared using one-way ANOVA. We tested the null hypotheses
that mean oyster height and weight on the 20-cni and 40-cm plat-
forms was equivalent to mean height and weight of oysters de-
ployed on bottom. Oxygen concentration, temperature and salinity
on bottom and 40 cm above bottom were plotted in an attempt to
show a relationship between various environmental parameters
and survival and growth.

RESULTS

First Deployment

Oysters grew from 15.2 mm to more than 50 mm during the
first year. On August 12, 1998 there was no significant difference
in height between treatments (Table 1 ). At the subsequent two
sampling dates, oysters on the 4()-cm high platform were signifi-
cantly longer than oysters on bottom (P < 0.05). The sample col-
lected on November 24 suggested equal growth of oyster juveniles
on bottom and 40 cm above bottom. Howe\cr, oysters on bottom
were thin and elongated (coons). Oysters collected 40 cm above
bottom were more circular and deeply cupped. These ob.served

Effects of Hypoxia and Anoxia on Oyster Juveniles

TABLE L

Height (mm) of oysters deployed on bottom and 20 cm and 40 cm
above bottom (mean ± SE) at Fish River Reef on July 30, 1998

Date

Bottom

20 cm

40 cm

30 July 1998

12 Aug. 1998
08 Sep. 1998
06 Oct. 1998
19 Jan. 1999
03 May 1999
08 June 1999

13 Aus. 1999

15.20 ±0.44
15.10 + 0.39
19.70 ±0.47
26.40 + 0.88
52.24 ± 1.97
52.28 ± 2.09
56.40 ± 1.45
56.22+ 1.80

15.20 ±0.44
16.50 ±0.55
22.20 ±0.71
26.90 ± 0.85
42.92 ± 1.58
52.97 ± 1.89
56.57 ± 1 .86
5 1 .37 ± 2.04

15.20 ±0.44
15.90 ±0.25
22.20 ± 1.05
31.50 ± 1.29
52.06 ± 1 .66
56.06 ± 1.43
54.20+ 1.64
54.24 ± 2.02

morphological differences remained throughout the .study. On
January 19, 1999, oysters on the 20-cm platforms were signifi-
cantly shorter than oysters at the other two levels, but this differ-
ence disappeared in subsequent samplings. Height, length, width,
and condition inde.x of the oysters collected on June 8, 1999,
approximately 1 yr after planting, are presented in Table 2. The
data suggest that meat weight and condition of oysters at 40 cm
above bottom were significantly greater than weight and condition
of oysters on bottom. Oyster survival on June 8, 1999, was similar
on bottom (84.5%) and 40 cm above bottom (74.9%) (P < 0.0.5).
Dermo intensity varied between 2 and 3 on the Mackin (1962)
scale. During routine visual inspections on July 6, most oysters
appeared to be alive. On August 13, 1999 all the oysters at the
three treatment levels were found dead.

Algal and bryozoan fouling was rare throughout the study. No
predators were observed from August 1998 until June 1999. On
June 8, 1999, egg cases of the oyster drill, Strainonita haenuis-
toma. were found on the bags and juvenile drills were found within
the bags at all subsequent inspections. Occasional blue crabs were
also encountered after June 8, 1999. No signs of predation were
observed on the shells deployed outside the bags.

There were no differences in oxygen, temperature and salinity
measurements on bottom and 40 cm above bottom during the
study. Temperature ranged from a low of 12 °C in February to a
high of 32 °C in August. Salinities generally ranged from lows of
5 ppt to highs of 15-20 ppl. Spikes above and below these ranges
occurred but were infrequent. Oxygen concentrations at both
depths were also similar, generally ranging from lows of 0.5 mg
L~' to highs of 5 mg L~' and greater. There were several instances
during which oxygen concentrations on bottom were lower than 1
mg L"' while oxygen concentrations at 40 cm above bottom were
greater than 1 mg L ~'. These instances were usually of short
duration, rarely lasting more than 24 h.

On three occasions, oxygen concentrations at the two depths
were near 0 mg L~' for extended periods of time. Between Feb-
ruary 25 and February 27, 1999 (Fig. 2) oxygen levels were near
zero. Wind speed during that period varied between 1.6 km h '
and 19 km h~' and wind direction varied on an hourly basis. Water
temperature during this period ranged between 13.5 °C and 15 °C.
On July 16, 1999 (Fig. 2) oxygen levels dropped to near 0 mg L"'
and did not rise above 0.5 mg L"' until July 22. Wind speed ranged
from 0 km h ' to 15 km h ' changing provenance constantly, and
water temperature was about 28 °C. On August 4, oxygen levels
dropped to zero where they remained until August 9 (Fig. 2). Wind
speed and direction were variable and temperature was above 30 °C.

Throughout the study, including the three extended low D.O.
events, oxygen level dropped when salinity increased (Fig. 2).
Even a 5-h rise in salinity coincided with a dip in oxygen concen-
tration. As soon as salinity levels fell, oxygen concentrations rose
again. The lowest salinity encountered during the study was 4.4
ppt on February 22, 1999, coinciding with a bottom oxygen con-
centration of 9.31 mg L"'. The highest salinity encountered was
29.8 ppt on September 21, 1999, coinciding with bottom oxygen
concentrations of 0.18 mg L"'. Water profiles depicted in Figure 3
indicate a strong pycnocline on July 21, 1999, during a period of
anoxia near the bottom. Stratification was observed during hypoxic
events and absent when bottom waters were aerobic.

Second Deployment

The oysters deployed on August 6, 1999 had grown by ap-
proximately 19 mm during the first 83 days ending on November
I, 1999 (Table 3). There was no significant difference in height
between treatments (P < 0.01) at any sampling event. On Novem-
ber I, 1999 survival was 38% on the bottom, 66%- at 20 cm, and
69% at 40 cm above bottom. On February 22, 2000 there was no
significant difference in oyster height among treatments (P <
0.02). Oyster heights were 44.9 mm, 50.4mm and 49.6 mm on
bottom, 20 cm and 40 cm, respectively. Survival was 29% on
bottom, 48% at 20 cm, and 52% at 40 cm. Seventy-three percent
of the dead oysters had a predator hole on one of their shells.

Inspections of the bags during samplings revealed that the bags

l»*^ I —

W\

^

1 "

i\

A R J"— sy^

c ,,

r ^

\ t , y*' ' IfVit

! ,„

^^

J&pi.-j/ vv^

L ,,

1 "ym

„ ,Nivft

^ \l 1 \ 1

4 I

, 2

Figure 2. Bottom temperature (°C), salinity (ppt) and oxygen concen-
tration (mg L"') at Fish River Reef during low oxygen events in 1999.

812

Saoud et al.

TABLE 2.

Height (mm), LW (g), SW (g), DW (g), and CI of oysters grown for 1 yr on bottom and 40 cm above bottom at Fish River Reef in Bon

Secour Bay, Alabama"'

Height

LW

SW

DW

CI

Bottom
40 cm

57.00 ± 3.95
54.20 ± 2.25

21.82 ±3.08
23.12 ±2.88

15.46 ±2.06
16.44 + 2.08

0.46 ± 0.09
0.57 ± 0.06

72.45 ± 4.72
87.36 ± 3.53

Measurements are means ± standard error.

* Abbreviations: LW, live weight: SW. shell weight: DW, dry tissue weight: CI, condition index.

on bottom had become covered with a layer of mud and silt while
the bags on the platforms remained clean. No morphological dif-
ferences were observed between oysters at the various depths.
Oxygen concentration was near zero the first two days that the
oysters were deployed but did not go lower than 0.5 mg L"' for
more than a few hours between August 6, 1999 and February 22.
2000.

DISCUSSION

This study documents periodic long-term hypoxic events on the
eastern side of Mobile Bay. Oxygen concentrations were ineasured
every half-hour, which allowed documentation of all diurnal fluc-
tuations in oxygen concentrations. Water parameters on bottom
and 40 cm above bottom were equivalent throughout the study.
Austin (1954) and Ryan (1969) reported flood tides moving east-
ward and northward along the eastern side of the Bay and ebb tides
moving southward along the western side. According to May
(1973) and Turner et al. (1987), density stratification causes the
isolation of bottom waters in Mobile Bay, and high biochemical
oxygen demand (BOD) in the sediment leads to oxygen depletions.
Based on these reports, we believe that during periods of stratifi-
cation, salt water entering Mobile Bay with high tide remains
below the pycnocline and gets depleted of oxygen as it moves
towards Fish River Reef. These anoxic conditions are different
from jubilees that occur north of FRR and are a result of atmo-
spheric conditions that cause the upwelling of anoxic waters that
form in low-lying areas of the bay bottom.

Dtpthtm)

I'lyiire ,^. Water column pniiHe of dissolved nxyncn (niK L~'), salinity
(|)|)t) and lemperalurv ( C) at I'ish River Reef on 21 ,|ul> ]')')').

Two-day hypoxic and anoxic conditions coincident with low
temperatures (February 1999, Fig. 2) did not cause oyster mortali-
ties. Five-day anoxic conditions in conjunction with high tempera-
tures (28.5 °C) such as occurred in July 1999 (Fig. 2) caused oyster
die-offs. Oxygen depletions in 1971 were blamed for oyster die-
offs in Mobile Bay (May 1972). Eckmayer (1979) suggests that
oxygen depletions might have been the cause of oyster mortalities
on planted beds in Bon Secour Bay although no direct evidence
was provided. Lenihan and Peterson (1998) associated mass mor-
tality of oysters with extended periods of anoxia in the Neuse
River estuary in North Carolina. Other researchers have reported
that oysters can survive several days of hypoxic and/or anoxic
conditions (Sparks et al. 1957, Baker and Mann 1992). In the
present study, oysters survived anoxia for 3 days in February 1999
but did not survive a 6-day anoxic period in July 1999. There was
a difference in the duration of the two anoxic events, but maybe
more importantly, there was a difference in water temperature.
These findings corroborate those of various authors who found that
oyster tolerance to anoxia decreased with increasing temperature
(Dunnington 1968, Andrews 1982. Shumway and Koehn 1982.
Stickle et al. 1989). Stickle et al. (1989) report LT,„ (lethal tem-
perature) values of over 28 days for oysters in anoxic conditions at
temperatures of 10 °C and only 3 days for oysters at 30 °C. Oysters
buried in anoxic sediments survived for more than 5 wk at tem-
peratures less than 5 °C but only 4 days at temperatures greater
than 25 °C (Dunnington 1968). Therefore, it appears unlikely that
a viable oyster population could be inainlained at Fish River Reef
due to periodic anoxic events coincident with high water tempera-
tures in the summer.

Temperature and salinity on bottom were consistently similar
to temperature and salinity 40 ctii above bottom, and ranged from
1 1.8 to 32.8 C and 4.4 to 29.8 ppt. respectively. Variations in
oxygen concentrations at both depths al.so follow a similar pattern
over time. Although oxygen concentration on bottom was nor-
mally lower than oxygen concentration 40 cm above bottom, the
differences were small. The lower oxygen tension near bottom was

TABLE 3.

Height (mm) of oysters deployed on bottiini and 20 cm and 40 cm
above bottom (mean ± SE) al Fish River Reef in 1999

Date

Bottom

20 cm

40 cm

06 Aug. 1999
08 .Sep. 1999
27 .Sep. 1999
01 Nov. 1999
22 Feb. 2()()()

17.9±0..Vi
27.03 ± 0.93
28.40 ± 1 .35
36.28+ l..^()

44.9 ± 2.4

1 7.9 ± 0.36
26.80 ± 0.84
30.69 ± 0.99
.W..56 + 1 .57

SOA ± 2.2

17.9 ±0,36
27.25 ±0.91
30.00 ± 1 .09
38.08 ± 1.26

49.6 ± 1 .5

I

Effects of Hypoxia and Anoxia on Oyster Juveniles

813

probably due to high BOD at the water sediment interface, and low
mixing due to boundary layer effects. The difference in oxygen
tension at the two depths was more pronounced between October
3 and 6. IWS. most likely due to neap tides and calm winds which
did not induce much mixing of the bottom waters shortly after
Hurricane Georges had caused the deposition of a lot of sediment.
This hypoxic event appears to have temporarily reduced the
growth of oysters deployed on bottom and 20 cm above bottom,
more than it reduced the growth of oysters 40 cm above bottom.
Widdows et al. ( 1989) and Baker and Mann (1992, 1994), attribute
such a reduction in growth during a hypoxic event to a reduced
feeding rate. Less than 2 months later, the oysters on bottom had
undergone compensatory growth and had caught up with oysters
on the 40-cm platforms.

Vertical profiles of the water column suggest periodic stratitl-
cation due to a pycnocline between 1 m and 2 m depth (Fig. 3;
Table 4). The data does not support the presence of a thermocline.
Others have reported similar low-oxygen events as a result of
pycnoclines in Mobile Bay (May 1972, Turner et al. 1987,
Schroeder et al. 1990) as well as in Chesapeake Bay (Breitburg
1990, Sanford et al. 1990). These events are said to occur in
stratified waters when high salinity bottom waters are exposed to
high BODs in bottom sediments. Vertical mixing, vertical advec-
tion, and water mass movements within shallow estuaries such as
Mobile Bay are wind driven (Ward 1980, Wiseman et al. 1988). In

TABLE 4.

Presence or absence of a halocline and/or thermocline at Fish River
Reef from June through October 1999

Halocli

ine

Thermocli

ine

Date

Present

Absent

Present

Absent

2/5/99

+

+

25/5/99

+

+

3/6/99

+

+

8/6/99

+

+

9/6/99

+

+

15/6/99

+

+

16/6/99

+

+

21/6/99

+

+

22/6/99

+

+

30/6/99

+

+

6/7/99

+

+

13/7/99

+

+

21/7/99

+

+

26/7/99

+

+

2/8/99

+

+

11/8/99

+

+

17/8/99

+

+

23/8/99

+

+

31/8/99

+

+

8/9/99

+

+

15/9/99

+

+

24/9/99

+

+

29/9/99

+

+

12/10/99

+

+

19/10/99

+

+

26/10/99

+

+

1/11/99

+

+

the absence of a strong unidirectional wind, the water column may
stratify creating a hypoxic layer near bottom. In February, July,
and August 1999, when the anoxic events in this study were re-
corded, wind velocities were variable and wind direction changed
constantly. The formation of waves is dependent on a unidirec-
tional wind with ample fetch and duration. Wind conditions during
these periods were not conducive to the formation of waves large
enough to mix the water column. Baker and Mann (1992) and
Osman and Abbe (1995) associated hypoxia with a reduction in
postlarval growth of C. virginica. A slower growth of juvenile
oysters prolongs their susceptibility to disease and predation by
crabs, drills, and fish (Dittman et al. 1998, Grant 1996). No signs
of predation were detected on the oysters deployed outside the
bags in the summer of 1998. In June 1999, we noticed some oyster
drills on deployed oysters but total mortality of oysters in July
prevented documenting potential effects of predation.

The 1 -year growth rate of oysters in the present study was 3.25
mm month"'. Anderson (2000) raised oysters in suspended culture
in Mobile Bay, approximately 3 km south of FRR and reported
growth rates similar to those reported herein. In both studies, oys-
ter growth rates in Bon Secour Bay were less than the 4.65 mm
month"' reported by Supan (1983) in Mississippi Sound. Various
workers have discussed the importance of factors such as location,
timing of larval settlement, genetics and year-to-year environmen-
tal variation on growth (Newkirk et al. 1977, Losee 1979, Crosby
et al. 1991, Dittman et al. 1998, Bataller et al. 1999). With such
variability in oyster growth rates, we believe that growth rate
determinations would only be relevant when averaged over several
growing seasons.

Traditional methods of estimating oyster growth are by mea-
suring changes in height. Height measurements were not a good
indicator of growth in the present study. Walne ( 1958) and Hilbish
( 1986) discuss the inadequacy of using linear measurements as a
sole indicator of growth in bivalves while Losee (1979) multiplied
length and width of oysters and used changes in square area as an
index of growth. In the present study, oysters deployed at 40 cm
above bottom initially grew faster than oysters deployed on bottom
but, within 5 months, the average height of oysters at both depths
was similar. However, morphological observations suggest that
oysters at 40 cm were more rounded and cup-shaped than those on
the bottom. Moreover, data in Table 2 suggest that although the
average height of the oysters at 40 cm was shorter than the average
height of oysters on bottom, oysters on bottom had less dry meat
weight and a smaller average condition index. We propose that
oxygen stress and/or siltation stress caused the oysters maintained
on bottom to grow into coons. Probably, the silt settled through the
oyster bags, and in situations where there were no void spaces
under the bags (on bottom), the silt gathered in the bag. The bags
on the platforms were lying on shell and the silt was washed down
into the shell. Although the oy.sters were lying horizontally in the
bags, and increased height would not allow them to grow out of the
sediment, it appears that the reaction to adverse conditions is to
grow into coons, regardless of orientation.

Results of the present study suggest that even with restoration
efforts such as cultch and oyster deployment, it is unlikely that the
relic oyster reef at the Fish River site can be restored to a healthy
and productive oyster reef. Moreover, the low oxygen events ob-
served at FRR could be occurring at other relic reefs in Bon Secour
Bay, thus the need for investigations similar to the present work
before restoration is attempted.

il4

Saoud et al.

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Joiiriuil of Shellfish Reseurch. Vol. 19, No. 2, SI 5-8:0, 2()()().

DWARF MALES IN THE PUELCHE OYSTER (OSTREA PUELCHANA, D'ORB.):
DIFFERENTIAL MORTALITY OR SELECTIVE SETTLEMENT?

MARCELA PASCUAL

Laboratoiio de Mohiscos.

Instituto de Biologfa Marina y Pesqueru

CC 104,

(8520) San Antonio Oeste, Argentina

'Alte. Storni.

ABSTRACT Oslrea puelchana is the only ostreid for which a "dwarf male" phenomenon has been described. Adult females 055
mml "carry" small individuals settled on a expansion of the anterior margin of the concave shell. This phenomenon co-occurs with sex
reversal. These small oysters mature as males and have their growth severely retarded as a result of interaction with the carrying female
oyster. The non-random distribution of small males is. at least in part, a consequence of the higher survival rate of settlers on the
platform of adult females, where they are sheltered from the foraging activities of chitons. In the present work "carriage" was studied
in wild oysters from three natural grounds and in fanned oysters. The results show that in farmed oysters, carriage begins at a larger
size than in wild oysters, suggesting that the attraction effect exerted by the adult oysters on the larvae ready to settle is related to age
rather than to the size of the oyster. Settlement is constrained by the previous occupation of the platform. The settlement and/or survival
of new recruits is inversely related to the presence and size of previous recruits. Recruitment on dead and living oysters, and the
settlement of O. puelchana larvae on adults of O. puelchana and O. edulis were experimentally assessed. The results of both
experiments suggest: ( I ) the specificity of the carriage phenomenon and (2) the existence of an active selection of (he platform by the
larvae. Thus, the carriage phenomenon may be explained by two simultaneous mechanisms: the selective mortality of recruits settled
on the shells, and the differential larval settlement through a process of active selection.

KEY WORDS: oysters. Ostrea puelchana. dwaij males, settlement, mortality

INTRODUCTION

The Patagonian flat oyster. Ostrea puelchana. is the only os-
treid for which a "dwarf male" phenomenon has been described
(Calvo and Morriconi 1978, Pascual et al. 1989). Besides sharing
the general breeding pattern with the rest of Ostreas species, O.
puelchana shows a unique system in which larvae setting on a
expansion of the internal surface of the anterior margin (the shell
platform) of the concave shell of adult females (Fig. 1 ), mature as
male oysters at about 2 mm of shell size. Growth in these males is
severely retarded as a result of interaction with the carrying female
oyster (Pascual et al. 1989).

During the .settlement season oyster larvae settle on all avail-
able hard surfaces but preferentially on the shells of living oysters
(Pascual and Zampatti 1995). In natural grounds, the number of
recruits settled during the settlement peak is significantly higher on
living oysters than on dead oysters showing that a chemically
mediated adult-larval interaction triggers settlement in this species
(Pascual and Zampatti 1995). Larval settlement on live oysters
occurs on the outer surface of the shells and on the anterior plat-
form of the concave shell, where they are sheltered by the flat
shell. The concentration of small individuals is much higher in the
inner surface of the platform than on the external surface of the
shells. Calvo and Morriconi (1978) suggested that the high con-
centration of small individuals on the platform could be due to a
localized settlement response triggered by a chemical released by
the females.

Most recruits settling on the surface of live oysters die during
their first month of life (Pascual 1997). This fact led to an alter-
native explanation for the non-random distribution of small indi-
viduals, namely that settlement is random, but spat that settle on
the internal platform have a higher survival rate (Pascual 1997).
Experimental work carried out later demonstrated that, in the case
of the puelche oyster, grazing by chitons is an important mortality
source for oyster recruits that settle on the outer shells of oysters

(Pascual 1997). Shelter from chitons may result in higher survival
rate of settlers on the shell platforin of adult females (Pascual
1997).

Settlement of spat on the outer surface of the shells of live
oysters is independent of the oyster's size. This pattern suggests
that each oyster attracts, on average, a constant number of larvae
(Pascual and Zampatti 1995) and that this effect is maintained
during the entire lifespan. Settlement of spat on the internal shell
platform, on the other hand, does not occur until the oyster reaches
55 mm in size (Calvo and Morriconi 1978). This unambiguous
pattern strongly suggests the existence of a mechanism of active
selection by the larvae or an attraction by the female carrier oyster.

In this study, I explore the hypothesis of the existence, in Os-
trea puelchana, of a differential larval settlement on the platform
through a process of active selection, a mechanism that can also
explain the carriage phenomenon. First, 1 present novel informa-
tion concerning the pattern and timing of carriage in wild and
fanned oysters, and the spatial arrangement of settlers on the plat-
form of wild oysters in one of the main oyster grounds. Second, I
compare larval settlement between oyster species, and between
live and dead Ostrea puelchana.

MATERIAL AND METHODS

Carriage in Oysters of Three Natural Grounds: Banco Reparo, Las
Grulas, and Bajo Oliveira

The oyster ground at Banco Reparo occupies an area of 30 km"^
on the NW of San Mati'as Gulf, Argentine Patagonia (40°40'S;
63°.^0'W; Fig. 2). The bottom is sandy and covered partially by
pebbles and mollusk shells. Water currents are strong, and depth
ranges from 2 to 3 m at low tides. Maximum oyster density in this
ground is 3.2 individuals per m" (Pascual 1993).

The oyster ground studied at Las Grutas is located in open
waters on the NW coast of the gulL 15 kin south of Banco Reparo

815

816

Pascual

Figure 1. Ostrea puekhana: female oyster carrying tliree dwarf males
on the anterior margin of its concave shell (shell platform).

Figure 2. San Mati'as Gulf: location of the main Puelche oyster
grounds. 1 = Banco Reparo; 2 = Las Grutas: 3 = Bajo Oliveira.

width). Nine hauls (towing time: 10 min; swept area per tow: 2,700
nr ) were performed over the high-density area of the ground (Pas-
cual 1993). One sack of oysters (approx. 40 kg) was randomly
chosen from the total catch of each haul. All the oysters were
measured (total height), sacrificed and the number and size of
dwarf males in the shell platform of each oyster were recorded.
Two of the nine samples randomly were chosen for a more detailed
sampling. Recruits (spat settled during that season) on the outer
shells of oysters were counted and measured under binocular mi-
croscope; the oysters were then opened and recruits and dwarf
males on the shell platfomi of oysters were counted and measured.

(40°48'S; 65°0.'i'W; Fig. 2). The bottom consists of coarse .sand
and shell, occasionally interrupted by limestone platforms. Tidal
currents are weaker than in Banco Reparo and depth ranges from
2.5 to 6 m at low tide. The oyster ground occupies an area of
approximately 2 km". Maximum densities (22.0 oysters per nr)
are found in sandy channels and depressions (Pascual 1993).

The oyster ground at Bajo Oliveira is the largest of the San
Mati'as Gulf oyster grounds. It is located at the NW of the gulf
(40°5rS; 65°05'W; Fig. 2). The bottom is predominantly sandy
and flat, covered by pebbles and mollusk shells. The oyster ground
occupies an area of SS km" and it lies in north-south direction with
depths ranging from 10 to 20 m (Pascual 1993). Maximum oyster
densities (0.8 individuals per m'), recorded in previous surveys,
arc found in the northwest portion of the ground.

Samples were collected on April 1987 from Banco Reparo and
on November 1987 Irom Las Cirutas. At each site, a lOO-m transect
was drawn across the central densely covered area of the ground.
All individuals found in a 2-m wide path along the transect were
collected by divers. Oysters were measured (height: maximum
distance in mm from the umbo to the opposite margin) and the
number and si/e of dwarf males settled on the shell plallorm of
each carrier oyster was recorded.

At Bajo Oliviera, a survey was performed on May 1 988 with a
research vessel equipped with a comiiKTcial ilrcdge (2..^ iii dredge

Carriage in a Stock of Farmed Oysters

The oysters used in this experiment were collected on artificial
collectors placed on the oyster ground of Las Grutas during the
1983 through 1984 settlement season. In April 1984, juveniles
(mean height = 26.7 mm: SD = 4.3: n = 775) were tranfeired for
growth-out (Pascual and Bocca 1988) to Banco Gar/as, a protected
site within San Antonio Bay (Fig. 2). The oysters were placed in
plastic mesh bags tied to off-bottom racks, anchored to the bottom.
Over a 50- mo period, the oysters were measured on a monthly or
bimonthly basis. At each sampling date, 30 individuals were ran-
domly collected. Size increments and spat recruitment on the in-
ternal shell platform were recorded.

Selecliiity (if iMrvul Seltlement

Kxperiment I : Settlement of Ostrea puelchana larvae on O.
piielchana and Ostrea edulis

This experiment was designed to evaluate the level of speci-
ficity of larval seltlement on the outer shells and in the shell
platform of the puelche oyster. Adult individuals o\ Ostrea cdiilis
(Luropean Hat oyster) and O. piu'klMiui were exposed to the re-
cruitment of a lot of pediveligers of O. puekhana. The experiment
was carried out in the hatcherv of Roncc-les-Bains (IFRFMER.

Dwarf Males in the Puelche Oyster

817

France) in May 19S9. The oysters of both species (n = 12) used
in the experiment had similar size (mean height O. piieUhana: 85.4
mm ± 4.6; mean height O. ediilis: 85.6 mm ± 4.8). Four sieves (40
X 15 cm) were placed in each of three raceways. Two oysters (one
Puelche oyster and one European oyster) were placed in each
sie\e. During the experiment the oysters were fed with an Isoch-
lysis galbiiiui diet and were maintained in an open water circuit.
The puelche oyster larvae were produced in the same laboratory.
The experiment begun when 50% of the larvae in the larval tanks
reached the pediveliger stage. A batch of 50.000 larvae was intro-
duced in each sieve. Oysters were sacrificed at day 4. when free
larvae were no longer observed swimming in the sieves. The spats
settled on the outer shells and on the shell platform of each oyster
were counted and measured using a compound microscope.

Experiment 2: Comparison of Larval Settlement on Dead and
Live Oysters

This experiment was carried out at Las Grutas oyster ground
during the 1990 through 1991 settlement season. Oysters from the
natural ground were collected, sorted to get a uniform size stock
(mean height = 76.5 mm; SD = 4.8; n = 186), cleaned from
epibionts with a wire brush, and allocated randomly into two
groups (treatments) of 93 individuals each. All oysters in one
group were sacrificed, and the empty clean shells were tied to-
gether with a plastic band. Each treatment was arranged in three
replicates of 31 oysters, each placed in a separate culture lantern.
Mesh size guarantied predator exclusion. The six lanterns were
suspended from a long line, alternating treatments. The structures
remained suspended at mid water during the entire settlement sea-
son (December 28, 1990 to April 22, 1991).

At the end of the season, the number and size of the recruits
settled on the outer shells and shell platform of each oyster (live or
dead) were recorded. Carriage was expressed as the proportion of
recruits settled on the platform in relation to the total number of
recruits settled per "substratum oyster." Data were arc-sine trans-
formed for analysis. The number of recruits settled on the shell
platform and outer shells in live and dead oysters was statistically
analyzed with a Nested Anova test (Sokal and Rohlf 1969).

RESULTS

Carriage in Oysters of Three Natural Grounds: Banco Reparo, Las
Grutas and Bajo Oliveira

Samples obtained in Banco Reparo and Las Grutas enabled us
to estimate the full size structure of the population. Figure 3 shows
the size frequency distribution of the whole population (free oys-
ters and dwarf males), in the two sites. These size distributions are
bimodal, one mode representing the dwarf males settled on the
shell platform of carrier oysters (mean = 11.7; SD = 6.9; n =
263. for Banco Reparo. and, mean = 8.2 mm, SD = 6.3; n = 418
for Las Grutas), and the second representing free oysters (mean =
74.1 mm; SD = 13,6; n = 597 for Banco Reparo and mean =
66.7 mm, SD = 18.7; n = 722 for Las Grutas).

Carriage of small males begins at a size of 55 mm at Banco
Reparo, and at 50 mm at Las Grutas. In both grounds, the per-
centage of carriage (number of carrier oysters/total number of
oysters larger that 50 mm x 100) in the population increases as the
size of the oyster increases (Fig. 4). Carrier oysters in both grounds
hold a maximum of six epibiotic males per carrier oyster, 61% of

Banco Reparo

o

z

LU

15
O
LU

a:

300

Las Grutas

Bajo Oliveira

5 20 35 50 65 80 95 110 125 140

SIZE (mm)

Figure 3. Size frequency distributions of the complete oyster popula-
tion, composed by free oysters (carrying or not carrying dwarf males),
and dwarf males fixed on the shell platform of carrier oysters. Graphs
represent the oyster populations of Banco Reparo (above). Las Grutas
(center), and Bajo Oliveira (below).

the carriers holding one epibiotic male, in Banco Reparo, and 68%
in Las Grutas.

The oyster population of Bajo Oliveira is composed of large
individuals (Fig. 3). The population of free oysters has a mean size
of 89 mm (SD = 13.4; n = 1550). The complete population,
including dwart' males, is clearly bimodal. The mode representing
dwarf males has a mean size of 9.1 mm (SD = 6.5; n = 532).

The percentage of carriage is very high in this ground, an
88.28% of the oysters larger than 50 mm carry dwart' males on the
platform. The oysters carry a maximal number of five dwarf males
per carrier oyster. Of the carrier oysters, 63% carry two or more
dwarf males. A detailed observation of new settlers (recruits of the

818

Pascual

80 100

TOTAL HEIGHT (mm)

Figure 4. Percentage of carriage (proportion of carrier oysters/total
number of oysters >S0 mm x 100) in relation to tlie oyster's size at the
oyster grounds of Banco Reparo (above) and Las Grutas (below).

season) showed that oysters had a very low recruitment on the
shells, while recruitment on the platform was very high. A maxi-
mum of 52 settlers fixed on the platform of carrier oysters was
recorded (Fig. 5).

Oysters settled on the platform were divided into dwarf males
(age >1 y) and newly settled spat (recruits of the last season).
Figure 6 shows the relationship among the number of spats newly
settled (recruits of the last season) on the shell platform and the
size of previously settled dwarf males (weighted by its number).
The number of new settlers decreases as the size of the previously
settled dwarf males increa.ses.

Carriage in a Stock of Farmed Oysters

Recruitment on the platform was delected when the experimen-
tal lot reached a mean size of 70.6 mm (SD = 7.9: n = 164) and
an age of 48 mo. Carrier oysters in the lot represented a 2."^% of the
total stock and were in a size range of 6.^ to 82 mm (mean height
= 73..^ mm; SD = 8: n = 38).

Selectivity of iMrval Settlement

ExpcrimunI I: Scdkmenl of Ostrea puelchana larvae on ().
puek'hana and Ostrea ediills

Mean settlement on the shell platform ot the puelche oysters
was. at the end of the experiment, 1 .66 spat per oyster (Table 1 ).
The F.uropean Hal oysters did not show settlement along the an-
terior margin.

Settlement on the shells was heavier in the European oysters
(Table I) even when non-significant differences were detected (I
= 1.42; P > .05). The size of recruits, settled on both species, did
not show ditferences among species.

O

z
O

LU

4 8 12 16 20 24 28 32 36 42 46 50

Number of new settlers fixed on the shells per oyster

12 16 20 24 28 32 36 40 44 48 52

Number of new settlers fixed on the platform per oyster

Figure 5. Settlement of spat on living oysters during the 1988 settle-
ment season at the oyster ground of Bajo Olivelra. Graphs show the
number of spat newly settled on the outer shells per oyster (above), and
the number of spat newly settled on the shell platform per oyster
(below).

Experiment 2: Comparison of Larval Settlement on Dead and
Living Oysters

Larvae did settle on the outer shells and shell platform of oys-
ters assigned to both treatments and replicates (Table 2). However,
settlement on the shells of dead oysters was significantly lower
than on the shells of living oysters (Nested Anova: F = 59.85; P

Number x mean size of dwarf males (mm)

Figure 6. Pattern of settlement of .spat on the shell platform of oysters
of the ground at Bajo Oliveira. Relationship among (he number of
newly settled spat (recruits of the last season) and the size of dwarf
males previously settled (weighted by its number), per ojster.

Dwarf Males in the Puelche Oyster

819

TABLE 1.

Results of a experiment designed to evaluate the level of specificity

of larval settlement on the outer shells and the shell platform of the

puelche oyster.

O. puelchana

O. ediilis

No. of spat on outer shells per oyster

Mean ± SD

268.5+ 135

347.5 ± 1 36.4

n

12

12

No. of spat on shell platform per oyster

Mean ± SD

!.7± 1.1

0

n

12

12

Size of spat

Mean height (in mm) + SD

2.5+ 1.4

2.8 ± 1.3

n

324

359

Adult individuals of O. puelchana and O. edtilis were exposed to the
recruitment of a batch of pediveligers of Ostrea piieUluiiui.
SD = standard deviation.

< .005). In addition, the number of recruits settled on the platform
was significantly higher in live oysters (Nested Anova: F = 32.16;
P < .005).

The recruits which settled on living oy.sters reached a greater
mean size (8.7 mm; SD = 3.9; n = 303) than those settled on
dead oysters (6.6 mm; SD = 3.7; n = 176) (t = 1 1.22; P <.005).

DISCUSSION

The probability of carrying dwarf males on the shell platform
increases as the size of the female oyster increases. From the time
the oyster reaches a size of 50-55 mm. the size at which carriage
begins, the shell platform gradually widens. The epibiotic male's
own growth additionally produces a hollowing in the platform,
which (once the small male detaches) becomes more favorable for
the settlement of new recruits (Pascual 1993). This correlation
between the development of the shell platform and the oyster's
size is reflected in the number of dwarf males carried per oyster:
in Banco Reparo and Las Grutas. 61% and 68%, respectively, of
the carrier oysters carry 1 dwarf male, while in Bajo Oliveira.
where the mean population size is higher, 63% of the oysters carry
two or more dwarf males.

The pattern of recruitment on the shell platform analyzed in
oysters of Bajo Oliveira. suggests that settlement is constrained by

TABLE 2.

Results of a experiment carried to evaluate larval recruitment on
living and dead oysters.

No. spats on

No. spat on shell

outer shells

platform

Treatment

Lantern

n

(mean ± SD)

(mean ± SD)

Living oysters

1

28

3.10 ±2.43

2.44 ± 1.69

2

28

3.60 + 2.96

1.88 ± 1.70

3

31

4.06 ±2. 15

2.I2± 1.78

Dead oysters

1

29

1.72 ±1.55

0.64+ 1.00

2

29

2.I0± 1.89

0.82 ±0.64

3

29

2.62 ± 2.05

0.62 ± 0.55

Values are mean numbers of recruits settled on the outer shells or shell

platform.

SD = standard deviation.

the previous occupation of the platform. The settlement and/or
survival of new recruits are inversely related to the presence and
size of previous recruits.

Post-settlement mortality is very high during the 3 mo follow-
ing recruitment and similar among grounds (Pascual 1997). As it
was demonstrated previously, grazing (or bulldozing) by epibiotic
chitons is an important mortality .source for oyster recruits that
settle on oysters (Pascual 1997). The low recruitment on shells at
Bajo Oliveira, together with the intense settlement on the platform,
suggests that those differential mortality agents indeed operated on
recruits.

In natural grounds, carriage is a character that is expressed
unambiguously once the oyster exceeds 50 mm in size, strongly
matching the size of sex reversal to the female phase in normal
conditions (Calvo and Morriconi 1979). The debut of "carriage" in
farmed oysters took place at a mean size 1 5 mm higher than that
of wild oysters of the .same locality, suggesting that the attraction
effect could be related to age rather than to size of the oyster.

The results obtained from experimentally comparing recruit-
ment on dead and living oysters confirm those reached while
evaluating the recruitment in natural grounds (Pascual and Zam-
patti 1995) and indicate that settlement on the platform is a re-
sponse to an active attraction effect produced by the live oyster and
not merely a result of a random recruitment process.

The specificity of the carriage phenomenon was proved by
experimentally evaluating the inability of the European oyster, O.
ediilis. for "carrying" epibiotic oysters on the platform. The speci-
ficity of the carrying phenomenon is supported by the fact that
recruits of the congeneric O. spreta, which co-occurs with O.
puelchana in Patagonian waters, were never observed settled on
the platform of puelche oysters or "carrying" epibiotic oysters
themselves.

The high density of spat settled on the outer shells of both
Puelche and European oysters could be explained by the large
number of larvae offered in the experiment. In contrast, the rela-
tively low number of spat .settled on the shell platform of puelche
oysters may be related to the reproductive stage of the oysters used
in the experiment, since there is some evidence suggesting that the
attraction effect exerted on the larvae ready to settle is weaker in
already spawned females (Pa.scual, unpublished). However, this
topic is currently under investigation.

Calvo and Morriconi (1978) suggested the existence of a
chemical attractant triggering a settlement response of the larvae
that settle on the shell platform of adult oysters. The match be-
tween the initiation of the female phase (.sex reversal) and "car-
riage" (Morriconi and Calvo 1978) suggests a relationship between
this phenomenon and the reproductive cycle. A chemical linked to
gonadal maturation may trigger the differential settlement of lar-
vae on the platform. Alternatively, increased carriage could be the
result of the development of the platform, which enlarges with the
age of the female oyster, as well as of the hollowing of the plat-
form by the growth of dwarf males. The big platform of old oysters
from Bajo Oliveira, which carried dwarf males of maximal sizes,
is consistent with this scenario.

The development and adaptive significance of the carriage of
dwarf males by females of the puelche oyster can be conceptual-
ized from three viewpoints.

Functional. Fertilization occurs in two alternative ways in this
species (via free males or dwarf males). The functionality of
carriage is based on two features of this mechanism: the archi-
tecture of the relation female carrier-carried male, and the syn-

820

Pascual

chronization in gonadal maturation in both partners. The result
is an increase in fitness for both sexes (Calvo and Morriconi
1978, Pascual et al. 1989),

Developmental. Male dwarfness is a result of retarded growth rate
of males by the interaction with a female carrier (Pascual et al.
1989). As a result, epibiotic males that remain attached to the
platform have a long lifespan, with lowered predation risks and
less sperm loss.

Topological. The relatively high concentration of individuals on
the platform of oysters >50 mm, as compared to the surface of
the shells can be explained by two mechanisms: selective mor-
tality of recruits and active selection of larvae.

The non-random distribution of small males is, at least in
part, a consequence of the higher survival rate of settlers on the
shell platform of adult females, where they are sheltered from
the foraging activities of epibiotic chitons (Pascual 1997). This
ecological contingency may have provided a selection pressure
favoring development of selective settlement behavior.

Larval attraction by adults is a process already described for
O. piielchano (Pascual and Zampatti 1995) and other oyster

species (Crisp 1967, Veitch and Hidu 1971, Bonaret al. 1990).
Active selection of the platform by the larvae could be regarded
as a special case of the general process of attraction that adults
exert over larvae ready to settle. However, the fact that carriage
occurs only when the oyster exceeds 50 mm in size, while
attraction to the larvae ready to settle is independent of the
oyster's size (or age), suggests the existence of two different
mechanisms.

Summing up, the carriage phenomenon may be explained by
two simultaneous processes; differential survival of the recruits
settled on the platform, which provides a refuge against graz-
ing, and the differential larval settlement through a process of
active selection.

ACKNOWLEDGMENTS

I thank M. A. Pascual, L. Orensanz, and two anonymous re-
viewers for critical readings and helpful suggestions on the manu-
script, and to Henri Grizel for advise and facilities at LPGIM
(IFREMER). This work was financially supported by the Interna-
tional Foundation for Science (Grant No. Ay0704).

LITERATURE CITED

Bonar, D, B., S. L. Coon, M. Walch, R. M. Weiner & W. K. Fitt. 1990.

Control of oyster settlement and metamorphosis by endogenous and

exogenous chemical cues. Bull. Mar. Sci. 46: 484^98.
Calvo, J. & E. R. Morriconi. 1978. Epihiontie et protandrie chez O.^trea

puelchaiw. Haliotis 9: 85-88.
Crisp. D. J. 1967. Chemical factors inducing settlement in Cra.sso.ttrea

virginica Gmelin. J. Animal Ecol. 36: 329-336.
Morriconi, E. R. & J. Calvo. 1979. Cicio reproductivo y aiternancia de

sexos en Ostrea puelchana. Physis 38: 1-17.
Pascual, M. S. & A. Bocca. 1988. Cultivo experimental de la ostra puelche,

Ostrea puelchana D'Orb., en el Golfo San Mati'as. Argentina, pp. 329-

345. In: J. Verreth. M. Carrillo. S. Zanuy & E. A. Huisman (eds.).

Aquaculture Research in Latin America. Pudoc Wageningen, The

Nethedands.
Pascual. M. S,, O. Iribarne, E. ZampaUi & A. Bocca. 1989. Female-male

interaction in the breeding system of Osvea piielcluma. J. E.xp. Mar.
Biol. Ecol. 132: 209-219.

Pascual, M. S. 1993. Contingencia y adaptacion en la ecologia reproductiva
de Ostrea puelchana. Doctoral dissertation. Mar del Plata Natl. Univ.,
Argentina. 183 pp.

Pascual, M. S. 1997. Carriage of dwart' males by female puelche oysters:
the role of chitons. / E.xp Mar. Biol. Ecol. 212: 173-185.

Pascual. M.S. & E. Zampatti. 1995. Chemically mediated adult-larval in-
teraction triggers settlement in Ostrea puelchana: applications in hatch-
ery production. Aquaculture 133: 33^4.

Sokal, R. R. & J. Rohlf 1969. Biometry. The prmciples and practice of
statistics in biological research. Freeman & Co. Ed. 776 pp.

Veitch, F. P. & H. Hidu. 1971. Gregarious setting in the American oyster
Crassnstrea virginica Gmelin. 1 . Properties of a partially purified "set-
ting factor." Chesapeake Sci. 12:173-178.

Journal of Shellfish Rcscurch. Vol. 19, No. 2, 821-826, 2000.

COLLECTION OF PEARL OYSTER (FAMILY PTERIIDAE) SPAT AT ORPHEUS ISLAND,

GREAT BARRIER REEF (AUSTRALIA)

ANDREW C. BEER' - AND PAUL C. SOUTHGATE'

' Aqiuicidture, School of Marine Biology and Aquaculture
James Cook University
Queensland 481 1 Australia
'Fisheries WA
P.O. Box 774, Carnarvon
Western Australia 6701 Australia

ABSTR.ACT The aim of this study was to identify ihe pearl oyster species recruiting to spat collectors (50% shade mesh in
polyethylene mesh bags) and to examine temporal and depth differences in recruitment at Pioneer Bay Orpheus Island, Great Barrier
Reef, Australia. Over 24,900 bivalve spat were counted during the 12 months of study (March 1995 to March 1996). In excess of
21,600 pearl oyster spat were collected from three genera of the family Pteriidae: Pinctada. Pleria. and Electroma. The majority of
this total (20,378) was the non-commercial species Eleclroma papilliomicea (Lamarck 1819). However, collection of eight species of
Pinchula (2,628 spat) and five species of Pleria (approximately 1,200 spat) included species presently used in commercial pearl culture
operations throughout the Indo Pacific, ntitably Pinciada niargarilifera. Piiichula fiicata. Pinctada alhina. Pinctada maxima, and Pteria
penguin. All species collected showed seasonal variation in recruitment intensity. Some species showed brief and temporally isolated
types of spawning and associated recruitment pulse of temperate species, whereas the majority displayed peaks in recruitment intensity.
A total of 23 1 P. niargarilifera spat were collected during the study, with the recruitment peak ( mean density 7. 1 ± 1 . 1 spat per collector
unit) recorded between May and June 1995. No significant difference in recruitment densities between 2 m and 6 m were recorded for
P. margaritifera. Pt. penguin settled at the highest densities at 6 m in March/April 1995 (mean recruitment per collector ± SE 46.2 ±
6.7). A total of 1,000 Pt. penguin spat were collected during the study, and a significant proportion of these settled on the outer mesh
bag of the spat collector.

KEY WORDS: Pearl oyster, spat collector, Pteriidae. Pinctada. Pleria, Electroma

INTRODUCTION

Pearl oysters from the family Pteriidae are exploited in various
cultured pearl production ventures throughout the world. The four
major species used for cultured pearl production are Pinciada
maxima (Australasia), Pinctada fiicata (Japan, China, India, and
Sri Lanka), Pinctada margaritifera (French Polynesia, Cook Is-
lands, and Australia), and Pteria penguin (Japan. Thailand. Tonga,
and Australia).

Although hatchery methods for the production of pearl oysters
have been developed over recent years (Gervis and Sims 1992,
Southgate and Beer 1997), collection of spat from the wild still has
an important role in pearl culture operations. As a relatively low
cost and low technology method, wild spat collection may provide
spat for developing fanning operations (Friedman and Bell 1996),
for established pearl culture industries (Coeroli et al. 1984, Sims
1992), and for stock assessment or to estimate species diversity
(Knuckey 1993). Spat collection also provides a method of col-
lecting small fragile and cryptic species that are otherwise unob-
tainable. Reseeding and stock enhancement programmes (Hortle
and Cropp 1987, Robins-Troeger and Dredge, 1993, Saudeco et al.
1994) also may be based on spat collection.

Spat collection can be a very effective way to accumulate pearl
oyster stock (Crossland 1957); however, efficient spat collection
requires information on seasonal patterns of spat recnjitment. Re-
cruitment variability, or failure to collect spat, has been identified
as a risk that could have high economic costs for pearl culture
industries reliant on wild spat collection (Rose and Baker 1994);
however, assessment of temporal and spatial trends in spatfall may
reduce these risks. To maximize collection of bivalve spat, local-
ized differences in spawning activity (Tranter 1958a, Tranter
1958b. Tranter 1958c, Tranter 1958d. Tranter 1958e. Rose et al.

1990). substrate material preferences (Scoones 1990, Friedman
and Bell 1996). the effects of spat collector position in the water
column (Robins-Troeger and Dredge 1993). and location (Fried-
man and Bell 1999) must be recognized and exploited.

Spawning in bivalves is often associated with temperature ex-
tremes or other environmental changes (Quayle and Newkirk
1989). Species from temperate regions generally exhibit more dis-
crete and regular spawning seasons and generalizations applied to
bivalve reproductive cycles from temperate regions are not always
applicable to tropical species (Gervis and Sims 1992). Reproduc-
tive seasonality in pearl oysters was described by Tranter (1958c)
as "relative breeding intensities" with "major breeding season(s)."
In many species of pearl oysters, spawning and subsequent spatfall
may occur more than once in the year (Tranter 1958a, Tranter
1958b, Tranter 1958c, Tranter 1958d. Tranter 1958e, Wada et al.
1995, Behazi et al. 1997).

There are few published data on the collection of pearl oyster
spat in Australia. The primary objectives of this study were: (1) to
identify the species of pearl oysters that recruited onto spat col-
lectors in Pioneer Bay. Orpheus Island; (2) to determine the sea-
sonal trends in recruitment and; (3) to determine the effect of spat
collector depth on the density of recruitment.

MATERIALS AND METHODS

Location of Study Site

Orpheus Island (18°35'S. 146''29'E) is within the Palm Island
group of high continental islands approximately 80 km north of
Townsville and approximately 20 km offshore (Fig. 1). Pioneer
Bay is on the leeward (western) side of Orpheus Island (Fig. 1).
The surface longline used in this study was located at the northern

821

822

Beer and Southgate

A

i Pelorus Island

Pioneer Bay i ' ^ i.

*\ r Orpheus
\'^ ' Island

/'

Fanlome
Island

Curacoa
Island

HffSO-

Great Palm
Island

0^.

,-'>5

^

\ 18-50'

Figure 1. Location of Orpiieus Island spat collection site within the
Palm Islands, 20 l<ni offshore and 80 lim from Townsville, north
Queensland. The surface longline (A) is 50 m from the reef flat in
10-13 m deep water and adjacent to James Cook University's Orpheus
Island Research Station (•).

end of Pioneer Bay. approximately 50 m from the reef flat with a
depth under the longline of 10-13 m. This study began in March
1995 and concluded in March 1996.

Collection of Spat

Ten spat collectors were deployed on the longline. five at each
depth of 2 m and 6 m. Collectors were attached to a 7-m length of
rope, weighted at the end. Each spat collector consisted of two 40
X 70 cm mesh bags (polyethylene mesh 1.5 mm fiber size, 8x5
mm mesh size) with three 0.5 nr sheets of 5()9( shade mesh
(0.5-mm fiber size. 2 x 1-mm mesh size). The collection material
(shade cloth and mesh bag combination) was chosen because of
availability, low cost, durability, rc-useability. and successful use
of these materials in hatchery trials with pearl oysters {Southgate
and Beer 1997).

Collectors were changed every 4 weeks, at which time they
were washed with high-pressure seawater to remove sediment and
loose spat. Both shade mesh and mesh bags were inspected manu-
ally and spat removed with a scalpel. The water used to wash the
substrates was sieved through .^-min and 1 .7-mm mesh screens.
Spat caught on the 1. 7-mm screen were preserved in 10% buffered
formalin for later counting and identification using a stereo-
dissector microscope. Spat were identified using keys (Hynd 1955.
Takemura & Okulani 1955. Takemura &. Okulaiii 1958). informa-
tion from previous studies (Shirai 1994, Lamprell and Healy
1998). Validation of species identification was obtained from ju-
veniles grown out from collected spat (unpublished data).

Analysis

Recruitment ilata lor each species were analyzed with unisari-
alc. Iwo-lactor ANOVA examining the factors of depth and lime.

Monthly recruitment data required log,,, (x-i-1) transformation and
was examined with Cochran's test for homogeneity of variance
(Zar 1996). Recruitment data for visible and sieved portions were
pooled. From the family Pteriidae, the following taxa were
counted: Pinctada imirgaritifera. P. fiicata. P. albina, Pleria pen-
guin, and Electroma papillionacea.

Temperature

Temperature was recorded at 2 and 6 m throughout the study
and the maximum, minimum, and mean seawater temperature re-
corded in Pioneer Bay. Figure 2 shows the changes in sea water
temperature with a mean summer maximum of 28.5 °C (March
1995) and a rapid decline to the winter mean minimum of 21.7 °C
(August 1995) returning to 29.4 °C in summer (February 1996).

RESULTS

Species Present at Pioneer Bay. Orpheus Island

A total of 20 species of pearl oysters from three genera
(Pinctada. Pteria. and Electroma) were recorded in this study.
Table I presents a summary of the recorded recruitment peaks for
the major species during this study. Eight species of Pinctada were
recorded from a total of 3,972 spat. P. inargaritifera (Linnaeus
1758), P. fiicata (Gould 1850), and P. albina (Reeve 1857) ac-
counted for 27% of the collected spat: however. P. macidata
(Gould 1850). P. maxima (Jameson 1901). P. cliemnitzii (Phillipi
1849). and P. nigra (Gould 1850) also formed 4% of the spat
collected. Unidentified Pinctada spat accounted for 69% of the
total. Eight species of Pteria were recorded during the study. Pt.
penguin (Roding 1798) was the most common ( 1.000 spat. 83.3%
of genus total), however. Pi. cypesyllns (Dunker 1872). Pi. falcata
(Lamarck 1819), Pi. <()/((wa- (Dunker 1872). Pi. lata (Gray 1845),
Pt. bernhardiareddte 1939). Pt. cooki (Lamprell and Healy 1997),
and Pt. chinen.sis (Leach 1814) were also collected. The total col-
lection for the genus exceeded 1.200 spat.

Electroma is a widespread, common, but little known genus
within the Pteriidae. At Orpheus Island, four species were present:
£. alacorvi (Diliwyn 1817), E. malleii.s (Reeve 1857). E. zebra
(Reeve 1857). and E. i>apillionacea (Lamarck 1819), The former
three species were present in very low numbers contrasting with E.
papillionacea.

Recruitment

Total Pinctada Recruitment

Between March 1995 and March 1996. the mean (± SE) num-
ber of Pinctada sp. spat per collector ranged from 1 .3 + 0.4 in

32 T

30

28

26

24

22

20

t'

1 1 1 1 1 1 1 1 1 1 1 1 1 1

JF FM MA AM MJ JJ JA AS SO ON ND DJ JF FM
Month
Figure 2. Mean, maximum, and minimum seawater temperature re-
corded in Pioneer Bay between March I995 and March 19M6.

Collection of Pearl Oyster Spat

823

TABLE I.
Summary of pearl oyster spat collection at Orpheus Island between March 1995 and March 1996.

Peak

Depth Effect

Maximum Mean

Total Number (and %

Total Collected

Settlement

and

(± SE) Settlement

of total) Collected at

March 1995-

Species

Time

'Preference'

per Collector

Peak Settlement

March 1996

Pinaada maigariiifera

May-June

No

7.1 ± LI

71 (30.7%)

231

P. fucata

Feb-March

-

-

324(39.5%)

820

P. alhina

Jan-Feb

-

-

132(45.0%)

293

Pinaada sp.

Feb-March

-

-

1570(59.7%)

2628

Pleria penguin

March- April

Yes 6 m

46 ± 6.7

462 (46.2%)

1.000

Elearoma papilUonacea

Oct-Nov

Yes 6 m

335.7 ± 38.4

3357(16.5%)

20.378

September/October 1995 to 194.5 ± 10.3 in February /March 1996.
Figure 3 shows the monthly combined totals for the major
Pinctada taxa. P. fucata was the most common commercial
Pinctada species recruiting to collectors with a total of 820 spat
collected between November 1995 and March 1996. P. alhina was
present in all months except May-June and September-October.
Although there was recruitment throughout the year, the species
composition recorded each month varied considerably.

A high proportion (69%) of the overall collection totals were
species of Pinctada that were unable to be positively identified
when collectors were examined after 4 weeks deployment. A total
of 2.628 unidentified Pinctada spat recruited to collectors and
were pre.sent in all months with the greatest mean (± SE) recruit-
ment in February/March 1996 of 157 ± 9.7 per collector.

Pinctada margaritifera

P. margaritifera showed distinctly bimodal recruitment with a
significant difference in densities through time (P < 0.001) (Fig.
4). A total of 231 P. margaritifera spat were collected with the
maximum in May/June (7.1 ±1.1 per collector unit). Depth did not
have a significant effect on recruitment density, primarily due to
the low recruitment density and variability in most months. How-
ever, in May-June when recruitment was highest and spat were
recorded in all collectors, mean recruitment at 2 m and 6 m was 9.8
± 0.5 and 4.4 ± 0.7 spat per collector, respectively.

Pteria penguin

Recruitment of Pt. penguin showed clear seasonal and spatial
trends. Mean recruitment per collector was highest between Mar-

ch-April (46.2 ± 6.7) and April/May (31.8 ± 6.3) (Fig. 5). coin-
ciding with a decline in seawater temperature. A total of 1.000 spat
were collected with 78% recruiting to collectors between March
and May. Recruitment was significantly higher at 6 m (P < 0.001)
with a total of 702 at an overall mean yield of 1 1.7 ± 2.7 spat per
collector. At 2 m. a total of 298 spat were collected at 4.96 ±1.1
per collector. Many Pt. penguin spat were recorded on the outer
surface of collectors during months of high recruitment. Two-
thirds of the spat that settled at 6 m were attached to the outer mesh
bag.

Electroma papilUonacea

Throughout the study, E. papilUonacea was the most abundant
species with a total of 20,378 spat collected. There was a signifi-
cant difference between the density of recruitment at 2 m and 6 m
{P < 0.001 ) with 9.243 spal recorded at 2 m and 1 1.135 at 6 m.
Recruitment between months was significantly different (P <
0.001 ) with peak recruitment in October/November (335.7 ± 38.4
per collector) (Fig. 6) and a maximum density in one collector of
843 spat. This was the highest number of spat per collector of any
bivalve species during the study.

a

E

i

a

Figure 3. Total monthly recruitment of Pinctada sp., P. fucata, P. al-
bino, and P. margaritifera to spat collectors in Pioneer Bay, Orpheus
Island, between March 1995 and March 1996.

FM MA AM MJ JJ JA AS SO ON ND DJ JF FM

Month
Figure 4. Mean P. margaritifera spat recruitment per collector (± SE),
between March 1995 and March 1996.

824

Beer and Southgate

o 30-

FM MA AM MJ JJ JA AS SO ON ND DJ JF FM
Month

Figure 5. Mean Pt. penguin spat recruitment per collector (± SE),
between March 1995 and March 1996.

DISCUSSION

This study recorded a relatively high species diversity of Pteri-
ids in Pioneer Bay, Orpheus Island. Eight of the nine species of
Pinctada recorded in Australian waters (Lamprell and Healy 1998)
were collected in this study. Similarly, 13 species within the genus
Pterin occur in mainland Australia, of which 12 are tropical and 8

E 150

ffl

rfl

AS SO
Month

Figure 6. Mean /;. papillinnacea spat recruitment per collector (± .SK),
between March 1995 and March 199h.

were collected during this study. All five Elearoma species re-
corded from north Queensland are present at Orpheus Island. Spe-
cies diversity was considerably higher in this study than in similar
studies in northern Australia. For example, spat collection in the
Northern Territory recorded nine species of Pteriids (four
Pinctada, three Pteria, and two Electroma) (Knuckey 1995), and
a similar study on the Great Barrier Reef (Dayton et al. 1989)
recorded seven species of Pteriids (seven Pinctada and one Pte-
ria). Pearl oyster species diversity recorded from spat collection
from locations such as the Solomon Islands (Friedman and Bell
1996) and the Cook Islands (R. Braley, personal communication)
is considerably lower than that recorded in this study.

Patterns of spat recruitment over the course of this study chal-
lenge conventional wisdom relating to the recruitment of bivalves
in the tropics. Recruitment peaks and clearly defined seasons dem-
onstrated by some species (e.g., Pt. penguin) contrast with the idea
of relatively continuous spawning throughout the year for tropical
bivalves (Gervis and Sims 1992). Seasonal variation in seawater
temperature is sufficient to suggest that there are significant dif-
ferences between summer and winter and wet and dry sea.sons.
Therefore, it would be reasonable to assume that discrete and
regular spawning seasons should exist for .some taxa. However,
this study also showed recruitment throughout the year for species
such as E. papillionacea and P. margaiitifera. with seasonal
peaks, supporting the observation of "relative breeding intensities"
(Tranter 1958c).

The bimodal spat recruitment of P inargariiifera reported in
this study corresponds with the earlier work in Australia by Tranter
{ 1958d). who reported spawning activity in two distinct cycles; in
the spring-summer (September to February) and autumn-winter
(March to August). Highest recruitment of P. inaigaritijera in
Pioneer Bay during May to July corresponds with the secondary
spawning season in the winter reported by Tranter ( 1958d). During
this study, winter recruitment provided the most P. niargaritifera
spat per collectors; however, more spat could be collected over the
summer months as the recruitment period is sustained. Subsequent
observations at Orpheus Island showed that winter spatfall is more
intense than in summer, but of shorter duration (8-10 weeks).

Spat collection of P. inargaritifera has been the main .source of
oysters for culture operations in the Red Sea (Crossland 1957),
French Polynesia (Coeroli et al. 1984). and islands in the south
Pacific (Gervis and Sims 1992). In French Polynesia ( 14 °S), Co-
eroli et al. (I9S4) reported year-round spal collection with peak
recruitment between August and October coinciding with the win-
ter low in sea water temperature (26 to 27 °C). Bimodal recruit-
ment with spring maximum and lower summer recruitment re-
corded in the Solomon Islands (Friedman et al. 1998. Friedman
and Bell 1999). is alTected by seasonal changes lo water tempera-
lure and the monsoonal wet/dry season cycle. P.wargaritifcia spat
collection in Donogab Bay on the Sudanese coast of the Red Sea
(21°N) was an outstanding success with annual collection of 4.5
million spal in 1921. However, P. inargariiifera spawning in the
Red Sea was rcslriclcd to an annual summer event \\ ith "no trace
of gonad in the aulumn or winter" (Crossland 19571. The study by
Crossland (1957) showed ihal spal colleclion can he extremely
cffeclive as a seed colleclion method, provided accurate seasonal
pallerns of reproductive acli\ ily are known.

RecruilmenI of other species of Pinctada with commercial
value (/-". alhiiia and P. fiicuta) followed the .sea.sonal trends found
in Torres Strait, Northern Australia (Tranter 1958b, Tranter 1958c,
Tranler I958e). The total number of P. alliina spal collected in

Collection of Pearl Oyster Spat

825

Pioneer Bay was low (293) and varied from zero in May-June to
a maximum in January-February of 132, Although P. alhiiui re-
cruited throughout the year, gonad staging and spat collection in
the Torres Strait area (10°S) of Australia, demonstrated "relative
breeding seasons" where the heaviest spatfall occurred between
June and August during the annual water temperature minima
(Tranter 1958b).

Previous studies on reproduction in P. fticata in Australia
(Tranter 1958e. Sumpton et al. 1990). reported two spawning pe-
riods within the main breeding season of December to May. The
peaks in reproductive activity were termed the "summer spawn-
ing" in January-February and "autumn spawning" in April-May;
the latter was the more consistent of the two. Similar bimodal
reproductive activity has been reported for P. fiicata in the Persian
Gulf (Behzadi et al. 1997). However, spatfall of P. fiicaki has been
recorded year round in Northern Australia with highest spatfall
occurring between January and March (Tranter 1958e). Early sum-
mer recruitment has been recorded for P. fiicata populations in
subtropical Australia (Sumpton (Vn/. 1990) with up to 142 spat per
collector. This is similar to the pattern of P. fiiciiki recruitment
observed in this .study and these data indicate a broad spawning
season from November to March for P. fucata in north Queen-
sland. The reproductive seasonality of P. fucata varies with lati-
tude and populations from different genetic stocks (Wada 1995.
Behzadi et al. 1997). Although P. fucata provides approximately
1Q% of world cultured pearl production (Shokita et al. 1991), spat
collection and culture of P. fiicata is not commercially exploited in
Australia at present.

The number of P. ma.xima spat collected during this study was
very low. This contrasts the heavy P. maxima spatfall reported at
Pandora Reef (Fig. 1) by Dayton et al. (1989). This is surprising
given the proximity of Pandora Reef to Orpheus Island (12 nau-
tical miles); however, there are differences in turbidity between
these two locations and the inshore habitat at Pandora Reef may be
more suitable to P. maxima (Yukihira et al. 1999). Four years of
subsequent observations showed continued low recruitment of P.
ma.xima spat in Pioneer Bay (A. Beer, unpublished data 1999).

Differential recruitment at 2 m and 6 m was more clearly
shown by Pt. penguin than any other species collected. Further
research has found this spatial pattern consistent over several spat
collection seasons, the majority of spat collected at 6 to 8 m (A.
Beer, unpublished data 1999). Numbers of Pt. penguin spat were
greater on the outside of the collectors, behavior previously re-
corded for Pt. penguin (Gervis and Sims 1992). Pt. penguin at
Orpheus Island generally settle and grow on longline ropes and

moorings at depths in excess of 4 m and the natural host for this
species is the "Black Coral" (Order Antipalharia). a gorgonian
common in high current, 10- to 20-m deep water.

Summer-autumn spawning peaks have been reported for Pt.
penguin in Tonga (Malimali 1995. Tanaka 1997) with heavy settle-
ment between March and May 1995, and further research at Or-
pheus Island has provided similar results. The clearly defined
spawning season of Pt. penguin at Orpheus Island contrast with
Pteria sterna in Mexico which spawns throughout the year
(Ramirez et al. 1992); however, the depth trends vary with season.
Pt. sterna settled at highest densities at relatively shallow depths (4
to 7 m) in winter (Caceres-Martinez et al. 1992) and deeper (1 1-
15 m) in summer (Caceres-Martinez et al. 1992, Monteforte et al.
1995).

E. papillionacea was the most abundant of all the bivalves
collected and may present a considerable negative factor for col-
lection of commercial species of pearl oysters. E. papillionacea is
very small (<16 mm) with a life span of three to five months (A.
Beer, unpublished data 1999). It is not a commercial species and is
likely to have considerable economic importance as a significant
fouling species on spat collectors.

In summary, 20 species from three genera (Pinctada, Pteria.
and Electroma) recruited onto spat collectors during this study:
eight species of Pinctada [P. margaritifera, P. fucata. P. albina. P.
maculata. P. mcLxima. P. cliemnitzii. and P. nigra); eight species of
Pteria (Pt. penguin, Pt. cypesyllus . Pt. falcata, Pt. cotumix, Pt.
lata. Pt. bernhardi. Pt. Cooki. and Pt. chinensis); and four species
of Electroma (£. alacor\'i. E. malleus. E. zebra, and E. papilliona-
cea). This study also demonstrated that spat collection in Pioneer
Bay. Orpheus Island, has the potential to supply significant num-
bers (4,000 Pinctada sp. and 1,200 Pteria sp.) of juvenile pearl
oysters for pearl oyster culture operations.

ACKNOWLEDGMENTS

This project was funded by the School of Biological Sciences,
James Cook University with assistance from the Australian Centre
for International Agricultural Research (ACIAR), (Project No.
9131 ). Many thanks to Professor John Lucas for his assistance and
a number of people who assisted with the field work. The staff at
Orpheus Island Research Station provided logistic and practical
support. Sincere thanks are extended to Mr Kevin Lamprell of the
Queensland Museum. Dr Richard Willan from the Northern Ter-
ritory Museum, Darwin and Dr Francisco Borrero from INVE-
MAR. Columbia for assisting with species identification.

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Journal of Shellfish Ri'scunh. Vol. 19, No. 2, 827-834. 2000.

IN VITRO KILLING OF PERKINSUS MARINUS BY HEMOCYTES OF OYSTERS

CRASSOSTREA VIRGINICA

ASWAN! K. VOLETY'* AND WILLIAM S. FISHER^

College of Arts and Sciences
Florida Gulf Coast University
10501 FGCU Boulevard
Fort Myers. Florida 33965-6565
~U.S. Environmental Protection Agency
Office of Research and Development

National Health and Ecological Effects Research Laboratory
Gulf Ecology Division
One Sabine Island Drive
Gidf Breeze. Florida 32561-5299

ABSTRACT A colorimetric mircobicidal assay was adapted, optimized, and applied in experiments to ciiaracterize the in vitro
capacity of eastern oyster (Crassoslrea virginica) hemocytes to kill cultured isolates of Pcrkinsiis manmis. a protozoan parasite causing
a highly destructive disease of oysters in U.S. Atlantic and Gulf of Mexico coastal waters. //; vitro challenges showed that hemocytes
from two geographically distinct oyster stocks (Florida and Rhode Island) were able to reduce viable P. marimts cells by 2i% to 90%.
depending on the P. marinus isolate. Variability in killing was most likely due to differences in susceptibility among the 7 cultured
isolates of P. nuuimis. which ranged in ongin from Long Island Sound (Connecticut) to Laguna Madre (Texas). Hemocytes from
oysters collected monthly from Escambia Bay, Florida, exhibited a killing capacity that ranged from 21% to 90% (average 57%)
throughout a year-long period, with highest killing measured from October to December. Application of this technique demonstrated
the in vitro capacity of hemocytes to kill cultured P. marinus. but does not necessarily reflect their ability under natural conditions
where the disease is widespread.

KEY WORDS: Perkinsus marinus. eastern oysters. Cras.sostrea virginica. bivalve defenses, cellular defense, invertebrate immunol-
ogy, bactericidal activity

INTRODUCTION

For nearly 50 years, eastern oyster (Crassoslrea virginica)
populations along the U.S. Atlantic coast and Gulf of Mexico have
been infected by a highly virulent and transmissible pathogen Per-
kinsus marinus (Mackin et al. 1950. Andrews and Hewatt 1957,
Quick and Mackin 1971, Andrews 1988. Burreson et al. 1994).
Recently the disease has spread into the northeastern U.S. (Ford
1996), a region previously considered too cold to support P. mari-
nus. Prevalence and intensity of P. marinus throughout its geo-
graphical range are highly influenced by temperature and salinity
(see reviews by Andrews 1988. 1996. Bun-eson and Ragone-Calvo
1996, Soniat 1996). In the Gulf of Mexico, prevalences approach
100% year-round in areas with salinities above 6 ppt (Soniat
1996). In the Chesapeake Bay. prevalences approach 100% during
dry. summer periods, but decline during the cool, wet winters
(Andrews 1988. Burteson and Ragone-Calvo 1996). Once estab-
lished, infections will retard oyster growth (Andrews 1961. Payn-
ter and Burreson 1991) and may ultimately be lethal. The high
prevalence combined with high mortality have had a devastating
effect on eastern oyster populations, populations that are not only
an economic asset, but serve critical ecological roles as benthic
substrate, secondary producers, and a link between pelagic and
benthic food webs (Kennedy 1996).

Among marine invertebrates, oyster antimicrobial defenses
have been relatively well studied (see Fisher 1988). Hemocytes are
considered the primary line of oyster defense, largely because of
their ability to phagocytose and destroy invading microorganisms.

♦Corresponding author.

but also becasue they function in inflammatory response, wound
repair, and encapsulation (Cheng 1979, Fisher 1986). However,
the effectiveness of these putative defense mechanisms against P.
marinus is questionable since oysters do not appear to suppress
progression or transmission of the disease (Anderson 1996). Some
investigators have found, using electron microscopy, that hemo-
cytes appear to degrade intracellular P. marinus (La Peyre 1993,
Bushek et al. 1994). while others report that P. marinus cells are
able to survive and multiply within eastern oyster hemocytes (Per-
kins 1996). Studies to elucidate these hemocyte-P. marinus inter-
actions were previously limited by the difficulty of isolating the
pathogen from host tissues. Development of continuous culture
techniques for P. marinus (Gauthier and Vasta 1993. Kleinschuster
and Swink 1993. La Peyre et al. 1993. La Peyre 1996) now enables
a closer examination of this interaction and may lead to a better
understanding of the inability of oyster defenses to prevent this
disease.

A colorimetric technique to estimate killing of bacteria by oys-
ter hemocytes was recently introduced (Volety et al. 1999a). Bac-
teria and hemocytes were incubated separately and together in
wells of a microtiter plate during a challenge period, followed by
a grow-out period where surviving bacteria were allowed to mul-
tiply to numbers that could be detected after reaction with a tet-
razolium salt |3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2-H-tetrazolium], or MTS. The MTS
was added with phenylmethasulfazone (PMS) to the microtiter
plate wells where they were enzymatically reduced by living cells,
in proportion to their numer. to yield a colored, water-soluble
formazan. The absorbance of this reduction product was used to
estimate numbers of live bacteria, which was then converted to a

827

VOLETY AND FiSHER

killing index, i.e. the percent of bacteria killed or rendered non-
reproductive by the hemocytes. Much like bacterial plate counts,
the assay does not distinguish bacteria survival from their ability to
multiply. Nonetheless, a bactericidal/microbicidal terminology has
been adopted as a convenient generalization. Successful applica-
tion of the bacterial killing assay (Genthner et al. 1999) prompted
further research to adapt the technique to evaluate P. iiniiiiuis
killing by oyster hemocytes.

In this study the MTS/PMS colorimetric technique is adapted,
optimized, and subsequently applied in two experiments intended
to characterize the variability of oyster heinocyte capacity to kill P.
marinu.s. Previous investigators (Dungan and Hamilton 1995) have
documented that living P. marinus cells can reduce MTS/PMS
tetazolium salt to a formazan product that is proportional to the
living biovoluiTie (and approximate cell number). The first experi-
ment compares hemocytes from two geographically different oys-
ter stocks in their ability to kill 7 different strains of cultured P.
marinus. and the second experiment documents monthly changes
in killing ability of hemocytes from oysters at a single field loca-
tion during a year-long period.

METHODS AND MATERIALS

Development and Optimization of the Microbicidal Assay for
P. marinus

Oysters were collected from Bayou Texar, an inlet of Escambia
Bay, Florida, between August 1998 and July 1999 and transported
in coolers to the laboratory of the U.S. Environmental Protection
Agency Gulf Ecology Division (GED) to be used for a series of
experiments. On days of collection, ambient salinities ranged from
10 to 16 ppt and ambient water temperatures ranged from 10 to
29 °C. At GED, oysters were held in 1,900-L holding tanks
equipped with a tlow-through raw seawater delivery system for no
longer than I wk prior to experimental use (for the seasonal stud-
ies, experiments were performed on the day of collection).
Through the entire holding period, salinity ranged from 13 to 20
ppt and temperatures 10 to 30 °C. Hemocytes in whole
hemolymph were collected from the adductor muscle through a
notch in the oyster shell using a syringe fitted with a 23-gauge
needle. Hemolymph was placed in polypropylene tubes and sub-
merged in an ice bath to reduce hemocyte clumping. For some
studies, hemolymph samples were pooled from several (4—10) oys-
ters and lor others, hemolymph samples from individual oysters
were used.

A single isolate of cultured /'. nnirinus was used for all experi-
ments involved in the development and optimization of the assay.
This isolate (YR-VA) was originally cultured from infected oys-
ters in the York River, Virginia, in 1992. It was held aseptically for
7 y with successive bimonthly culture transfers using Dulbecco's
modified Eagle's medium and F-12 HAM's nutrient mixture
(DM1-;M/I-12; Gauthicr and Vasta 199.^). Parasite cultures for all
studies were less than 2 wk old anil upon microscopic examination,
YR-VA cells appeared healthy with a si/e distribution ot 4 to 40
|xm and the majority of cells at 4 to 10 |j.m. Densities of P. maiiims
in all experiments were determined by microscopic hcmocylome-
ter counts.

The killing assay was performed in a manner similar to that
described for bacterial killing by oyster hemocytes (Volety et al.
1999a) using formation of colored forma/an (reduced from a tet-
razoliuni salt) to dislins;uish living from dead (or non-

proliferating) cells. Briefly, the assay involved incubation of oyster
hemocytes and YR-VA, both separately and together for a 3-h
challenge period, a grow-out period of surviving YR-VA in P.
marinus cell culture medium, and a formazan formation period
with MTS/PMS solution. Formazan, the reduction product of
MTS/PMS, was measured at 490 nm using a microplate reader
(Bio-Tek Instruments Model 31 1-SX). Replicate wells (4-8) were
measured and results averaged for calculation of percentage kill-
ing, or killing index (KI), as follows:

KI(%) = [1 -(Aj.

Ah)/(Ap,„)| X 100

Equation subscripts refer to absorbances measured from wells with
hemocytes only (H), P. marinus only (Pm), or both (H -H Pm). All
experiments were performed at 20 ppt salinity, a condition suitable
for both P. marinus and oyster hemocytes.

To optimize the miciobicidal assay for P. marinus. some of the
same conditions used for bacterial killing (Volety et al. 1999a)
were initially adopted. However, the slower growth rate of P.
marinus necessitated a longer grow-out period (22-24 h) to attain
numbers high enough for colorimetric sensitivity. From this initial
protocol several different procedural modifications were examined
(see below) to determine the influence of these factors on assay
results and to characterize the most responsive methodology. As
they were identified, the most responsive conditions (higher kill-
ing) were assumed for subsequent experiments. In all cases, ab-
sorbance was measured at 490 nm. Where appropriate, ANOVA
was used to determine the difference in KI resulting from different
formazan formation periods, P. marinus.hemocyle ratios, volumes
of medium, challenge durations, and assay temperatures. As nec-
essary to meet the assumptions of ANOVA, data were transformed
either by log,,, or arcsin. When significant differences in means
were found using ANOVA, Tukey's multiple comparison test was
employed to resolve the differences due to treatments. All statis-
tical analyses were performed using SAS (Statistical Analysis Sys-
tems, Gary, NC).

Formazan Formation Period

Cells of P. marinus (YR-VA) were harvested from culture
flasks, counted with a hemocytometer, and diluted with DMEM/
F12 cell culture medium (Gauthier and Vasta 1995) to a density of
5 X 10" mL"'. This was then diluted serially to obtain 6 different
cell densities (0%. 6.37r, 12.5%, 25%, 50%, and 100%) used to
simulate a range of cells surviving a challenge with hemocytes.
Aliquots (100 |jlL) from each density were placed in microtiter
wells (N = 8 replicate wells) and held for 22 to 24 h at 28 °C.
After this grow-out period, 20 p.L of MTS/PMS were added. Ab-
sorbance w as measuretl after 1 , 2, and 3 h at 28 °C.

Challenge Ratio of VR-VA to Oyster Hemocytes

Three dllTcrenI ratios ii\ P. marinus cells to oyster hemocytes
were exannned to determine eltects on assay results. Hemocytes
from 10 oysters were pooled and for each challenge ratio, 1x10'
hemocytes were placed in 8 replicate microtiter wells with appro-
priate numbers of YR-VA to achieve ratios of 10: 1 , 5: 1 , and 2.5: 1
/'. marinus cells per oyster hemocyte. Hemocytes were diluted
with sterile-filtered (0.22 pim) sea water at 20 ppt salinity (FSW).
Experiments were performed in a final volume of 100 |xL FSW.
The challenge period was 3 h at 17 "C, followed by a 22-h grow-
out and a 3-h forma/an formation period, both at 28 °C.

In vitro Killing of P. marinus

829

Volume of Sea Water during Challenge Period

The influence of FSW on the assay was examined by compar-
ing the results obtained with four different vokimes (25. 50. 100.
and 200 (xL). Hemocytes from 1 0 oysters were pooled and for each
test volume, 1 x 10'' hemocytes were placed in 8 replicate micro-
titer wells with 1 x 10*" YR-VA (challenge ratio = 10:1 P. marinus
per oyster hemocyte) in FSW. The challenge period was 3 h at
17 °C. followed by a 22-h grow-out and a 3-h formazan formation
period, both at 28 °C.

Challenge Duration

The length of time that P. marinus were expo.sed to oyster
hemocytes was also examined. Hemocytes from 10 oysters were
pooled and for each challenge duration tested, 1 x 10'^ hemocytes
were placed in 8 replicate microtiter wells with 5 x 10'^ YR-VA
(challenge ratio = 5:\ P. marinus per oyster hemocyte) and 100
p.L FSW. The challenge period was varied from 1 to 5 h at 17 °C.
followed by a 22-h grow-out and a 3-h formazan formation period.
both at 28 °C.

Assay Temperature

To determine the effect of temperature on hemocyte killing
activity, the challenge portion of the assay was performed at five
different temperatures (5. 12. 17. 29. and 37 °C). Hemocytes from
1 0 oysters were pooled and for each challenge temperature, 1x10*^
hemocytes were placed in 8 replicate microtiter wells with 5x10'
YR-VA (challenge ratio = 5:1 P. marinus per oyster hemocyte)
and 100 |j.L FSW. The challenge period was followed by a 22-h
grow-out and a 3-h formazan formation period, both at 28 °C.

Comparison of KI with Direct Counts

Hemocytes from each of 10 individual oysters were placed in
five wells ( 10°^ per well) in a 96-well microtiter plate. P. marinus
(YR-VA; 5x10' per well) were added to the hemocytes for a 3-h
challenge at 17 °C and to replicate wells in a separate plate that
contained no hemocytes. At the end of the challenge period. P.
marinus culture medium (DMEM/F12) was added for 22 h at
28 °C, enabling the surviving cells to proliferate. After the grow-
out period. 4 of the 5 replicate wells received 20 p.L MTS/PMS for
3 h at 28 °C prior to measurement of absorbance. The fifth repli-
cate well was aspirated thoroughly and a 50% concentration of
trypan blue dye was added to the wells for 5 to 10 min. The
contents were aspirated and 2 aliquots placed on a hemocytometer
where living P. marinus cells were counted at 40x. Dead cells take
up trypan blue dye and appear blue, whereas live cells are not
stained. The average number of live P. marinus from the two
counts was subtracted from the live average in the control (no
hemocyte) wells and the percentage dead calculated for each oys-
ter. (Because some hemocytes may have engulfed and masked
some P. marinus from exposure to trypan blue, the percentage
dead may be somewhat underestimated.) Killing index, calculated
for each oyster from the average of four wells measured colori-
metrically, was compared wth the percentage dead (by count). A
paired / test was used to determine whether significant differences
(P € 0.05) existed between means (;; = 10) of Kl obtained by
tetrazolium salt reduction and direct count methods, and Pearson's
correlation analysis was used to compare the methods.

Susceptibility of Different Cultured P. marinus Isolates to
Hemocyte Killing

Seven cultured P. marinus isolates were tested against oyster
hemocytes (Table 1). These included isolates originally cultured
from infected oysters collected from Long Island Sound, Connecti-
cut (LI-CT), Delaware Bay, New Jersey (DB-NJ), Oxford, Mary-
land (OX-MD), Mobjack Bay, Virginia (MB-VA), York River,
Virginia (YR-VA), Barataria Bay, Louisiana (BB-LA), and La-
guna Madre, Texas (LM-TX). Cultures of all these isolates were
initiated from infected oysters between June 1991 and October
1992. These isolates have been used to investigate mechanisms of
infection (Volety and Chu 1995). host-parasite interaction (Bushek
and Allen 1996b. Bushek et al. 1997), different races off. marinus
(Bushek and Allen 1996a), population genetics (Reece et al. 1997),
and potential virulence factors (La Peyre et al. 1996). Since iso-
lation, cultures have been maintained on P. marinus culture me-
dium (Gauthier and Vasta 1993. Gauthier and Vasta 1995) with
transfers every 2 to 4 wk. Cultures for all experiments were less
than 2 wk old and all cells appeared healthy upon microscopic
examination.

Two geographic sources of oysters (stocks) were used to com-
pare susceptibility of different P. marinus isolates. Oysters were
collected from Bayou Texar in Escambia Bay (FL) on May 6 and
May 10, 1999 where ambient water temperature was 23 to 26 °C
and salinity was 13 to 16 ppt. They were held overnight in tanks
with running sea water (25 °C and 21 ppt salinity) prior to assay.
Oysters were also collected from Bissel Cove, Rhode Island on
June 15, 1999 where ambient temperature was 20 °C and salinity
was 30 ppt. Oysters were shipped overnight in a cooler with ice to
the GED laboratory where they were acclimated to 20 °C and 20
ppt over 5 d prior to assay. Two trials were performed for each
oyster stock. For each trial, 4 to 5 pools of hemocytes drawn from
3 to 5 oysters were challenged with P. marinus. The assay was
performed simultaneously for all 7 P. marinus isolates and all
pools of hemocytes from a single oyster stock. The protocol con-
sisted of a 3-h challenge period at 17 °C with a 5:1 ratio of P.
marinus per oyster hemocyte. followed by a 24-h grow-out period
and a 3-h formazan formation period at 28 °C. KI was calculated
for each pool of hemocytes and results for each pool in a trial were
averaged. Because the two oyster stocks were collected at different
times of year, each was considered a separate experiment. Two-
way ANOVA was performed separately for each oyster stock to
determine differences in KI due to main effects of P. marinus
isolate and trial number and Tukey"s multiple comparison test was
applied if significant differences were found.

TABLE I.

P. marinus in vitro isolate cultures.

Isolate

Geographic Source

ATCC Code

LI-CT

Long Island Sound, CT

50508

DB-NJ

Delaware Bay. NJ

50509

OX-MD

Choptank River. MD

50439

MB-VA

Mobjack Bay. VA

50510

YR-VA

York River. VA

—

BB-LA

Barataria Bay. LA

505 1 1

LM-TX

Laguna Madre. TX

.50512

830

VOLETY AND FiSHER

Seasonal Killing Ability of Oyster Hemocytes

Oysters were collected monthly from Bayou Texar (Escambia
Bay. Florida) and transported in coolers to GED. Ambient water
salinity and temperature measurements were made at each time of
collection. Ten oysters were arbitrarily selected and hemolymph
drawn from the adductor muscle as described above. Hemocyte
densities were determined and the microbicidal assay was per-
formed using hemocytes from each individual oyster and the P.
murinus isolate YR-VA. Assay protocol was the same as that used
for examining the susceptibility of different P. mariinis isolates.
Rectal tissues from the sampled oysters were processed with fluid
thioglycollate medium to determine infection intensity of P. inari-
mis (Ray 1966). and individuals assigned a value based on the
Mackin scale (Craig et al. 1989). Several simple linear regression
analyses were used to determine the association of independent
variables temperature and salinity with monthly mean hemocyte
densities and KI. Pearson's correlation analyses (SAS, Cary, North
Carolina) were conducted to evaluate the relation between monthly
mean hemocyte densities and KI and P. murinus infection intensity
and KI.

RESULTS

Development and Optimization of the Microbicidal Assay for
P. marinus

Preliminary results indicated that a 22- to 24-h grow-out period
of P. marinus cells was needed to allow sufficient proliferation to
detect changes in absorbance. Cells of P. marinus incubated with
MTS/PMS showed an increase in absorbance at higher densities
(Fig. I ). Regardless of the period allowed for formazan formation,
absorbance was highly correlated with cell density (r = 0.96-
0.97). The slope of the relationship increased with increasing for-

mazan formation. Killing indices obtained using challenge ratios
of 2.5:1 (KI = 80.0%) and 5:1 (KI = 15.2%) P. marinus ceWs per
oyster hemocyte were statistically greater {P = 0.0001) than that
derived from the 10:1 ratio (KI = 17.0%. Fig. 2). The KI from
microtiter wells containing 100 (jlL FSW (KI = 66.5%) was sig-
nificantly higher {P = 0.0001) than indices obtained from wells
with higher and lower volumes (Fig. 3). No significant differences
were detected in KI among challenge periods that were I, 2, 3, or
5 h (Fig. 4). but the 4-h challenge was lower than all other chal-
lenge durations {P = 0.001). Although there were no statistically
significant differences in KI among assay temperatures of 5, 12,
17, and 37 °C, KI at a challenge temperature of 29 °C was signifi-
cantly lower (P = 0.0001 ) than those obtained at 5. 12, and 17 °C
(Fig. 5).

Killing indices for 10 individual oysters, using the formazan
technique, averaged 61.4% (±12.1 SD) as compared to an average
57.1%' (±15.4 SD) estimated by direct counts made after incuba-
tion in DMEM/FI2 cell culture medium. The greatest discrepancy
between methods for an individual was 27%, yet discrepancies of
1% or less were found for 3 of the 10 oysters. There was no
statistical difference between the average P. marinus killing for
any of the 10 individuals as measured by the two techniques (P =
0.29) and the results were positively correlated (r = 0.628, P <
0.05).

Susceptibility of Different P. marinus Isolates to Hemocyte Killing

The Mobjack Bay P. marinus isolate (MB-VA) was consis-
tently the most susceptible to oyster hemocyte killing, with KI for
individual oysters ranging from 73 to 91% and isolates BB-LA,
DB-NJ, and LI-CT (individual KI = 20-46%) were among the
least susceptible, regardless of oyster origin (Fig. 6). Average KI
for both oyster stocks against all cultured isolates was 48%. There

1.00

0.75

O

H 0.50 -

• 1 hr incubation
O 2 hr incubation
T 3 hr incubation

^

0.25

0.00

100

% Viable cells

Figure 1. Absorbance readings (490 mil) (il miiniliter wells coiilalniiin /'. marinus (VR-VA) trophozoites at densities of (1, 6.3, 12,5. 25, 50, and
100% of a 5 X 10' cell inoculum after a 22-h grow-out period followed bv a 1-h (r = 0.97), 2-h (r = 0,96), and .Vh (r = 0.97) incubation with
MTS/PMS reagent.

In vitro Killing of P. marinus

831

P martmts: hemocyte ratio

Figure 2. Mean KI (±SD) for in vitro challenge ratios of 2.5:1, 5:1, and
10:1 P. marinus trophozoites (YR-VA) per oyster hemocjte. Different
letters designate statistically different means (Tukey's multiple com-
parison. P < 0.05).

was no significant difference due to trial in the Rhode Island oyster
challenges (ANOVA, P = 0.08) and Tukey's multiple comparison
test found KI of isolate MB-VA to be significantly higher than all
other isolates. A significant difference due to trial was detected for
Florida oyster challenges (ANOVA, P = 0.001).

Seasonal Kilting Ability of Oyster Hemocytes

The capacity of oyster hemocytes to kill YR-VA varied over
the sampling period, with monthly average KI ranging from 2 1 to
90% (Fig. 7) and an average 57% across all months of the year-
long monitoring period. Killing index was lowest in July and Au-
gust (average KI = 21^1%) and highest in October through
December (average KI = 75-90%i). Correlation analysis found KI
to vary inversely with salinity and temperature measured at the
collection site at the time of collection. Simple linear regression
analysis of KI with salinity was significant (r = -0.75, P =
0.006), whereas that with temperature was not (r = -0.50, P =
0.1 1). Hemocyte density was not clearly associated with KI (r =
-0.26. P = 0.42) nor temperature (r = 0.51, P = 0.09), but was

Challenge time (hr)

Figure 4. Mean KI (±SD) for in vitro challenges between oyster
hemocytes and P. marinus (YR-VA) ranging from 1 to 5 h for the
challenge period. Different letters designate statistically different
means (Tukey's multiple comparison, P < 0.05).

weakly associated with salinity (r = 0.59. P = 0.04). Prevalence
of P. marinus was 100% throughout the study period and infection
intensity ranged from 2.5 to 4. 1 (scale of 0-5). Neither individual
oyster KI nor monthly average KI was significantly as.sociated
(Pearson's) with infection intensity.

DISCUSSION

An in vitro microbicidal assay, previously developed to esti-
mate susceptibility of bacteria to killing by eastern oyster
hemocytes (Volety et al. 1999a), was adapted and used to charac-
terize the capacity of hemocytes to kill P. marinus cells. In vitro
challenges showed that hemocytes from two geographically dis-
tinct oyster stocks were able to eliminate 25 to 90% of P. marinus
cells grown from cultures isolated at 7 different locations along the
Gulf of Mexico and U.S. Atlantic coast (Fig. 6). This finding,
coupled with the year-round average of 57%- KI of one isolate by
hemocytes from Florida oysters (Fig. 7), implies that hemocytes
may have some capacity to combat this destructive disease agent.
Such an implication is supported by at least a few reported obser-

25 50 100 200

Volume of medium (ul)

Figure 3. Mean KI (±SD) for in vitro challenges between oyster
hemocytes and P. marinus (YR-VA) using 25 to 200 fiL sea water in
microliter wells. Different letters designate statistically different
means (Tukey's multiple comparison, P < 0.05).

Temperature ( C)

Figure 5. Mean KI (±SD) for in vitro challenges between oyster
hemocytes and P. marinus (YR-VA) ranging from 5 to 37 °C for the
challenge period. Different letters designate statistically different
means (Tukey's multiple comparison, P < 0.05).

832

VOLETY AND FiSHER

LI-CT DB-NJ OX-MD MB-VA YR-VA BB-LA LM-TX

DB-NJ OX-MD MB-VA YR-VA BB-LA LM-TX

, mannus i

solatcs

Figure 6. Mean KI (±SD) for 7 different P. marinus isolates challenged
in vitro with hemocjtes from oysters collected in Escambia Bay,
Florida (top) and Bissel Cove, Rhode Island (bottom). Bars represent
the combined average of two trials and for Rhode Island oysters (bot-
tom), there was no difference between trials (ANOVA). Different let-
ters designate statistically different means (Tukey's multiple compari-
son, P < 0.05).

vations of phagocytic destruction of P. marinus in tlcld-collected
oysters (La Peyrc 1993, Bushek et ai. 1994).

However, further investigation is required to determine whether
oyster hemocyles can provide a meaningful defense against P.
mciriniis in nature. One of the most crucial questions related to
evidence provided by this study is whether cultured P. marinus
isolates accurately retain the vigor of naturally occurring /'. mari-
nus. Through repeated transfers and lack of contact with oyster
tissues, cultured parasites may have lost the ability to protect them-
selves against host defenses, typically accomplished through
masking (Mauel 1984. Hall and Joiner 1991 ) or repellent chemical
.secretions (Le Gall et al. 1991. Yoshino el al. 199.1. Volety and
Chu 1995). Perhaps as a consequence, several investigators have
reported low virulence of cultured isolates (La Peyre et al. 199.3.
Gaulhier and Vasla 199.1, Volely and Chu 1994. Chinlala et al.
1995).

Other factors must also be reconciled between natural chal-
lenges and the in vitro assay conditions presented here. Unlike
natural infections where several different sizes and stages of P.
marinus exist in oysters (Perkins 199(1). the cultured cells were
relatively homogenous (4-10 ^^.m diameter! and generally lacked

O 25

»^^

1

/ ' ''

V,

\

/ \ ^1
/ \ '' /
' \ '

L-^

-

V

/

\ 1 /

\\

/

^

V

V

V

/ \ /

-

• ■ Temperature

— •— Salinity

Aug-98 Sep Oct Nov Dec Jan-99 Feb Mar Apr May Jun Jul

I

ALg-98 Sep Oct Nov Dec Jan-9'> Feb Mar ^ pr May Jun Jul

Sampling month

Figure 7. Monthly monitoring of oysters in Escambia Bay, Florida
during a 1-y period in 1998 and 1999: (top) ambient water tempera-
ture and salinity measured at the time of oyster collection; (bottom)
mean hemocyte density (±S1)) and Kl (±SD) calculated from indepen-
dent tests on 10 individual oysters.

hypnospores and tomonts because of their growth on nutrient-rich
medium. The in vitro chemical environment was unlike natural
conditions because the challenge medium did not include
hemolymph. Finally, killing in natural conditions may be intlu-
enced by a variety of external conditions such as temperature,
salinity, or the nutritional and gamelogenic status of the oyster.
Ultimately. P. mariiuts is known to persist, and even tlourish in
oysters from Mexico to Maine (Burreson et al. 1994, Ford 1996).
So, whatever killing capacity oyster hemocytes may have, their
ability under natural conditions is insufficient lo offset P. marinus
rates of multiplication (Powell el al. 1996) and to eliminate disease
from the oyster population.

An importanl technical difference exists between Ihis iiielhod
lor /'. marinus and the bactericidal assay (Volety et al. 1999a). In
ihe /'. marinus method, the post-challenge cell culture medium
does not affect hemocyte survival, so Ihe 22- to 24-h grow-out
period for P. marinus multiplication may simultaneously allow
continued hemocyte killing. This probably created (he relatively
consistent Kl (except for the 4-h duration) found when the chal-
lenge period was varied from 1 to 5 h (Fig. 4). An addilional 22-h
period for killing may have overshadowed differences that oc-
curred between 1 and 5 h. An extended killing period may al.so
explain the lack of significant differences in Kl at assay tempera-
lures of 5 lo 29 "C (Fig. 5). After the .3-h challenge period in these
experiments, a grow-oul period proceeded for an additional 22-h at
2S C. conditions sufficiently similar to 29 ' C (o mask any effects
of lemperalure differences applied for only .3 h. Because of the

In vitro Killing of P. marinus

833

TABLE 2.

Recommended protocol to estimate killing of P. marinus by
ovster hemoo tes.

1. One week prior to assay, scan a P. murliuis culture (1-5 x 10''
cells/mL) in 1:2 DMEM/F-12 nutrient mixture (Gauthier and Vasta
1995) from a culture no more than 1 -mo-old. Maintain cultures at
28 °C.

2. Collect hemolyph from the adductor muscles of oysters using a 1
to 3 mL syringe fitted with a 23-gauge needle. Place the
hemolymph into polypropylene tubes in an ice bath to reduce
heniocyte aggregation. Estimate hemocyte numbers using a
hemocytometer and calculate the volume of hemolymph needed to
yield I x 10"^ hemocytes for each microliter plate well.

3. Before adding oyster hemolymph, add 100 jjiL FSW
(filter-sterilized. 20 ppt) into the wells of a microtiter plate. To the
H and H 4- P wells, add the volume of hemolymph needed for I x
10'^ hemocytes. Centrifuge the microtiter plate at \(iOg for 10 min
to ensure hemocyte adhesion in a monolayer.

4. Gently remove hemolymph plasma/FSW from the microtiter plate
wells using a multichannel pipet (leave the hemocyte monolayer
moist). Quickly add 100 p,L FSW to control blank wells and H
only wells. Add 100 |j.L FSW containing 5 x 10'' P. marinus to
H + P and P only wells.

5. Centrifuge microtiter plate at I60(; for 10 min to ensure contact of
P. marinus with hemocytes.

6. Maintain microtiter plates in a humid chamber at 17 °C for 3-h
challenge period.

7. Add 100 p,L P. marinus cell culture medium (DMEM/F-12) to all
wells. Maintain the plate at 28 °C for a 22 to 24 h P. marinus
grow-out period.

8. Add 20 \xL MTS/PMS reagent to each well and incubate for 3 h at
28 °C to allow color development.

9. Measure absorbances of the wells at 490 nm using a microplate
reader set to subtract the absorbance of blank wells from sample
wells.

10. Calculate KI as follows:

Kl {%) = [1 - (A„,|,,„ - A„)/(Ap„,)l X 100

likelihood of simultaneous killing during the parasite grow-out
peiiod, a separate challenge period may be duplicative. Alterna-
tively, future iterations of the technique could concentrate on a
means to kill or disable the hemocytes at the end of the challenge
period (without damaging the parasites).

Hemocytes withdrawn from two geographically distinct stocks
of oysters (Florida and Rhode Island) exhibited similarities in their
ability to kill different isolates of P. nuuiiuis (Fig. 6). Hemocytes
from both oyster stocks were more able to kill MB-VA and less
able to kill BB-LA, DB-NJ, and LI-CT. Because of this similarity,
variations in the results appeared to stem from differences in sus-
ceptibility of the isolates rather than differences in the killing
ability of hemocytes from the two oyster stocks. There was no
evidence of geographic differences in killing ability and no evi-
dence to account for observations (Bushek 1994, Bushek and Allen
1996b) that Atlantic coast isolates of P. marinus (DB-NJ and
MB-VA) generate higher infection intensities than Gulf coast iso-

lates (BB-LA and LM-TX). It is possible that different culture
histories influenced the condition and vigor of the various cell
lines, but susceptibility may also be influenced by genetic vari-
ability among races of P. nuirinits (Bushek and Allen 1996a, Reece
et al. 1997).

Florida oyster hemocytes were capable of killing P. marinus
YR-VA throughout the year-long monitoring period (Fig. 7). KI
was lowest during July and August, but climbed dramatically
through December. If this seasonal pattern is typical, these data
imply a mechanistic link for reduced killing capacity with high
temperature, high salinity, and post-reproductive condition (An-
drews 1988. Burreson and Ragone-Calvo 1996, Soniat 1996). As-
sociation of killing capacity with intensity of natural P. marinus
infections was not found, yet interpretation of such data should be
viewed with caution since it is likely that only oysters with rela-
tively low infections survive during the warm summer months
(Fisher et al. 1992).

The greater capacity of Florida oyster hemocytes to kill P.
nuirinus during the winter (Fig. 7) contrasts with their low winter
bactericidal capacity for Vibrio parahaemolyticus and Lysleria
monocytogenes (Genthner et al. 1999, Volety et al. 1999b). In
addition, the positive correlation between oyster hemocyte density
with bactericidal activity (Volety et al. 1999b) was not observed
here. These discrepanices may signal different /;; vitro hemocyte
killing mechanisms for bacteria and P. marinus.

The results presented here demonstrate unequivocally that oys-
ter hemocytes in vitro have the capacity to kill P. marinus. More-
over, the technique that was developed provides an efficient and
defendable tool for examining interactions of oyster defenses with
P. marinus and other microorganisms. The protocol recommended
from optimization studies (Table 2) is relatively simple, inexpen-
sive, and can be easily performed with numerous repetitions. Simi-
lar responses were obtained with hemocytes from two distinct
oyster stocks and there was good correspondence between the
colorimetric technique and direct microscopic counts. We antici-
pate that this technique will be used in the future to investigate
potential differences between hemocyte killing capacity and kill-
ing ability in natural conditions. It may also play a key role in
elucidating other host-parasite interactions, such as the role of
external environmental factors, mechanisms of action, and differ-
ences in defense responses to different microbial invaders.

ACKNOWLEDGMENTS

Oysters from Rhode Island were collected by Mamita Chintala
of the U.S. Environmental Protection Agency (EPA). Culture iso-
lates were generously provided by David Bushek (LI-CT, DB-NJ,
MB-VA. BB-LA, and LM-TX), Chris Dungan (OX-MD). and
Frank Perkins (YR-VA). Determinations off. marinus infection
intensity were provided by Jim Winstead (EPA). We are grateful
for constructive comments provided by Leah Oliver (EPA) and
journal reviewers. This work was conducted while A. K. Volety
was a National Research Council Associate at the U.S. EPA. This
is Gulf Ecology Division Contribution 1 107.

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Jourmil of Shellfish Research. Vol, 19. No. 2. 8.15-839. 2000.

A METHOD FOR PRESERVING OYSTER TISSUE SAMPLES FOR FLOW CYTOMETRY

HUIPING YANG,' - TOM GALLIVAN," XIMING GUO,' ^ and
STANDISH K. ALLEN, JR."

^Haskin Shellfish Research Laboratoiy. Institute of Marine and Coastal

Science, Rutgers University. 6959 Miller Avenue. Port Norris,

New Jersey 08349
'Experimental Marine Biology Laboratory, Institute of Oceanology,

Chinese Academy of Sciences. 7 Nanhai Road. Qingdao. Shandong

266071. People 's Republic of China

ABSTRACT Flow cytometry (FCM) is a powerful method for ploidy determination which has become important because of the
increasing use of triploids in aquaculture. Tissue samples for FCM can he biopsied and kept fresh or frozen in a staining solution
containing dimethyl sulfoxide (DMSO). Samples can be stored in the stain/DMSO at -80 "C indefinitely, or shipped on dry ice to a
flow cytometry lab. But ultracold freezers and overnight shipping are not always available, for example, at rural labs and hatcheries.
We investigated several methods of preserving FCM samples that do not involve freezing. Three different tissues, gill, mantle, and
hemolymph from diploid and triploid Pacific oysters. Cnissostreii gigiis Thunberg. were preserved by different methods, including
pre-treatments and different fixatives. Gill was the best tissue for FCM analysis, and ethanol (75%) was the preferred fixative.
Hypotonic treatments before fixation promoted nucleus-dissociation needed for FCM. The recommended protocol for preserving gill
tissue is to dissect or biopsy a piece gill tissue (-0.5 cm"), treat with 0.075 M KCl for 10 min and fix in 75% ethanol that is changed
once. Before FCM. the fixed tissue is washed once using a phosphate buffered saline (pH = 6.8) and transferred to a staining buffer
containing 10% DMSO. The stained sample is frozen and thawed, votexed. aspiarted. and filtered before analysis. This method can
also be used for preserving D-stage larvae and gill tissue samples of other bivalve species.

KEY WORDS: Triploidy. polyploidy. DNA content, flow cytometry, fixation, oyster, mollusc

INTRODUCTION

Chromosome set manipulation is an important field in shellfish
genetics and breeding. Triploid molluscs are important for aqua-
culture because of their sterility, high meat yield, and quality
(Chew 1994). Tetraploids are valuable because they can produce
100% triploids by mating with normal diploids (Guo et al. 1996).
Polyploid induction has been studied in over 20 molluscan species
so far (Beaumont and Fairbrother 1991. Guo et al. 1999). During
chromosome set manipulation, ploidy determination is a critical
and necessary procedure. Methods for ploidy determination in-
clude chromosome counting, comparing nuclear size (Utting and
Child 1994), electrophoretic assay (Allen et al. 1982). fluorescence
staining (Komaru et al. 1988, Uchimura et al. 1989), and flow
cytometry (FCM) (Allen 1983). FCM is by far the most powerful
method for ploidy determination because of its speed, simplicity,
and accuracy. It measures DNA content by recording the fluores-
cence intensity of nuclei stained with a DNA-specific dye, such as
4,6-diamidine-2-phenylindole (DAPI) or propidium iodide (PI).
Ploidy can be determined by analyzing thousands of cells in a few
minutes.

Normally, samples for FCM analysis need to be prepared fresh
or kept frozen in a staining buffer containing dimethyl sulfoxide
(DMSO). While taking samples is relatively straight forward in
shellfish, storing them until they reach a flow cytometry laboratory
is often difficult. This is especially true for remote laboratories and
hatcheries. Shipping samples from remote places often results in
severe degradation because tissues can not be kept cold during

'Present address: Aquaculture Genetics and Breeding Technology Center,
Virginia Institute of Marine Science, College of William and Mary, Glouc-
ester Point. VA 23162.
■"Corresponding author. E-mail: xguo@hsrl.rutgers.edu

transport. Individual cells such as hemolymph or isolated nuclei
can be fixed for FCM without problem. Solid tissue samples, when
fixed, are problematic for FCM. There are several existing meth-
ods for isolating nuclei for FCM from fixed tissues, mostly vari-
ants of the Hedley method (Hedley et al. 1983. Hedley 1994).
These protocols are designed for clinical applications and involve
laborous rehydration, enzyme treatments, and washes, and are not
practical for rapid ploidy determination of a large number of
samples.

The purpose of this study was to find a simple and effective
method to preserve tissue sainples for FCM analysis without freez-
ing and laborous post-fixation treatment, allowing tissue samples
to be stored, transported, and analyzed easily. Three separate ex-
periments were conducted using diploid and triploid Pacific oys-
ters. Crassosuea gigas Thunberg, to test different tissues, pre-
treatinents, and methods of fixation. Here we report a simple pro-
tocol for tissue preservation and nucleus isolation, which is
effective for rapid FCM analysis of ploidy in molluscs.

MATERIALS AND METHODS

Pacific oysters used in this study were 2-y-old diploids and
triploids produced by crossing diploids and tetraploids (Guo et al.
1996). FCM analysis was conducted on a Partec Ploidy Analyzer
using a staining solution consisting of 10 mg/L DAPI dissolved in
a nucleus isolation buffer ( 10 niM Tris, 146 niM NaCl. adjust pH
to 7.4, 2 mM CaCU, 22 mM MgCK, 0.1% Triton X-100. and
0.005% bovine serum) with 10% DMSO (DAPI/DMSO, modified
from Guo et al. 1993). The following method of sample prepara-
tion, which is routinely used in our laboratory, was used as the
normal control method in this study. Briefly, the tissue sample
(-0.5 cm") is placed directly into I mL of DAPI/DMSO in a
1.5-mL centrifuge tube and frozen at -80 °C until use. Before
FCM, the frozen sample is thawed at room temperature, vortexed.

835

836

Yang et al.

aspirated 5 times with a 1-cc syringe (25-gauge needle), and fil-
tered through a 25-(jLm screen. We conducted three experiments to
evaluate different aspects of tissue preservation and preparation.

Experiment 1: Direct Fixation versus Pre-Treatment

Hemolymph. gill, and mantle tissues were dissected from 15
diploid and 15 triploid oysters. Hemolymph (0.2-0.3 mL) was
drawn from the adductor muscle using a 25-gauge needle, split into
2 aliquots, and kept on ice. Gill and mantle tissues were cut into
three pieces (-0.5 cm~) and kept on ice. One piece/aliquot was
prepared by the normal method (frozen) and used as a control. The
others were prepared for FCM by the following methods.

Method 1: Direct Fixation

Hemolymph (0.1-0.15 mL). gill, and mantle tissues (-0.5 cm")
were each immersed in 1 mL of Camoy's fi.\ative (3:1. absolute
methanokacetic acid) in 1.5-mL tubes. Gill and mantle were fur-
ther cut into 3 pieces to facilitate complete fixation. Fixative was
changed once and then samples were stored at 4 °C for 1 wk.

Method 2: Hypotonic Pre-Treatment

Gill and mantle tissues (-0.5 cm") were treated with a hypo-
tonic solution (0.075 M KCI) for 20 min and then cut into 3 pieces
in Carnoy's fixative that was changed once. Samples were stored
at 4 °C for 1 wk.

Before FCM, the fixed samples were washed once with phos-
phate buffered saline (PBS, 0.025 M KH^POj, pH = 6.8), trans-
ferred to 1 mL of DAPI/DMSO. votexed, aspirated five times, and
then filtered through a 25-(jL,m nytex screen. Wash and fixative
change were made by pelleting the cell/tissue suspension with
centrifugation ( 1 min at 2,000g) and replacing the supernatant.

Experiment II: Pre-Treatments

Gill tissue was dissected from 5 diploid Pacific oysters, and cut
into -0.5 cm" pieces. Each piece was treated in the following way
before fixation in Camoy's: (1) hypotonic treatment with de-
ionized water for 5, 10, 15, 20, 25, and 30 min; (2) hypotonic
treatment with 0.075 M KCI: treatment times were 10, 15. 20, 25,
and 30 min; (3) treatment with detergent, 0.5'7r Triton X-100 dis-
solved in ().S5% NaCl. for 10. 15, 20, 25, and .30 min: (4) no
treatment, tissue was directly fixed in Carnoy's; and (5) tissue was
frozen in DAPI/DMSO (the control method). After pre-treatments,
each gill sample was cut into 3 to 5 pieces in Camoy's fixative that
was changed once. These samples were stored at 4 °C for 1 wk.
Before FCM. tissue samples were washed once with PBS (pH =
6.8), vortexed in 1 niL of DAPI/DMSO for 10 to 15 sec, aspirated
five times with a 1-cc syringe (25-gauge needle), and then filtered
through a 25-fjLm nytex screen.

Experiment IJI: Fixatives

In this experiment we tested three different fixatives: Carnoy's,
75'/f ethanol. and 10^* formalin. Gill tissue from 3 diploid Pacific
oysters were dissected and cut into equal pieces (-0.5 cnr). each
piece was treated with 0.075 M KCI for 5, 10. 15, or 20 min and
then cut into 3 pieces in 1 of the 3 fixatives. After changing
fixatives once, samples were stored at 4 "C for I wk.

For FCM, samples were washed once with PBS (pH = 6.S).
frozen in I niL of DAl'l/DMSO al -80 C tor I h, thawed al room
temperature, vortexed for 15 to 20 sec, aspirated 5 times with a
25-gaugc needle, and filtered through a 25-^JLm nytex screen.

Data Collection

FCM records, in rapid succession, the fluorescence intensity of
nuclei, which are suspended in the DAPI/DMSO staining solution.
The FCM histogram so obtained describes the distribution of fluo-
rescence signals from nuclei by a frequency distribution histogram.
The peak position of the histogram as measured by channel num-
bers on the horizontal axis reflects the relative DNA content/
nucleus, and the number of nuclei recorded is shown on the ver-
tical axis (counts). The term peak position (in channel numbers) is
used to describe the position of the histogram along the x-axis in
this report.

For this study the flow cytometer was set to obtain at least
10,000 counts at a rate of 400 to 500 cells/nuclei per second. FCM
histograms were analyzed using the curve-fitting program, Mod-
fitT"', to estimate peak position and coefficient of variation (CV) of
frequency distribution histograms. The peak position to a control
peak of known ploidy level was used to determine ploidy level of
a given sample. CV was used to evaluate the quality of the distri-
bution peak that is affected by sample preparation. A large CV is
an indication of poor sample quality caused by preparation. Dif-
ference in peak position and CV was compared by two-sampled I
test or ANOVA using the statistics package, SYSTAT 6.0 (Wilkin-
son 1996).

RESULTS

Experiment I: Pre-Treatment versus Direct Fixation

The peak position and CV of hemolymph, gill, and mantle
tissue from diploids and triploids prepared by freezing (the control
method) and fixation in Camoy's without (Method 1) or with
(Method 2) the hypotonic pre-treatment are presented in Table 1.

For hemolymph, a 0.2-niL sample was enough for FCM and
histograms were of good quality. Peak position of directly fixed
hemolymph. however, was significantly (P < 0.001) higher than
that of frozen samples in diploids and triploids (two-sampled ;
test). With the control method the ratio of peak position of triploid
to diploid was 1.54. With direct fixation, the 3n/2n ratio was 1.61,
primarily because of a shift of the triploid peak position to the
right. Hypotonic pre-treatment was not used for hemolymph cells
considering that they are single cells.

Gill tissues from diploid and triploid oysters were prepared by
freezing and fixation with or without hypotonic pre-treatment. Di-
rect fixation did not change peak position of diploids and triploids
compared with the control method (Table 1). Hypotonic pre-
treatment before fixation increased the peak position of diploids (P
= 0.024), but not triploids. Direct fixation with Carnoy's affected
the quality of the histogram and resulted in higher CVs than the
control method. Comparatively, pre-treatment with the hypotonic
solution did not affect the quality or CV of the histograms. An
important difference between fixation with and without pre-
Ircalmcnl was the number of nuclei produced. Samples pre-treated
with the hypotonic treatment produced higher concentrations of
nuclei than the directly fixed samples. Sometimes it was dilficult
to obtain enough nuclei (lO.OOO) for FCM from directly fixed
samples.

For mantle tissue, the peak position of directly fixed samples
did not differ from that of frozen samples for diploids and triploids.
With the h>potonic pre-trcatnienl. peak positions were the same as
with the control method for triploids. hut higher tor diploids (P =
0.002). Mantle tissue, when directly fixed, resulted in broad his-

A Method for Preserving Oyster Tissue Samples for Flow Cytometry

837

TABLE 1.

Peak position and C^ (mean ± SD) of FCM histograms when different tissues of diploid and triploid Pacific oysters were prepared by
freezing (control), direct fixation in Carnoy's, and a hypotonic pre-trcatment before fixation (n = 15).

Diploid

Triploid

Peak

Hemolymph

Gill

Mantle

Hemolymph

Gill

Mantle

Position

Freeze control

50.2 ± 2.6

49.6 ± 1.8

48.6 + 4.0

77.4 ±2.6

73. 1 + 3.9

72.9 ± 3.2

Direct fixation

55.9 ± 3.4

48.6 ±2.8

50.0 ± 4.2

90.1 ±3.6

73.1 ±7.4

75.9 ±5.0

P value*

<0.001

0.624

0.296

<0.001

1.000

0.142

Pre-treatnient

—

52.4 ± 3.8

52.0 ± 2.8

—

76.4 ± 4.8

74.6 ± 4.3

P value*

—

0.024

0.002

—

0.248

0.513

CV

Freeze control

12.0 ±2.0

11.2+ 1.6

1 1.7 ± 1.9

12.4 ± 1.4

10.5 ±1.4

12.2 ±1.7

Direct fixation

10.3 ±2.6

15.7 ±2.7

15.8 ±5.0

10.7 + 2.0

13.9 ±2.1

14.8 ±2.5

P value*

0.058

<0.001

0.004

0.014

<0.001

0.002

Pre-treatment

—

9.8 ± 1.7

12.5 ±2.8

—

11.0 ±1.4

12.7 ±1.6

P value*

—

0.136

0.810

—

0.729

0.765

*P \alues are from luo sample ; tests between treatment groups and the control.

tograms. CVs from direct fixation were significantly higher than
that from the control method for both diploids and triploids (Table
1). CVs from pre-treated mantles were the same as that from the
control method. When fixed, mantle tissue rarely produced ad-
equate numbers of nuclei for FCM, with or without the pre-treatment.
All trials with mantle tissue were subsequently discontinued.

Experiment II: Different Pre-Treatments

Because the pre-treatment in Experiment I affected peak posi-
tion in two cases, different pre-treatments were further tested and
compared with direct fixation. Peak position and CV of gill tissues
pre-treated with different treatments and for different durations
before fixation are presented in Table 2. Varying the time of hy-
potonic treatment did not change the peak position or CV in any of

the three pre-treatments, as suggested by ANOVA. The mean peak
position and CV (across pre-treatment durations) of each pre-
treatment were compared to that from the control method. Only the
Triton X- 1 00 treatment differed from the control method and re-
sulted in lower peak positions and higher CVs than the control.
Pre-treatment with KCI or H,0 did not affect peak position and
quality.

Sufficient numbers of nuclei were obtained from all pre-
treatments. Again, it was sometimes difficult to obtain enough
nuclei with direct fixation.

Experiment III: Different Fixatives

In this experiment, three fixatives, Carnoy's, 75% ethanol. and
10% formalin, were tested, using gill tissues from diploids, with

Peak position and CV (mean

TABLE 2.

SD) from FCM histograms of gill tissue of diploid Pacific oysters preserved in Carnoy's after different
pre-treatments for 5-30 min (h = 5).

Pre-treatment

Peak

time (min)

0.075 M KCI

De-ionized water

0.5'7r Triton-100

Direct fixation

Freeze control

Position

0
5

50.6 + 2.2

55.4 ± 1.2

54.2 ±0.6

10

56.6 ±1.7

52.7 ± 2.4

50.7 ± 3.8

15

56.6 ± 2.3

51.6 ±3.9

50.9 ± 3.6

20

54.5 ±1.8

52.1 ±2.9

49.6 ±2.0

25

55.8 ±1.4

50.4 ± 2.4

49.9 ± 4.6

30

56.4 ±1.0

52.5 ± 0.6

48.4 ± 3.9

ANOVA P value

0.361

0.717

0.822

Overall mean

55.98

51.64

49.90

55.42

54.20

/ test P value

0.642

0.226

0.007

0.928

CV

0
5

12.2 ±2.6

11. 7± 1.2

9.7 ±0.7

10

12.0± 1.4

11.1 ±0.9

13.5 ±2.2

15

13.1 ±2.6

11.3± 1.4

12.7 ±2.3

20

12.6 ±2.2

11.3± 1.5

13.3 ±1.3

25

12.1 ± 1.8

11.6±1.5

13.0 ±2.3

30

11.6±1.8

12.2 ±1.0

13.6 ±3.7

ANOVA P value

0.573

0.861

0.979

Overall mean

12.27

11.60

13.23

11.70

9.66

t test P value

0.282

0.359

0.01 i

0.561

838

Yang et al.

TABLE 3.

Peak position and CV (means ± SD) from FCM hitograms of gill tissue of diploid Pacific oysters preserved in different fixatives after

pre-treatment with 0.075 M KCI for 5-20 min in = 3).

Peak

Pre-treatment
time (min)

Carnoy

Ethanol (75%)

Formalin (10%)

Freeze control

Position

CV

0

—

—

5

52.7 ± 2.0

53.3 ±2.1

10

51.5 ±3.7

52.2 ±1.2

15

52.0 ± 0.9

52.3 ± 1.6

20

52.9 ± 1.8

51.8± 1.8

ANOVA P value

0.873

0.721

Overall mean

52.28

52.41

/ test P value

0.881

0.915

0

—

—

5

12.7 ±0.6

10.5 ±0.1

10

12.2+1.3

9.8 ± 1.6

15

13.9 ±2.8

9.9 ± 0.4

20

10.8 ±0.9

9.2 ± 0.4

ANOVA P value

0.179

0.277

Overall mean

12.42

9.86

t test P value

0.026

1.000

44.5 ±5. 1

44.3 ±1.5

45.7 ± 2.3

47.3 ± 2.6

0.674

45.45

<0.00l

I2.8± 1.0

14.9 ± 1.3

14.5 + 0.6

13.2 ±0.7

0.090

13.86

<0.00l

53.3

53.34
9.9

pre-treatments with 0.075 M KCI for 5, 10, 15. or 20 min. Again,
the dtiration of hypotonic treatment did not affect peak position
and CV for all three fixatives (Table 3). Of the three fixatives, only
formalin affected FCM and produced significantly lower peak po-
sitions and higher CVs. Peak positions from Carney's and 75%
ethanol fixation were unchanged relative to the control method.
FCM histograms from ethanol-fixed samples were comparable in
quality to that from the control method (Figure 1).

DISCUSSION

FCM is a powerful technique for ploidy determination in shell-
fish (Allen 1983. Chaiton and Allen 1985. Guo et al. 1993). and
there is an increasing demand for ploidy analysis in shellfish re-
search and production. Access to FCM for most shellfish labs and
hatcheries is limited by difficulties in the storage and transporta-
tion of fresh or fro/en samples. We had several experiences where
important FCM samples degraded during shipment. We have pre-
viously attempted direct fixation with Camoy's solution without
success. The problem is that cells are tightly packed in fixed tis-
sues, and nuclei cannot be easily dissociated. While there are pro-
tocols for nucleus isolation from fixed materials for clinical appli-
cations, they are too laborous for rapid ploidy analysis of large
numbers of samples.

There are several ways to dissociate and isolate nuclei from
fresh and fixed tissues, including mechanical, en/ymatic. and
chemical treatments (Song and l.i 1992. Hedlcy 1994). Mechanical
treatments such as frcc/e-and-thaw, vortexing, and aspirating are
commonly used for dissociation of nuclei and found to be helpful
in this study. Detergents can dissolve cell membrane and other
structural components and disintegrate cells or nuclei from tissues
( Vindelov 1977). In this study the detergent Triton X-IOO was used
to treat gill before fixation. Although it was effective in free-up
nuclei, Triton X-IOO reduced the peak position and increa.sed the
CV of treated samples. We did not lest en/ymc treatments as
described in most clinical protocols because we wanted to develop
a simple and inexpensive protocol.

As expected, direct fixation of oyster tissue samples made

nucleus isolation difficult. Directly fixed samples often failed to
produce enough nuclei for a normal FCM run (at 10,000 counts).
It also affected the quality of FCM histograms w ith increased CVs.
Direct fixation had no effects on peak position. We found that
pre-treatments before fixation made nucleus isolation easier.
Samples pre-treated with hypotonic solutions consistently pro-
duced sufficient numbers of nuclei. The hypotonic pre-treatment
had no effects on the peak quality or CV. Pre-treatment affected
peak position in two cases in Experiment I, which was not ob-

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Kitjure 1. KCM histograms of diploid and Irlploid Pacific <»stcrs,
Cra\s»\/r<>o gigas. (Al diploid and (B) Irlploid: gill tissue frozen in
1)\PI/I)MS() until anal>sis. IC'I diploid and iDl triploid: gill tis.sues
n.xcd in 75% ethanol and then stained with DAPl/DMSO.

A Method for Preserving Oyster Tissue Samples for Flow Cytometry

839

served in subsequent experiments and was probably accidental.
The level of variation in peal< position observed does not affect
ploidy determination. Because of the increased nucleus yield, we
recommend hypotonic treatment before fixation.

Fixation is a process by which tissues or their components are
fixed selectively at a particular stage to a desired state. The pur-
pose of fixation is to kill the tissue without causing much damage
or distortion of the components to be studied, such as nuclei for
FCM. Formalin has the ability to fix DNA through reacting with
related proteins (Sharma and Sharma 1980). It has been used to fix
fish erythrocytes to prepare FCM samples (Crissnian et al. 1979).
Also, it has been used in preparing shellfish FCM samples (Allen
1983) and yield acceptable cell suspension. In this study fixation in
formalin did not produce enough nuclei even with a hypotonic
pre-treatment. The peak position of formalin tlxed samples shifted
lower and the CV was higher than that with the control method.
Our results suggest that 10% formalin is not a good fixative for
preserving oyster gill for FCM.

Both ethanol (15%) and Camoy's fixative are acceptable for
preserving gill tissue for FCM. FCM histograms from ethanol
fixed samples were often the best with smallest CVs. Ethanol is
more readily available, more stable, and less corrosive than Car-
ney's fixative. Therefore, we recommend 75% ethanol over Car-
ney's fixative for preserving tissues for FCM.

Results of this study show that certain tissues are better suited
for FCM than others. Mantle was the most difficult tissue in pro-
viding enough nuclei for FCM. Hemolymph. while acceptable
when frozen, significantly changed peak positions after fixation in
Camoy's fixative. The best tissue for FCM. fixed or frozen, was
gill. It consistently produced large numbers of nuclei, which con-
firms our early experiences.

In summary, oyster tissue samples can be fixed for FCM analy-
sis. With pre-treatments aimed at nucleus dissociation, fixation
does not affect the quality of FCM and ploidy determination. With
several factors considered, we recommend the follow protocol for
fixing oyster samples for FCM: dissect a piece of gill tissue (-0.5
cm"), treat with 0.075 M KCI for 10 min, cut into multiple pieces,
fix in 75% ethanol, and change fixative once. The fixed sample can
be stored at 4 °C or room temperature and transported at ambient
temperature. Before FCM, the sample is washed once using phos-
phate-buffered saline (pH = 6.8) and transferred to a staining/
isolation solution with 10% DMSO. The sample is frozen-and-
thawed, votexed for 10 to 15 sec, aspirated 5 times with a 25-guage
syringe, and then filtered through a 25-|JLm nytex screen. Although
we did not test long-term storage, we were able to analyze samples
stored at room temperature for 6 mo. This protocol has worked for
adult zhikong scallop. ChUimys farreri. and oyster and scallop
larvae, and it is probably applicable to most molluscs.

ACKNOWLEDGMENTS

This study was conducted at Haskin Shellfish Research Labo-
ratory and was supported partly by grants from U.S. Department of
Agriculture, Sea Grant, and the New Jersey Commision on Science
and Technology. Dr. Yang is a visiting researcher from Institute of
Oceanology, Chinese Academy of Sciences, and her participation
is also supported by Grant 819-01-07 from China's National High-

Tech Development Program (863), the "100 Scholar" program of
the Chinese Academy of Sciences, and the China's Natural Sci-
ence Foundation (no. 39825121 ). This is publication number 00-17
of IMCS/NJAES.

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soft-shell clam: cytogenetic and allozymic confirmation. J. Hercility
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Beaumont. A. R. & J. E. Fairbrother. 1991. Ploidy manipulation in mol-
luscan shellfish: a review. / Shellfish Res. 10:1-18.

Chaiton. J. A. & S. K. Allen, Jr. 1985. Early detection of Uiploidy in the
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Chew. K. K. 1994. Tetraploid Pacific oysters offer promise to future pro-
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Crissman. H. A.. A. P. Stevenson. R. J. Kissane & R. A. Tobey. 1979.
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Guo. X.. G. DeBrosse & S. K. Allen. Jr. 1996. All-triploid Pacific oysters
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Guo. X.. W. K. Hershberger. K. Cooper & K. K. Chew. 1993. Artificial
gynogenesis with ultraviolet light-irradiated sperm in the Pacific oyster,
Crassostrea gigas. I. Induction and survival. Aquaculmre 113:201-
214.

Guo. X.. H. Yang. H. Que, Z. Wang. Z. Xu & R. Wang. 1999. Molluscan
cytogenetic biotechnology, pp. 101-125. In: X. Fan, S. Zhang, S. Qin
& X. Yan (eds.) Advances in Marine Biotechnology. Ocean Press,
Beijing.

Hedley. D.. M. Friedlander. I. Taylor. C. Rugg & E. Musgrove. 1983.
Method for analysis of cellular DNA content of paraffin-embedded
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Hedley, D. W. 1994. DNA analysis from paraffin embedded blocks, pp.
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Komaru. A.. Y. Uchimura. H. levama & K. T. Wada. 1988. Detection of
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Sharma. A. K. & A. Sharma. 1980. Chromosome Techniques: Theory and
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Song, P. & S. Li. 1992. Principle and Application of Flow Cytometry.
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Uchimura. Y., A. Komaru. K. T. Wada. H. levama. M. Yamaki & H.
Furata. 1989. Detection of induced triploidy at different ages for larvae
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Utfing. S. D. & R. D. Child. 1994. Genetic manipulation of the Manila
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I

Journal of Slu-llfish Ri-sfoirh. Vol. 19, No. 2, 841-844. 2000.

EVALUATION OF A GLUCOSE OXIDASE/PEROXIDASE METHOD FOR INDIRECT
MEASUREMENT OF GLYCOGEN CONTENT IN OYSTERS {CRASSOSTREA VIRGINICA)

SHELLEY A. BURTON,' ALLAN L. MACKENZIE,'
T. JEFFREY DAVIDSON,^ AND AUDREY C. FRASER^

^Department of Pathology and Microbiology, Atlantic Veterinary College,

University of Prince Edward Island, 550 University Avenue,

Charlottetown, PEI, CIA 4P3, Canada
'Department of Health Management, Atlantic Veterinaiy College,

University of Prince Edward Island, 550 University Avenue,

Charlottetown, PEI, CIA 4P3, Canada

ABSTRACT A colorimetric method for indirect measurement of glycogen concentration.s in ti.s.^ue homogenates of eastern oysters
{Crassostrea virginica) was evaluated. This method uses a conversion of glycogen to glucose by amyloglucosidase. The procedure was
optimized for extracting buffer pH (5.0) and amyloglucosidase concentration (5 mg/mL). Coefficients of variation (h = 10) for oyster
homogenates with mean glycogen concentrations of 84 and 242 mg/dL had within-run values of 3.29 and 3.66%. and between-run
results of 4.46 and 3.15'7r. respectively. When mean glycogen concentrations of thawed oyster homogenates were compared with those
of initial fresh homogenates. no significant {P < 0.05) differences were detected in samples thawed after 1 h. 1 day, 1 wk. or 1 mo.
Glycogen recovery percentages of 104.1. 103.7. and 104.5% were obtained with mi.xed solutions containing 111. 94. and 19 mg/dL
glycogen, respectively. The lower limit of sensitivity for the procedure was appro.ximately 14 mg/dL. The assay was considered to be
linear to 436 mg/dL. Lyophilized samples appeared to provide the most reliable determination of glycogen concentrations per gram
of tissue by avoiding variable water content in oyster tissues. Initial laboratory ranges for tissue glycogen based on wet (mean ± 2 SD:
l-4i mg/g) and dry (mean ± 2 SD: 19-145 mg/g) weights were determined with 49 second-year growth oysters obtained during July
1998 (Covehead. Prince Edward Island. Canada). It was concluded that the colorimetric assay offered a reliable indication of tissue
concentrations of glycogen in eastern oysters (C virginica).

KEY WORDS: Glycogen, oysters. Crassostrea virginica, method validation, colorimetric analysis

INTRODUCTION

Glycogen in oyster tissues has been evaluated in association
with growth (Kaufmann et aL 1994. Littlewood and Gordon 1988).
nutrition level (Whyte et al. 1990). and reproductive cycle (Little-
wood and Gordon 1988). Tissue concentrations of glycogen could
impact on disease resistance and commercial shelf life in this
species. Any method for glycogen determination in oysters must
undergo a validation procedure to ensure accuracy of results. Vali-
dation procedures are mandatory prior to the use of any assay in a
human or veterinary clinical chemistry laboratory (Peters and
Westgard 1986. Murray et al. 1993). It is logical to use the same
assurance of quality for assays used in laboratory investigations in
shellfish. Validation includes assessment of precision, linearity,
recovery, and sample stability for the reagent system (Peters and
Westgard 1986). The method (Carr and Neff 1984) most fre-
quently used to measure glycogen in shellfish tissues utilizes an
enzymatic conversion of glycogen to glucose with amyloglucosi-
dase, followed by measurement of glucose concentrations with
commercially available reagents. This method was recently vali-
dated for Myiihts ediilis tissues (Burton et al. 1997). but it would
be erroneous to extrapolate results to other shellfish species. The
purpose of the study reported here was to determine whether a
commercial oxidase/peroxidase assay for measurement of glucose
in human sera could be validated for indirect measurement of
glycogen in homogenates of eastern oyster (Crassostrea virginica)
tissues.

MATERIALS AND METHODS

Assay Procedure

Mature (6- to 7-cm shell length) oysters (C virginica) were
obtained from a lease in Covehead. Prince Edward Island. Canada.

They were removed from their shells, blotted dry and weighed,
then individually homogenized in ice cold trisodium citrate buffer.
The assay procedure has been described previously for marine
mussels (Burton et al. 1997). except that glycogen standards from
oysters (Glycogen type II from oyster, Sigma Chemical Co., St.
Louis. MO) rather than mussels were prepared. Briefly, following
individual homogenization of oysters in buffer, the samples were
heated in a boiling water bath, cooled, and rehomogenized. Ali-
quots were incubated with amyloglucosidase and the glucose con-
tent was determined using benchtop techniques and a commercial
glucose reagent. Enzyme-untreated (blank) aliquots and standards
were also prepared and analyzed using the same protocol.

Assay Optimization

A buffer volume of 100 mL for homogenization was chosen for
ease of use and to minimize error due to sample loss. To determine
whether the conversion of oyster glycogen to glucose could be
optimized, final buffer pH values of 4.5. 5.0. and 5.5 were evalu-
ated. To achieve this, six oysters were homogenized separately in
20 mL of 0.1 M trisodium citrate buffer (pH 5.0). After boiling
water bath incubation and rehomogenization, the samples were
divided into three aliquots of 5.0 mL each. To these aliquots. 20
mL of 0. 1 M trisodiuin citrate buffer of varying pH were added to
obtain final buffer pH values of 4.5, 5.0, and 5.5. The different
aliquots were analyzed as described previously. To determine
whether the concentration of amyloglucosidase could be optimized
for maximal conversion of oyster glycogen to glucose, amyloglu-
cosidase solutions with concentrations of 40. 20. 10. 5. I. and 0.5
mg/mL were prepared in 0.1 M trisodium citrate buffer (pH 5.0).
After the boiling water bath treatment and rehomogenization as
described above, 5.0 mL aliquots of homogenate from six separate

841

842

Burton et al.

oyster samples were each incubated with these amyloglucosidase
solutions. Enzyme-untreated (blank) solutions and standards were
prepared as previously described.

Assay Evaluation

To evaluate assay precision, within-run and between-run (day-
to-day) studies were conducted, and coefficients of variation (CV)
were calculated. Two oyster samples with mean glycogen concen-
trations of 84 and 242 mg/dL were analyzed 10 times to obtain
data for the within-run calculations. Aliquots of the same homo-
genates were frozen, thawed, and analyzed 10 times in separate
runs over a period of I month to obtain between-run precision. To
determine the frozen stability of oyster glycogen samples, an ad-
ditional six separate homogenates were analyzed for glycogen lev-
els (time zero) and aliquots frozen at -25 °C. These were thawed
after periods of 1 h, 1 day, 1 wk, and 1 mo, and the glycogen
concentrations were determined.

Recovery experiments were conducted to ensure that oyster
glycogen was being recovered and to evaluate the minimal con-
verted glycogen that could be reliably measured by the assay. To
accomplish this, oyster homogenates of known glycogen concen-
trations were diluted 1;9 with the commercial glucose standard (90
mg/dL) provided with the reagent kit, and the observed recoveries
were compared to the theoretical amounts. For minimum recovery
experiments, two oyster homogenates and 0. 1 M trisodium citrate
buffer (pH 5.0) were used. One homogenate was diluted (2-. 3-,
and 4-fold) with the second. A second set of mixtures (blanks) was
prepared using buffer and the second homogenate in the same
ratios. Both sets of mixtures were analyzed, and the glycogen
recovered was determined.

Linearity of the assay was evaluated by measuring the glycogen
concentrations of a set of serial dilutions (in 0.1 M trisodium
citrate buffer, pH 5.0) of an oyster homogenate sample with a high
glycogen concentration (436 mg/dL). The dilutions, based on per-
centages of the previous sample in the series (with expected results
in brackets expressed as mg/dL) were as follows: 100% (436),
75% (327). 66.7% (218), 50% (109), 50% (55). 50% (27). and
50% (14).

Initial laboratory ranges (mean ± 2 SD) for tissue glycogen
based on both wet and dry weights was determined with 49 sec-
ond-year growth oysters obtained during July 1 998 from the Cove-
head region of Prince Edward Island. Canada. To express the
glycogen amounts on a wet weight basis, the homogenates were
aliquoted (1 I niL) after the boiling water treatment and frozen at
-25 "C for 2 weeks. After thawing, the aliquots were analyzed for
glycogen content and expressed as mg/g wet weight (initial
shucked weight). To obtain the glycogen amounts on a dry weight
basis, u 2()-mL aliquot of each oyster sample was obtained follow-
ing the boiling water bath treatment and rehomogenization and
was dispensed into a 50 mL serum bottle (Wheaton '400" boro-
silicate glass; Wheaton. Millville. NJ). Samples were then lyoph-
ilizcd in a freeze dryer (Labconco Corp.. Kansas City. MO) and
stored in a refrigerator (4 °C). The lyophilized sample was
weighed (weight corrected for buffer salt content), reconstituted in
20 mL of deionized water and analyzed for glycogen contcni. The
glycogen concentrations were expressed as mg/g dry tissue.

Stalistical A nalysis

A computer software program (Minitab .Slalislical .Software
Inc., Version 9.1. .Stale Collciie, P.^) was used for statistical cal-

culations. All tests were performed at the P < 0.05 significance
level. Repeated-measures analysis of variance (ANOVA) calcula-
tions were performed to determine if differences existed in glyco-
gen concentrations due to changes in buffer pH and for different
amyloglucosidase concentrations. A repeated measures ANOVA
was also performed to see if significant differences were present
between glycogen concentrations of fresh and frozen aliquots in
the frozen stability assessment over a I -month period. A paired t-
test was used to test for difference between glycogen concentration
obtained with frozen (wet) and lyophilized (reconstituted) homo-
genates. Linear regression analysis was performed for comparison
between glycogen concentrations of the wet and lyophilized (re-
constituted) .samples, expressed as either mg/g or mg/dL and be-
tween wet and dry tissue weights.

RESULTS

Varying the buffer pH and changing the concentration of amy-
loglucosidase in the reagent mixture resulted in no significant (P £
0.05) optimization in glycogen concentrations in oyster samples.
The buffer pH of 5.0 and an amyloglucosidase concentration of
0.5% (5 mg/niL) previously reported by Carr and Neff (1984) and
used for marine mussel tissues (Burton et al. 1997) were therefore
used throughout the study.

In the precision study, the coefficients of variation (n = 10) for
oyster homogenates with mean glycogen concentrations of 84 and
242 mg/dL had within-run values of 3.29 and 3.66%, and between-
run results of 4.46 and 3.15%, respectively.

In the frozen stability assessment, the mean percentage differ-
ences in the glycogen concentrations of the thawed samples com-
pared to the fresh samples was 1.3, 2.7, 1.6, and 1.4%, for the 1 h,
1 day. 1 wk. and 1 mo samples, respectively. No significant (P s
0.05) differences were observed between fresh aliquots and
samples thawed after each time period. Therefore, glycogen in
frozen homogenates was concluded to be stable for at least I
month. Linearity was assessed to be at least 436 mg/dL (Fig. 1 ) as
serial dilutions of an oyster homogenate solution with a high gly-
cogen concentration (436 mg/dL) gave observed results within
3.7% of expected, including a value with an expected glycogen

500

CD E

a

400

300

T3
0)

O

t 200

100

Y = I.OOx + 0,22
r = 1.000

100

200

300

400

500

Theoretical oyster glycogen
concentration (mg/dL)

Figure 1. Linearity plot of a tissue homogenate of an oyster (('. vir-
f>iiiica} with a high glycogen concentration (4.'6 mg/dL) diluted in 0.1
M trisodium citrate huffer (pH SM). Ohserved glycogen concenlraticm
correlated closely (within 3.7%) with expected concentrations.

Indirect Glycogen Measurement in Oysters

843

concentration of 14 mg/dL. Recovery percentages of 103.7% and
104.1% were obtained using mixtures containing 94 and 1 1 1 mg/
dL. respectively (Table 1). Concentrations of 25 and 19 mg/dL
were acceptably recovered (94% and 104.5%, respectively) using
the minimum recovery experiments. However, solutions contain-
ing an expected glycogen concentration of 13.3 mg/dL had an
unacceptably low recovery of 27.1%. Based on the combined in-
formation from the minimum recovery experiment and the linear-
ity assessment, the lower limit of sensitivity for this assay was
considered to be approximately 14 mg/dL.

Initial laboratory ranges for oyster tissue glycogen concentra-
tions are presented in Table 2. Supporting data regarding wet and
dry weights for these 49 oysters are also provided. Linear regres-
sion analysis of the relationship between the converted glycogen
concentration (expressed as mg/dL in solution) in wet and dry
samples, showed significance (P < 0.05) with an r value of 0.98
(Fig. 2). Additionally, a paired t-test indicated no significant (P <
0.05) difference in these converted glycogen concentrations (in
mg/dL) between wet and dry samples. When expressed as glyco-
gen concentrations (in mg) per gram of wet or dry tissue, a sig-
nificant (P s 0.05) relationship was found, with an acceptable
r-value of 0.87 (Fig. 3). The relationship between the actual
weights of the wet and dry tissues (in grams) was significant (P s
0.05), with an r-value of 0.65.

DISCUSSION

The colorimetric assay evaluated in this study was determined
to be a reliable indirect indicator of tissue glycogen concentrations
in eastern oysters (C virginicu).

Two experiments were carried out to determine whether the
conversion of oyster glycogen to glucose could be optimized by
changing the buffer pH and/or the amyloglucosidase concentration
to levels other than previously published by Carr and Neff ( 1984)
in soft shell clams {Mya tnincata) and scallop adductor muscle
(Placopecten magellanecus) or Burton et al. (1997) in mussels
(Mytilus ediilis). Unit changes in pH (4.5-5.5) and a 80-fold in-

TABLE 2.

Laboratory ranges for oyster tissue glycogen based upon wet and

dry weights for 49 second-year growth oysters (Crassostrea virginica)

obtained during July 1998 from the Covchead region of Prince

Edward Island, Canada

Standard

Deviation Range

Variable Mean (SD) (mean ± 2 SD)

Wet weight (g)

2.63

0.55

1.53-3.73

Dry weight (g)

(J.80

0.14

0.52-1.08

Glycogen (mg/dL), wet samples

65.6

27.8

10-121

Glycogen (mg/dL),

lyophilized tissue

66.9

28.8

9-125

Glycogen (mg/g). wet weight

24.8

9.04

7-13

Glycogen (mg/g), dry weight

82.1

31.5

19-145

crease in amyloglucosidase (0.5-40 mg/mL) quantities resulted in
no significant change in glycogen concentration. Therefore, pre-
viously reported values for pH and amyloglucosidase were used in
the rest of the evaluation and are recommended for future glycogen
assays with oyster tissue.

The precision of this assay is acceptable, with coefficients of
variation of less than 3.7% and 4.5% for within-run and between-
run evaluations, respectively. These values are slightly higher than
those reported using the same technique in marine mussels (Burton
et al. 1997). This may be attributed to variability in the technical
skills of different laboratory personnel. The stability of the glyco-
gen levels in the frozen aliquots indicates that samples could be
frozen for at least one month, batched and analyzed in groups. If
glycogen concentrations are proven to influence disease resistance
and commercial shelf life in future studies, batch analysis would be
cost-effective and desirable from a marketing viewpoint. Recovery
experiments indicate that the oyster glucose produced after amy-
loglucosidase conversion responds in the assay identically to hu-
man glucose. Linearity of the system is excellent between 436 and

TABLE L

Recovery data for a colorimetric method for indirect glycogen
measurement in oysters [Crassostrea virginica)

Expected

Observed

Sample

Glycogen

Glycogen

Recovery

Description

(mg/dL)

(mg/dL)

Percentages*

Mixture 1

90.3

93.7

103.7

Mixture 2

106.8

111.3

104,1

Mixture 3

26.6

25.0

94.0

Mixture 4

17.7

18.5

104.5

Mixture 5

13.3

3.6

27.1

A = oyster tissue homogenate (93.2 mg/dL), B = commercial glucose
standard (90 mg/dL), C = oyster tissue homogenate (258 mg/dL), D =
oyster tissue homogenate (53.1 mg/dL), E = 0.1 M trisodium citrate buffer
(pH 5.0).

Mixture 1 = (1 volume A -h 9 volumes B), Mixture 2 = (1 volume C +
9 volumes B), Mixture 3 = ( I volume D + I volume C) - (1 volume E
-f 1 volume C), Mixture 4 = ( I volume D + 2 volumes C) - (1 volume E
+ 2 volumes C). Mixture 5 = (1 volume D ^- 3 volumes Cl - (1 volume
E + 3 volumes C).

* Recovery percentages = (observed concentration)/(expected concentra-
tion) X 100.

160 -

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c

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an -

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>^

r = 0.981

vt

J*

>.

_jfi

O

0 -

' 1

40

80

120

160

Oyster Glycogen Content
(mg/dl)-lyophilizeci tissue

Figure 2. Correlation and linear regression analysis of glycogen con-
centrations (mg/dL in solution) for 49 oysters (C virginica) obtained
from the Covehead region. Prince Edward Island, Canada in July
1998. Glycogen concentrations were obtained with wet and lyophilized
samples and have a significant (P < 0.05) correlation.

844

Burton et al.

.

/

c

40 -

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a>

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•

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CA

10 -

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0 -

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1 ' r

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50

100

150

200

Oyster Glycogen Content
(mg/g-dry tissue)

Figure 3. Regression analysis plot comparing glycogen concentrations
of 49 oysters (C. virgiiiica) in July 1998 from the Covehead region.
Prince Edward Island, Canada. Glycogen concentrations are ex-
pressed as mg glycogen/g-wet oyster weight and mg glycogen/g-dry
weight (lyophilizedl. A significant iP < 0.05) correlation was observed.

14 mg/dL (Fig. 1 ) with results from diluted samples within 3.7%
of expected concentrations. Minimal recovery experiments com-
bined with linearity assessment suggest that 14 mg/dL represents
the lower limit of sensitivity for the assay.

Comparison of the converted glycogen concentrations of the
wet and dry samples (expressed as mg/dL in solution), was per-
formed, using both regression analysis and a paired / test. The
relationship between these converted glycogen concentrations in
wet and dry samples was significant, with a high correlation co-
efficient of 0.98. The paired t-test also indicated no significant
difference in these converted glycogen concentrations (mg/dL) be-
tween wet and lyophilized homogenates. These results suggest that
lyophilization, as might be expected, does not affect the glycogen
levels in the tissue samples.

Correlation results for the glycogen concentration of oysters
expressed as milligrams of glycogen per gram of wet or dry tissue

(Fig. 3) was significant and had an acceptable correlation coeffi-
cient of 0.87. The correlation between the actual weights of these
wet and dry samples, while statistically significant, had a much
lower (0.65) correlation coefficient. As careful surface blotting
was done, this probably reflects variable water content within the
tissues of the wet weight samples. Overall, these results suggest
that the most accurate values are obtained with glycogen expressed
on a lyophilized dry weight basis. However, glycogen expressed
on a shucked wet weight basis could still be useful to commercial
growers desiring quick, albeit less accurate results, for assessments
such as shelf life prediction. A shucked wet weight glycogen
analysis can be performed in less than 24 h compared to 4—5 days
required for the dry weight determination. In this study, it was
convenient to process samples at room temperature over a 24-h
period. Carr and Neff ( 1984) report that samples can also be in-
cubated in a water bath (55 "C) for 2 h. This would appreciably
shorten analysis time in a commercial setting.

It should be noted that the boiling water bath incubation rep-
resents an important step in the procedure. Heating has been re-
ported (Carr and Neff 1984) to inactivate endogenous glycogena-
ses in shellfish tissues, which could alter the glycogen concentra-
tion obtained using enzymatic glucose analysis. More convenient
methods of heating samples, such as microwave use. remain to be
evaluated.

Initial laboratory range (Table 2) for tissue glycogen based on
both wet and dry weights was determined for samples from the
Covehead region of Prince Edward Island in July, 1998. More
work is required to derive reliable reference ranges for whole-body
glycogen concentrations in oysters for time of year, reproductive
activity and geographic region. In summary, indirect tissue glyco-
gen concentrations in the oyster (C viri;iiucci) were reliably de-
termined by this colorimetric assay . With this assurance of reli-
ability, further studies may show tissue glycogen concentration to
be valuable in assessing overall health status or in predicting shelf
life in eastern oysters.

ACKNOWLEDGMENT

Funding was provided by the Co-operative Agreement for Fish-
eries Development, Prince Edward Island, Canada.

LITERATURE CITED

Burton. S. A., A. L. MacKenzie, T. J. Davidson & N. MacNair. 1997.
Evaluation of a glucose oxidase/peroxidase method for indirect mea-
surement of glycogen content In marine mussels (Mylilii.s eiliilis). J.
Shellfish Res. l6:4.V'i-t.^9.

Carr, R. S. & J. M. NelT. 1984. Quantitative scmi-autonialed cn/ymutic
assay for tissue glycogen. Cum/). Hioihem. I'hysiol. 776:447— +49.

Kaufmann. M. J.. M. N. L. Seaman. C. Andradc & K. Buchholz. 1994.
.Survival, growth and glycogen conlcnl cil Pacific oyslcrs, Criixsosireii
f-if;cis (Thunherg. \T)?]. at Madeira Island iSuhlropical Atlantic). ./.
Shellfish Kes. \iMn-505.

Littlewood, D. T. J. & C. M, Gordon. 19S8. Sex ratio, condilion and

glycogen content of raft cultivated mangrove oysters Crassoslrea
rhizophorae. J. Shellfish Res. 7:.19.')-399.

Murray. W.. A. T. Peter & R. F. Tedaw. 1993. The clinical relevance of
assay validation. Comp. Com. Ed. Pnul. Vei. I.'i:166.'i-I67.'i.

Peters. T. & J. O. Wcstgard. 1986. Evalualion of methods, pp. 4 1()-423. In:
N. W. Tiel/ (cd.). Tcxthook of Clinical Chemistry. W.B. Saunders Co..
Philadelphia.

Whyle. J. N. C, J. R. Englar & B. L. Carswell. 1990. Biochemical com-
position and energy reserves in Crassoslrea gigas exposed to dilferent
levels of nutrition. Aquacullure. 90:157-172.

Journal of Shellfish Resecinh. Vol. 19. No. 2. 845-851. 2000.

SYNCHRONOUS OOGENESIS DURING THE SEMILUNAR SPAWNING CYCLE OF THE

TROPICAL ABALONE HALIOTIS ASININA

EDWARD J. JEBREEN, REGINA T. COUNIHAN, DON R. FIELDER,
AND BERNARD M. DEGNAN*

Department of Zoology and Entomology
University of Queensland
Brisbane. Qld 4072 Australia

ABSTRACT On the southern Great Barrier Reef, Haliotis asinina (Vetigastropoda: Pleurotomarioidea) synchronously spawn every
2 wk in a predictable fashion, allowing detailed analysis of reproduction, gametogenesis, and gonad development. Histological
examination of the ovaries of members of the Heron Reef population during this semilunar cycle reveals that oogenesis is also
synchronous and predictable, and requires more than two spawning cycles (i.e. >28 days) to complete. Shortly after a spawning event
the ovary comprises two cohorts of primary oocytes, one of which will be released at the next spawning event, and clusters of oogonia.
At this time there is a rapid proliferation and expansion of trabeculae, germinal epithelial, and oogonia. and a dramatic increase in the
size of the vitellogenic oocytes to be spawned at the next spawning event. Within 4 days these oocytes have filled the ovary. On the
day of the next spawning a lumen forms in the ovary as a result of localized degradation of trabeculae. The large primary oocytes
dissociate from the receding trabeculae, initiate maturation, and accumulate in the lumen; these oocytes become embedded in a jelly
coat layer. The next cohort of oocytes remain attached to the trabeculae. The jelly coat appears to be completely dissolved within 30
min of spawning. Comparison of the oogenesis and ovary development in H. asinina with other abalone species indicates that these
processes are very similar in tropical and temperate abalone. This suggests that insights into the regulation of reproduction and
spawning in H. asinina are likely to be applicable to other haliotids,

KEY WORDS: Gametogenesis. gastropod, germinal vesicle, haliotid, spawning cycle

INTRODUCTION

Reproductive cycles of marine invertebrates are often regulated
by a combination of exogenous and endogenous rhythms (Olive
and Garwood 1983). Temperature is often suggested as the main
environmental variable regulating abalone reproductive cycles
(Hahn 1989, Wells and Keesing 1989). Gametogenesis in the Japa-
nese abalone Haliotis discus hamuli has been linked quantitatively
with temperature. This species must experience a critical minimum
water temperature before gonad maturation is initiated (Kikuchi
and Uki 1974). Gonad maturation increases linearly with increas-
ing water temperature and the stage of maturity can be predicted
by the time spent above the critical minimum temperature (Uki and
Kikuchi 1984).

Reproductive cycles of different species and populations of
abalone vary significantly (Webber and Giese 1969, Shepherd and
Laws 1974, Hahn 1989, Hooker and Creese 1995, Wilson and
Schiel 1995). For example, the five species of abalone that inhabit
the waters along the southern Australian coast either spawn syn-
chronously during spring and summer (H. cyclobates and H. lae-
vigata), autumn and winter (H. ruber), or intermittently throughout
the year {H. roei and H. scalaris) (Shepherd and Laws 1974).
Webber and Giese (1969) observed that two populations of the
black abalone H. cracherndii. 1 1 km apart, spawn at different
times. This high degree of inter- and intraspecific variation sug-
gests that both intrinsic species-specific and local environmental
factors are important in regulating reproduction.

H. asinina, the largest of the tropical abalone species, is dis-
tributed throughout the coral reefs of the Indo-Pacific, including
the Great Barrier Reef (Talmadge 1963, Fallu 1991 1. In The Phil-
ippines, H. asinina are serial spawners, spawning asynchronously
year-round except during May and June (Capinpin et al. I998j. In

*Corresponding author.

Thailand, the spawning season of H. asinina is year-round except
April and May, and peaks in October and November when the
water temperature is lowest (Singhagraiwan and Doi 1992). Both
Capinpin et al, (1998) and Singhagraiwan and Doi (1992) re-
stricted their observations almost solely to H. asinina that were
housed in aquaria for months or ones that had been bred in cap-
tivity. Capinpin et al. ( 1998) did observe that recently captured H.
asinina (less than 6 wk) exhibited spawning patterns that were
more synchronous than longer-term captive abalone. H. asinina on
Heron Reef, Southern Great Barrier Reef exhibit synchronous
semilunar spawnings from October to April (Counihan et al. in
press). This highly predictable and rapid spawning cycle appears to
be unique amongst the haliotids and facilitates the analysis of
reproduction, gametogenesis, and gonad development, and the ex-
ogenous and intrinsic factors regulating these processes.

As a first step toward understanding gametogenesis and spawn-
ing in H. asinina we have undertaken a histological examination of
female ovaries during the semilunar spawning cycle. Previous
studies on the reproductive biology and gametogenesis in H. as-
inina have focused either upon determining the reproductive status
of wild-caught abalone (Capinpin et al. 1998) or classification of
gonads and gametes (Apisawetakan et al. 1997), Here we report
that oogenesis and ovary development in Heron Reef H. asinina is
synchronous, predictable, and tightly linked to the semilunar
spawning cycle. The different oocyte stages present within the
ovary can be classified into one of three cohorts that are develop-
ing simultaneously in the ovary. These three cohorts will be
spawned sequentially over the next three spawning events. These
data demonstrate that while a single spawning cycle in H. asinina
is extremely short, being 13 to 15 days long, oogenesis requires
more than two spawning cycles (i.e. >28 days) to complete.

MATERIALS AND METHODS

H. asinina were collected from the outer-coral algal subzone of
the reef flat of Heron Reef (23°27'S, l5r55'E) and kept at The

845

846

Jebreen et al.

University of Queensland Heron Island Research Station. Captured
abalone were maintained in flowing, ambient seawater and a natu-
ral light regime, and fed Grucilaiia spp. every 3 days. Between
November 4 and December 10. 1995, ovaries were excised from
female abalone daily. These were washed in 0.2 jj.m filtered sea-
water, left in Bouin's fixative for 6 days, washed three times in
70% ethanol over 3 days, dehydrated through a graded ethanol
series into xylene, and embedded in paraffin wax. Eight-
millimeter-thick transverse sections were cut at four equally par-
titioned sites along the ovary. Sections were transferred to glass
microscope slides, dried at 40 °C overnight, and stained with May-
er's haematoxylin and eosin. Spawned eggs were fertilized as de-
.scribed in Counihan et al. (1998). Live eggs, zygotes, and embryos
were whole mounted under raised coverslips. All light micro-
graphs were taken with an Olympus BX50 microscope with dif-
ferential interference contrast optics.

RESULTS

The 36-day series of sections taken from four equally spaced
regions of the H. asinina ovary revealed that oogenesis within an
individual female proceeded synchronously throughout the ovary.
Hence a section through any region of the ovary was representative
of the developmental state of the entire gonad. Ovaries that were
isolated from different females on the same day (i.e. at the same
time of the spawning cycle) were structurally very similar and
possessed oocytes at the same stage of oogenesis.

In a sexually mature females the green ovaries surround the
hepatopancreas. forming the conical appendage. Non-invasive
analysis of the conical appendage revealed that the extent of cov-
erage of the digestive gland by the ovary varied with season and
time in the semilunar spawning cycle (Singhagraiwan and Doi
1992. Capinpin et al. 1998). Microscopic analysis of the ovary
indicated that gross morphological changes in the conical append-
age during the spawning cycle correlated with changes in the struc-
ture of the ovary and the developmental state of oocytes within
(Figs. I and 2).

Oogenesis and ovary development were monitored during the
semilunar spawning cycle of H. usiinna in November and Decem-
ber. 1995. During this time of the reproductive season nearly all
sexually mature individuals were ripe and spawning regularly
(Counihan et al. in press). Inspection of ovaries that were fixed
during the spawning event revealed that three cohorts of oocytes
were present (Fig. I, A and B). These were classified according to
developmental .stage, with ( I ) oocytes that were in process of being
spawned being classified as mature oocytes (i.e. undergone ger-
minal vesicle breakdown); (2) large, late vilellogenic primary oo-
cytes attached to the trabeculae being classified as Cohort 1 oo-
cytes; and (3) small, early/pre-vitellogenic primary oocytes at-
tached to the irabcculae being classified as Cohort II oocytes.
Clusters of small (8-15 fxm) oogonia tightly associated with the
trabecular connective tissue were also present (Figs. I and 2 1. Al
spawning, ovary structure varied within individual females, with
trabeculae in most of the gonad projecting partially into the center
of the ovary from both proximal (side adjacent to the digestive
gland) anil distal (side adjacent to the epidermis) sides; the central
portion of this part ot the gonad lacked trabeculae (Fig. 2).

Toward the posterior tip of the ovary, trabeculae traversed the
entire gonad from the epidermal to the digestive gland side (Fig.
I A). Cohort I and II oocytes and oogonia were attached to the
trabeculae anil interspersed amongst each other (Fig. 1, A-D). The

base of Cohort I oocytes (i.e. side attached to the trabeculae)
corresponds to the future vegetal side, as the germinal vesicle (GV)
was located at the opposite end of the developing oocyte (Fig. IB).
Both Cohort I and II primary oocytes had intact GVs and nucleoli,
suggesting these oocytes were actively transcribing rRNA genes.
A single nucleolus was observed per GV, suggesting that single
nucleolar organizing region (i.e. rRNA gene cluster) was present in
the genome of//, asinina (Degnan et al. 1990). The GV remained
intact until the day of the spawning, at which time mature oocytes
became dissociated froin the trabeculae and predominantly local-
ized to the central cavity or oviduct (Figs. lA and 2). These oo-
cytes had undergone germinal vesicle breakdown (GVBD) and had
initiated meiosis to first metaphase.

The next morning approximately 8 h after spawning had fin-
ished, most of the trabeculae had increa.sed so as to span most of
the ovary from proximal to distal sides (i.e. the lumen had largely
disappeared; Fig 1 . C and D). We did not determine if this apparent
growth was because of cell proliferation in the trabeculae (i.e.
regeneration), changes in the shape of this tissue, or a combination
of these proces.ses. Cohort I and II oocytes and oogonia remained
associated with the trabeculae and spanned its entire length, sug-
gesting the structural modification of the trabeculae was partially
responsible for reorganization of the gonad. Active rRNA gene
transcription was detected in both cohorts of oocyte at this stage of
gonad development. There appeared to be little increase in size of
Cohort I and II oocytes at this stage.

A small number of unspawned mature oocytes were present in
the gonad after the spawning event had finished (Fig. 1. C and E).
At 8 h after spawning these oocytes were being engulfed by small
amoebocytic cells (Fig. IE). These cells were particularly abun-
dant in the jelly coat and appeared to phagocytize this acellular
material (Fig. IE). By 2 d after spawning we did not detect mature
oocytes or amoebocytes in the gonad, suggesting that the oocytes
had degenerated and been phagocytized by the amoebocytes,
which had subsequently migrated from the gonad. At this stage,
the distance between rows of trabeculae appeared to have de-
creased and the trabeculae had become thicker (Fig. IF). The yolk
content in Cohort I primary oocytes had increased slightly. These
oocytes had follicular cells surrounding the base (Fig. IG).

Fours days after the previous spawning, the ovary was packed
with yolky. polygonal Cohort I primary oocytes (Fig. I, H and I).
Cohort II oocytes were present interspersed amongst the large
Cohort I oocytes; oogonia were not readily observed in the sec-
tioned ovaries. The base of Cohort I oocytes that was attached to
the trabeculae had widened, giving the oocyte its polygonal shape
(Fig. II). A thin vitelline envelope or layer (5-10 (a,m thick) and
jelly coat were first observed around the Cohort I oocytes al this
stage. Between 4 and 1 3 d post-spawning stages we did not detect
any obvious differences in the size and shape of the Cohort I
gonads oocytes or in the overall structure of the gonad, except that
the vitelline envelopes and jelly coats were expanded in the older
oocytes (Fig. 1, J-L).

Twelve to 18 h before the next spawning, the gonad and Cohort
I oocytes began undergoing dramatic changes (Fig. 2. A and B).
Trabeculae became thin and no longer traversed the ovary from the
proximal and distal edges. Trabeculae first disappeared from the
middle of the ovary (Fig. 2A) and appeared to be receding from the
center of the ovary towards the periphery. Amoebocytic cells were
not associated with the tip of the receding connective tissue, which
increased in thickness towards the periphery (Fig. 2B). With an
increase in the thickness of the peripheral trabeculae. muscle cells

Oogenesis in Haliutis asinina

847

Figure 1. Oogenesis and ovary development during the semilunar spawning cycle of H. asinina. Part I, vitellogenesis. All photomicrographs are
haematoxylin-eosin stained transverse sections through the abalone ovary. A and B) Mid-spav\n. A section of the ovary where there is limited
trabecular degradation (i.e. where de novo oviduct formation has not occurred). Mature oocytes (M) are dissociated from trabeculae (t) while
Cohort I and II primary oocytes are attached. C-E) 8 h after spawning is completed. C and D) Trabeculae traverse almost the entire ovary.
Active rRNA gene transcription is occurring in the germinal vesicles (GV) of attached Cohort I and II oocytes as detected by the presence of
nucleoli (arrowheads). A small number of unspawned mature oocytes are present (arrow in C). Clusters of oogonia (og) are present on the
trabeculae. E) An unspawned oocyte with multiple small amoeboid cells in the jelly coat (arrowheads); these cells appear to be phagocytizing
the jelly coat. The micrograph in the bottom left corresponds to the box: many of the amoeboid cells have pseudopodia (arrowheads). F and G)
2 days post-spawning. The distance between rows of trabeculae decreases and the trabeculae have become thicker and more muscular. The yolk
content in Cohort I primary oocytes has increased. Follicular cells (arrows on some cells) surround the base of the oocyte. Unspawned mature
oocytes are never detected in this or subsequent stages. H and I) 4 days post-spawn. The ovary is packed with yolky Cohort I primary oocytes;
small Cohort II oocytes and trabeculae are still evident, although thinner than 2 days before. The vitelline envelope (ve) surrounding Cohort I
oocytes is approximately 5- to lU-pm thick. .1 and K) 1(1 days post-spawning. There is no obvious differences between 4 and 10 days post-spawning
ovaries or Cohort I oocytes, except the vitelline en\elope is thicker, being typically 10- to 15-pni wide in the older oocytes and jelly coat (jc) later
is obvious. L) 12 days post-spawning (2 days before next spawning). Cohort I primary oocytes are associated with trabeculae and are approxi-
mately the same size and possess the same histological characters as those in 4 and 10 post -spawning ovaries (see Fig. I, H-K), except the vitelline
envelope around the oocytes is thicker (approximately 12-17-nm thick). Scale bars: A, C, F, H, J, and L, 200 fim; B, D, E, I, and K, 50 jim; G,
25 fim.

848

Jebreen et al.

G

■ji ^5C ^* ^ .4^'* SL'f'at* •.#♦*" \#V^..-..

.t*?l

? i^:

^L -3.

Figure 2. Oogenesis and ovary development during the semilunar spawning cycle of//, asiiiina. Part II, trabeculae breakdown, oocyte matu-
ration, and tie novo oviduct formation. All photomicrographs are haematoxylin-eosin stained transverse sections through the abalone ovary. A
and B) 12 h before spawning. A) Trabeculae (t) are disappearing from the middle of the ovary. Cohort I oocytes are cither dissociated from or
attached to trabeculae, depending on location within the ovary; peripheral oocytes appear to be still attached (arrows). Oocytes associated with
trabeculae appear to still have intact germinal vesicles ((JV) with nucleoli, while a majority of released oocytes appear to be in the process of
maturation and germinal vesicle breakdown (arrowheads). 15) Higher magnification of the region where trabeculae appears to be degrading or
retracting (arrows) with Cohort I and 11 oocytes; jc, jelly coat. C-E) 4 to 6 h before spawning. Trabeculae degeneraticm continues with further
release of oocytes. Oocyte maturation is characterized first by the loss of the nucleolar organizing region (arrowheads), then by the absence of
a distinct germinal vesicle membrane (i.e. nuclear envelope; small arrows), and finally by a change in staining of nuclear material from light to
dark purple (large arrows). At this stage vitelline envelopes appear to be separating from the surfaces of the oocytes. F) Mature oocytes at
spawning. Oocytes arc embedded in a common Jelly coat and have completed germinal vesicle breakdown. (J) Ovary at spawning, with mature
oocytes in a jelly coat mass in the newly formed oviduct. The digestive gland (dg) is at l>ottoni and the outer epidermis (e) is at the top. Trabeculae
with associated Cohort I (previously defined as Cohort III and II oocytes project predominantly from the distal edge of the gonad (arrows). Scale
bars: A and C, 2(MI Mm: B, 5(1 pm; D, K.and F, KM) Mm: <;. 1 mm.

became appaiciil (Fig. 2B). Ciihorl I oocytes wei'e either dissoei-
atcd from or allaehcd to trabeeulae. depending on iheir location
within the ovary (Fig. 2A), with oocytes closer to the periphery of
ovary largely attached to the trabeculae. Oocytes that were no
longer attached to the trabeculae appeared to be undergoing malii-
ration and GVBD. while allached oocytes maintained intact GVs
with nucleoli. The first phase of GVBI5 was characterized by the
degeneration of the nucleoli, which initially took on a perforated
appearance. This was followed by the loss of a distinct GV meii)-
brane and a change in staining of nuclear material from light to
dark purple (Fig. 2, A-E). At this stage the \itelline envelope

appeared to become dissociated from the oocyte plasma mem-
brane. Shortly befoie spawning the mature oocytes (previously
Cohort I oocytes) coalesced in the center of the ovary and appeared
to become embedded in a common jelly coat (Fig. 2. F and G). The
\ilelline envelope appeared to be much thinner in the mature oo-
cytes compared to oocytes prior to maturation (Fig. 2. F and L^). At
spawning this mass of mature oocytes was released into the sea-
v\aier, where they dissociated into individual eggs (Fig. 3).

The spawned eggs were surrounded by a vitelline envelope that
was less than I p.m thick. A peri\itclline space was present be-
tween this en\elope and the oocyte. .Surrounding the entire egg

Oogenesis in Haliotis asinina

849

Figure 3. Fertilization of spawned eggs at 24 °C. All photomicro-
graphs are whole mounts of living eggs, zygotes or embryos. The ani-
mal pole is towards the top In all figures. A) Spawned mature oocyte
in metaphase of the first meiotic division. A thin vitelline envelope (ve;
less than 1 pm thick) surrounds the light green oocyte; these are sepa-
rated by a perivitelline space (ps) of about 20 pm. A jelly coat (jc) layer
surrounds the egg. The egg and jelly coat together have a diameter of
about 600 pm. B) Zygote approximately 15 min after fertilization. First
polar body (pb) is evident and a dark green cytoplasm begins migrat-
ing towards the vegetal hemisphere. The jelly coat is still about 600 pm
in diameter. C) Zygote approximately 25 min after fertilization. Cy-
toplasmic rearrangement continues such that the vegetal hemisphere is
dark green and animal hemisphere yellow. The jelly coat is no longer
detected. D) Two-cell embryo approximately 35 min after fertilization.
Two polar bodies are located on the animal side of the cleavage plane.
The vitelline envelope is present, but not the jelly coat. Scale bar: 100 pm.

was a jelly coat layer that was approximately 180 |jim thick (Fig.
3A). Within 15 min of fertilization the first polar body was de-
tected at the animal pole and within 25 min the jelly coat was no
longer evident (Fig. 3, B and C). During this period the egg cy-
toplasm underwent dramatic rearrangements such that the zygote
had a dark green vegetal hemisphere, yellow animal hemisphere,
and a white equatorial band.

DISCUSSION

Both gonad maturity and spawning behavior in H. asinina on
Heron Reef is seasonal, with fecund abalone spawning during the
summer from October to April. Analysis of spawning during the
summer reproductive season indicates that all individuals undergo
synchronous spawning, with gametes released approximately ev-
ery 14 d (this study. Counihan at al. in press). This predictable
semilunar spawning pattern is unique amongst the haliotids. H.
asinina populations in The Philippines and Thailand are fecund for
most of the year and captive populations exhibit asynchronous
spawning behavior (Singhagraiwan and Doi 1992. Jarayabhand
and Paphavasit 1996. Capinpin et al. 1998). Unpredictable syn-
chronous spawning of H. asinina occurs on Panagatan Reef, An-

tique, Philippines (Capinpin 1995) and recently captured H. as-
inina spawn more synchronously than longer-term captive abalone
(Capinpin et al. 1998).

In this study we exploited the predictable spawning cycle of H.
asinina from the southern Great Barrier Reef to obtain a detailed
understanding of the timing of developmental and structural
changes in abalone ovaries during the natural reproductive season.
Because all individuals in the population develop in synchrony we
were able to acquire a set of histological samples from individuals
at different times of the spawning cycle that together reflect the
timing of oogenesis and gonad development. Since in other aba-
lone the timing of natural spawning events is less predictable and
often asynchronous (Webber and Giese 1969, Shepherd and Laws
1974. Hahn 1989. Hooker and Creese 1995. Wilson and Schiel
1995), it is difficult to acquire a comprehensive developmental
time course by invasive methods. The H. asinina reproductive
cycle provides a tractable experimental system to investigate natu-
ral endogenous and exogenous factors controlling gonad develop-
ment and spawning behavior in abalone. Importantly, comparison
of ganietogenesis and gonad organization and development in H.
asinina with that existing in other abalone (reviewed in Hahn
1989) indicates that these processes and structures are nearly iden-
tical in tropical and temperate abalone. This suggests that insights
into the regulation of reproduction and spawning in H. asinina are
likely to be applicable to other haliotids.

Underlying macroscopic changes in ovary structure (i.e. those
observed by non-invasive techniques) are stereotypic changes in
trabecular connective tissue morphology and the oocytes. Ovarian
trabeculae degeneration and de novo formation of the lumen is
restricted to the area in the middle of the ovary near to the hepato-
pancreas and is considerably less than that observed for other
species of abalone (e.g. Tutschulte and Connell 1981 ). The rapid
reformation of trabeculae within 2 d of spawning probably results
from the reduced, localized nature of the pre-spawning breakdown.
Associated with the restructuring trabeculae and germinal epithe-
lium is a cohort of early primary oocytes (termed Cohort II oocytes
in this study) and cohort of larger vitellogenic primary oocytes
(Cohort I oocytes). Follicle cells present at the ba.se of Cohort I
oocytes, at the connection to the trabeculae, may be contributing
yolk to the developing oocytes (Dohmen 1983, de Jong-Brink et al.
1983, Hahn 1989, Voltzow 1994). These Cohort I oocytes appear
to have grown close to full size within 4 d of the previous spawn-
ing (compare Figs. 1, A and B with 1, H and I). In contrast the
cohort of smaller Cohort II oocytes has not increased proportion-
ally in size during the same period (compare Figs. I, A and B with
1, H and I: Fig. 4).

Analysis of the progression of both these oocyte cohorts
through a single 2-wk spawning cycle suggests that oogenesis
from a small primary oocyte (approximately 12-18 pirn) to mature
oocyte that will be spawned takes more than 28 d (Fig. 4). The
presence of different-sized oogonia shortly after a spawning event
suggests that some of these stem cells have begun oogenesis. To-
gether, these data suggest that the entire process of oogenesis is
slightly longer than 28 d and may be as long as 40 d.

The early/previtellogenic primary oocyte is actively transcrib-
ing rRNA genes, but not accumulating large amounts of yolk. The
rate of yolk deposition in these oocytes begins increasing around
the time of spawning of the older oocytes (i.e. about halfway
through oogenesis) and vitellogenesis appears to be completed 4 to
5 d later. During the next 8 to 9 d of gametogenesis there is no
obvious change in oocyte size or histospecific character; these

850

Jebreen et al.
maturation

maturation

160

I 120

E
n

■B

o

5.
u
o
O

<5 80

40

spawning
event

spawning
event

spawning
event

Figure 4. Oocyte growth and maturation in H. asinina during two consecutive spawning cycles. Maturation of Cohort I oocytes occurs 12 to 18
h prior to spawning. At any point in the spawning cycle the ovary contains two or three cohorts of primary oocytes and oogonial stem cells. Balls
at the end of the line represent oocytes that will be spawned; arrows represent oocytes that will continue to develop and grow.

older oocytes still appear to be actively transcribing rRNA genes,
as nucleoli are still present. During this period the packed polygo-
nal-shaped oocytes begin to produce a set of extracellular layers
that can be discerned by refractive properties under differential
interference contrast microscopy (Figs. 1 and 2). The inner refrac-
tive layer has been called the vitelline layer (Young and DeMartini
1 970, Lewis et al. 1 982), while the clear outer layer has been called
the chorion (Young and DeMartini 1970) or jelly coat (Lewis et al.
1982). We have used the term jelly coat, as this layer appears to be
very similar to an identically named structure in sea urchin eggs.
As in sea urchins the jelly coat is a transient layer associated with
spawned H. asinina eggs, disappearing shortly after fertilization.

Like many gastropods (Dohnien 1983. de Jong-Brink et al.
1983, Longo 1983). H. asinina and other abalone (Hahn 1989)
oocytes mature shortly before release, undergoing GVBD and re-
maining at metapha.se of the first meiotic division until fertiliza-
tion. We have observed that the onset of maturation does not begin
until the oocytes are dissociated from the trabeculae. some 12 to 18
h before spawning, although we did not determine if the process of
dissociation is the agent that induces the onset of maturation.
Nonetheless there must exist a mechanism to ensure that only large
Cohort I oocytes dissociate and not the immature Cohort II. as we
did not detect (he release of any small oocytes during oviduct
formation. This differential release of the older oocytes also occurs
in other abalone (see Tutschulte and Council 1981 ).

Attempts to induce spawning artificially in H. asinina with a
range of chemical and environmental factors that are known to be
effective in inducing other abalone to spawn (Morse et al. 1977.
Hahn 1989) has been problematic. Methods used for other abalone
(i.e. hydrogen peroxide sea water, ultraviolet- (UV) irradiated sea-
water, desiccation, and thermal shock) have been shown to be
ineffective for H. asinina (Singhagraiwan and Sasaki 1991, .Sing-
hagraiwan and l)oi 1992, Jarayabhand and I'uphavasit 199(i, Cap-

inpin et al. 1998, personal observations). We suggest that the pro-
tracted period between onset of maturation and spawning contrib-
utes to this lack of success. While the exact mechanisms by which
these treatments induce oocyte release from the trabeculae. oocyte
maturation and spawning behavior are unknown. Morse et al.
(1977) suggested that hydrogen peroxide and UV light activate
prostaglandin endoperoxide-forming cyclooxygenase which syn-
thesizes a product that induces spawning. It has not been deter-
mined if this activation is required for oocyte maturation, spawn-
ing, or both processes, although hydrogen peroxide or UV light
treatments typically result in mature eggs being spawned a few
hours later; H. rufescens, H. diversicolor diversicolor. and H. dis-
cus hannai spawn 2.5-5, -I, and -1.5 h, respectively, after treat-
ment begins (Morse et al. 1977. Takashima et al. 1978, Uki and
Kikuchi 1984). In H. asinina. hydrogen peroxide has been shown
to induce spawning behavior without the release of gametes (Cap-
inpin 1995). Together, these data suggest that the inability of well-
proven inducers of spawning to affect H. asinina may be because
oocyte maturation and spawning are temporally uncoupled com-
pared to other species.

We propose that ovulation and spawning in H. asinina requires
two steps, with both being associated with the tidal cycle. Ovula-
tion, which includes trabeculae degeneration. Cohort I oocyte
maturation, and dc novo formation of a lumen, begins during a
morning low tide that occurs around a new or full moon (i.e. spring
low tide). Once females have ovulated they are competent to
spawn. Spawning occurs at the next evening high tide, approxi-
mately 18 h later. While individual female H. asinina are able to
spawn over two consecutive evening high (ides (Counihan el al. in
press), we did not determine if there are two ovulation events.
While both ovulation and spawning are correlated with spring
tides, endogenous signals are sufficient to induce both these
events. H. asinina removed from natural lidal stimuli for a limited

Oogenesis in Haliotis asinina

851

period (i.e. up to about 6 wk) still spawn viable gametes in syn-
chrony with recently captured individuals (Counihan et al. in
press). These endogenous rhythms appear to be set and maintained
by lunar and tidal cycles.

ACKNOWLEDGMENTS

We thank Dr. Scoresby Shepherd for useful comments on the
manuscript, the staff of the Heron Island Research Station for their
assistance. Lina Daddow for technical assistance, and David Mc-
Namara for sharing unpublished data. This research was supported
by a University of Queensland New Staff Research Grant to
B.M.D.

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Journal of Shfllfisli Rcst-airh. Vol. 19. Ni). 2. 853-859. 2000.

POPULATION GENETIC ANALYSIS OF THE AB ALONE HALIOTIS FULGENS (MOLLUSCA:

GASTROPODA) IN BAJA CALIFORNIA, MEXICO

GERARDO ZUNIGA, SERGIO A. GUZMAN DEL PROO,
RAMON CISNEROS, AND GERARDO RODRIGUEZ

Departamento de Zoologia
Escuela Nacioual de Ciencias Biologicas-IPN
Pmlongacion de Carpio y Plan de Ayala s/n
Mexico D.F. 1 1340. Mexico

ABSTRACT Population genetic sti^cture was analyzed for Halimis fiilgens in the central part of Baja California, Mexico. The study
was carried out based on tlve abalone sites using electrophoresis analysis. Allozyme data for seven loci from individuals within ahalone
sites were obtained, and F statistics were used to as.sess population structure. Allozyme variation was low. but more similar than the
one reported in other haliotids. Malate dehydrogenase (MDH-1 and MDH-2) and glutamate oxalate transaminase (GOT) in some
population were fixed. There were not observed significant deviations from Hardy-Weinberg equilibrium among 31 comparisons made
over all loci and all populations. F statistics did not indicate departure from random mating or evidence of genetic structuring of the
H. fulgens populations, however f n- and f i^ data showed that inbreeding occurs inside abalone sites. Additionally, cluster analysis and
Mantel tests showed that genetic differences among abalone populations were not accumulative geographically. Data do not suggest
that H. fiilaeiis populations are genetically isolated from one another. The five populations formed a homogeneous group respect to
allele frequencies at six of the seven loci analyzed. Therefore, they cannot be considered as different stocks. The results suggest that
gene flow along the distribution of abalone banks could be variable depending of the coastal topography, dominant swells, and driving
winds, which determine the local coastal hydrodynamic and consequently the level of larval interchange among populations,

KEY WORDS: Allozymes, Haliotis fulgens. gene flow, gene variation, population structure, stock, Baja California

INTRODUCTION

The abalone {Haliotis spp,) is one of the highest value com-
mercial fishery resources worldwide, Haliotids are found in the
mild and cold waters of the Western Pacific, Indopacific. Eastern
Atlantic, and Califomian Current. In Mexico they are found along
the Pacific coast of the Baja California Peninsula where they are
commercially exploited. Seven species exist in this area and Hali-
otis fulgens Philippi constitutes 85% of the total catch. This species
is found between Santa Barbara, California and Isla Margarita.
Baja California. Mexico and is most abundant along the central
part of the peninsula between Isla Cedros and Punta Asuncion
(Guzman del Proo 1992).

Over the last two decades the total catch of worldwide abalone
fisheries has decreased (Shepherd and Brown 1993). The catch in
Mexico fell 5-fold between 1970 and 1985 and for H. fulgens the
catch decreased 3-fold during this period of time (Shepherd et al.
1991). This situation has been attributed mainly to over-
exploitation (Guzman del Proo 1992).

Aquaculture, life history, and population dynamics studies have
been carried out in Mexico to promote the recovery and sustain-
able yield of abalone fishery, however, no genetics studies have
been carried out.

Awareness of the importance of genetic studies for a rational
fisheries management has grown in recent years because an inad-
equate knowledge of genetic diversity and population structure
may impede any conservation or management program.

In this context, stock identification is a crucial aspect because
of the likelihood that a natural population may be composed of
spatially or temporally isolated stocks as a function of topography,
life history, behavior, larval dispersal, and historical circumstances
(Spangleret al. 1981). Thus in the abalone fishery a basic question
prevails: Do the abalone sites in each fishery region constitute the
same unit population or are they independent isolated units?

Allozyme analysis may be a good method to determine genetic

structure within and among site variation for a region. In haliotids
this kind of study has only been carried out in H. rubra Leach
(Brown 1991. Brown and Murray 1992) and H. laevigata Donovan
(Brown and Murray 1992). The results show small genetic differ-
ences between populations of H. rubra that accumulate with geo-
graphical distance, but suggests, due to a small-scale genetic het-
erogeneity, that local populations are predominantly recruited from
local stock. On the other hand, H. laevigata populations are ge-
netically different from each other even over small distances.

In this paper we describe the pattern of population genetic
structure in some H. fulgens sites from the Central Zone of Baja
California, Mexico using allozyme analysis. Specifically, we were
interested in whether these abalone sites could be considered the
same population unit or smaller independent units.

MATERIALS AND METHODS

Studv Area

This study was carried out in five sites along the Central Zone
of Baja California, Mexico. Three of them were located inside
Bahi'a Tortugas and relatively close to each other (2-5 km between
them). The other two sites were located in Bahia Vizcaino, at
Punta Eugenia and Malarrimo. toward the northern end of Bahi'a
Tortugas, separated from each other by 54 km, and 30 to 84 km,
respectively, from Bahia Tortugas (Figure I),

The characteristics of each site are as follows: Los Morros (27°
39' 14" N, 114° 52' 26" W) is at the southeastern end of Bahi'a
Tortugas, The bottom is rocky, rich in macroalgae, and mainly
covered by giant kelp beds (Macrocystis pyrifera). This site is
directly exposed to the waves and regularly exploited by fisherman
because of its high population density.

La Cantina (27° 40' 00" N, 1 14° 53' 55" W) is in the north-
western portion of Bahi'a Tortugas. It features large boulders and
sandy clearings, and the main vegetation is articulated coralline

853

854

ZUNIGA ET AL.

Punta Rugenia

Bahia Vizcaino

Figure. 1. Locations of the five sites sampled from Baja California
Peninsula.

algae mixed with scarce giant kelp plants. This reef is partially
sheltered from breaking waves. At this site, abalone density is low
and commercial catch is rare.

On the southeastern side of Bahfa Tortugas is La Bajada (27°
39' 30" N. 114° 50' 49" W). The sandy-stony bottom has poor
vegetation and giant kelp is absent. This is a site sheltered from the
breaking waves. Abulone is quite scarce and there is not a com-
mercial catch. Puma Eugenia (27° 50' 39" N. 115° 04' 35" W) is
located at the edge of Vizcaino Peninsula. It is quite similar to Los
Morros, with a stony-rocky bottom densely inhabited by M. /n-
rifera. Commercial abalone catch is important here.

Malarrimo (27° 47' 21" N. 1 14° 43' 10" W) is in the middle of
Bahi'a Vizcai'no. It has a sandy-stony bottom with beds of Phyl-
lospadix. coralline algae, and other red algae. Laminarial algae are
scarce. The commercial catch is moderate.

Collection and Electrophoresis

Total sample size was 102 adult organisms. A higher sample
size was not possible due to low commercial catch and the high
market price for each specimen. The population sample per site
varied from 18 to 22 organisms. Muscular tissue samples of each
organism were put in a buffer solution of 0.1 M Tris-HCl. pH 7.0.
and were immediately frozen in liquid nitrogen.

We used the starch-gel electrophoresis technique; enzyme
stains were modified from Gonzalez de Leon ( 1 986). Fragments of
tissue were homogenized at 4 °C, in a buffer solution of 0.1 M
Tris-HCl, pH 7.0 with a Glas-Col stirrer at 2,000 rpm for 7 min.
Each sample extract was stored at -70 °C until electrophoresis was
conducted.

A total of 1 2 enzymes were assayetl. but because separation and
interpretation of the elcctromorph banding lor all 1 2 enzymes was
not clear, we decided to analyze five enzymes which offered
clearly resolved bands. All migrated anodally. They were
glutamate oxalate transaminase (GOT, 2.6.1.1). malic cn/>nic
(ME, 1.1.1,40), malate dehydrogenase (MDH, 1. 1.1.371, lactate
dehydrogenase (LDH, 1.1.1.27), and leucine aminopeptidase
(LAP, 3.4.1 1.1 ). The clecirophorcsis was concluded when the mi-
gration of Ihe internal markers reached 7 to 8 cm from its origin.

Statistical Analysis

The eleclrophoretic data were analyzed using BIO.SYS-l
(.Swofford and .Selandcr I989i and NTSYS-PC (Rohll l')S4i. The

allele frequencies and the basic genetic parameters of heterozy-
gosity, polymorphism, and number of alleles per locus were cal-
culated for each population. Due to the difficulties encountered in
using chi-square distribution for small samples, we used the exact
probability test (Haldane 1954. Elston and Forthofer 1977, Weir
1 996) to test conformance of allele frequencies to Hardy-Weinberg
equilibrium ratio. In addition, we used the sequential Bonferroni
test to reduce the tablewide type-I error rate (Rice 1989). The
magnitude and direction of departure from expectations were
quantified by the fixation index (fjs). High levels of selling would
be reflected in high F|^; positive values, indicating heterozygous
deficiencies.

The geographic heterogeneity of allele frequencies at each lo-
cus was tested using jackknifed standard errors of the locus-
specific fsT estimates, jackknifing across the five populations
isensu Weir and Cockerham 1984). Then, a jackknifed standard
error for the overall F^-y estimate was determined by jackknitlng
across loci. Finally, we performed one-tailed t tests to test the
hypothesis that F^-^^ > 0.

The genetic population structure was analyzed by means of Fn-.
fis. and fsT statistics (Wright 1951, Wright 1978) to describe
non-random mating within and among populations. F^ is the total
reduction in heterozygosity of an individual due to the effects of
non-random mating and population subdivision combined. It may
be hierarchically partitioned to describe deviations from Hardy-
Weinberg expectations on different geographic scales. The parti-
tioning is ( I - Fiy) = (1 - f,s) (I- fsT*- where f,^ is the
reduction in heterozygosity of an individual relative to the sub-
population due to non-random mating and F^t is the total reduc-
tion in heterozygosity of an individual due to population subdivi-
sion. Also, F^j is a common statistic for describing average dif-
ferentiation among populations.

F statistics have been derived from three perspectives: as the
degree to which alleles, identical by descent, are distributed within
and among individuals and populations (Malecot 1969; Nei 1973);
as correlations between alleles in uniting gametes within and
among subpopulations (Wright 1978); and from a nested ANOVA
model where total allelic variation is partitioned among subpopu-
lations, among individuals within subpopulations, and between
pairs of alleles within individuals (Cockerham 1969. Cockerham
1973, Weir and Cockerham 1984). Although all of ihem yield the
same result and their interpretations are biologically equivalent, we
used Weir and Cockerham's F statistics because their method ex-
plicitly takes into account differences in sample sizes among the
populations tested. For the significance of F statistical, we report
the standard errors of the mean estimate based on the jackknife
procedure across loci, and the 99% confidence limits based on the
bootstrap analysis across loci (Weir 19961.

Levels of gene flow among populations v\ere inferred from
fsT This estimator is related to the number of migrants per gen-
eration {Njn). the historical average number of indi\iduals ex-
changed per generation between populations, by the equation F^j

1/(4 N^iii + I) (Wright 1951). However, we calculated gene
flow using the relation F^-, -1/(4 N,iii (n/n - 1 )" -i- 1 1 because it
takes into account the number of sampled populations (n) (Taka-
hata 1983, Takahala and Nei 1984, Chakraborty and Leimar I987|.
Although the relationship between F^^ and N,,m is based on an
infinite island model, the last equation provides a relatively robust
estimate of gene How for populations v\ith other population ge-
netic structure models (Crow and Aoki 1984, .Slalkin and Barton
I ^)89 ).

Genetic Analysis of Abalone Hauot/s fulgens

855

The D statistic (Nei 1972) was used to estimate the genetic
distance among populations. A phenogram based on Nei coeffi-
cients using UPGMA was generated. The branching points stan-
dard error was estimated by means of genetic distance data and
their variances. The clustering significance among populations was
accepted when the standard error bar at the branching point was
less than one-half of the branch length (Nei et al. 1985).

To find possible patterns of spatial differentiation, we have
compared the genetic and geographic distance matrices by Man-
tefs non-parametric test (Manly 1997). When the correlation be-
tween matrices of geographic distance and genetic distance was
significantly greater than the correlation between the geographic
distance matrix and randomized matrices of genetic distance, the
relationship was accepted as a significant one. Shuffling rows and
columns of the original matrix of genetic distance generated ran-
domized matrices. We used 5,000 iterations to build the random-
ization distribution to reach 95'* confidence intervals of the cor-
relation matrix.

RESULTS

Of the five enzymatic systems studied, seven loci were found:
MDH-1. MDH-2. LDH. ME. LAP-1, LAP-2. and GOT. Banding
patterns at each of them were consistent with Mendelian segrega-
tion. The allele frequencies at each locus were dominated by the
same allele in all sites and showed little variability between them
(Table 1).

Heterozygosity at loci varied widely within and among popu-
lations (Table 2). The mean direct count heterozygosity per popu-
lation ranged from 0.054 to 0.195. the mean number of alleles per
locus ranged from 1.7 to 2.0. and the percentage of polymorphic
loci was highly variable among samples ranging from 14% to

100%. The lowest values both of mean heterozygosity and poly-
morphism were found in La Bajada and the highest were found in
La Cantina (Table 2). The genetic diversity did not show a reduc-
tion or increase pattern consistent with geographic distance among
populations, or in moving from south to north.

Observed mean heterozygosities differed significantly from
those expected under conditions of Hardy-Weinberg equilibrium
(Table 1 ). Using the exact test we found six significant deviations
in gene frequencies of 31 comparisons made over all loci, all
populations (Table 3), and all heterozygous deficits. However, the
sequential Bonferroni adjustment of the P values showed that these
deviations Hardy-Weinberg equilibrium were the result of chance
alone.

The allele frequency variation across populations produced het-
erogeneous estimates of F^y ranging from 0.0064 to 0. 1 355. How-
ever, each of the locus-specitlc estimates of F^-^ except MDH-1,
was not significantly different than zero as revealed by t test uti-
lizing the jackknifed errors (Table 4).

F statistics did not indicate departure from random mating in
the H. fulgens populations (Table 5). f,s was large and highly
variable by more than a factor of 7 across loci, but only MDH-1
was statistically different from zero i.P < 0.01 ). F,y was less vari-
able and differed by no more than a factor of 3 across loci, only
MDH-1, ME, and LAP-2 were statistically different from zero
{P < 0.01). Finally, F^-^ across loci and overall populations was
small and not significantly different from zero (P < 0.01 ), except
MDH-1.

The average Nju. among all populations was 4.3. Gene flow
between population pairs was highly variable and ranged from
0.066 to infinity (Table 6). Low gene flow (Njn < 1 ) was observed
in Bahia Tortugas between La Bajada and Los Morros, and be-
tween La Bajada and La Cantina; however, Los Morros and La

TABLE \.
Allele frequencies at seven polimorphic loci in five populations of H. fulgens from Baja California, Mexico.

Populations

Allele

La Bajada

Los Morros

La Cantina

Malarrimo

P. Eugenia

MDH-1

MDH-

LDH

ME

LAP- 1

LAP-2

GOT

A
B

Hl

A

B

Hl

A

B

Ht

A

B

Hl

A

B

Hl

A

B

Hl
A
B
H,

0.952
0.048
0.095
1.000
0.000

0.810
0.190
0.190
0.976
0.024
0.048
0.976
0.024
0.048
0.952
0.048

1 .000
0.000

20
0.975
0.025
0.050
0.800
0.200
0.300
0.875
0.125
0.050
0.975
0.025
0.050
0.850
0.150
0.100
0.800
0.200
0.200
0.900
0.100
0.200

22
0.818
0.182
0.273
0.795
0.205
0.227
0.932
0.068
0.136
0.886
0.114
0.136
0.705
0.295
0.409
0.795
0.205
0.046
0.841
0.159
0.136

18
1.000
0.000

0.944
0.056
0.111
0.750
0.250
0.167
0.972
0.028
0.056
0.917
0.083
0.167
0.944
0.056
0.111
0.972
0.028
0.056

20
1.000
0.000

0.895
0.105
0.105
0.895
0.105
0.211
0.947
0.053
0.105
0.842
0.158
0.105
0.895
0.105
0.211
0.974
0.026
0.053

856

ZUNIGA ET AL.

TABLE 2.

Summary statistics (means and standard errors in parentheses where appropriate) describing genetic variation in five populations of H.
fulgens from Baja California, Mexico. A locus was considered polymorphic if the frequency of the common allele was 0.95 or less.

Population

Mean Sample Size
Per Locus

Mean No. of Alleles
Per Locus

Percentaje of Loci
Polymorphic

Mean Heterozygosity

Direct Count

Hardy-Weinberg Expected

La Bajada

21.0

(0.0)

Los Morros

20.0

(0.)

La Cantina

22.0

(0.0)

Malarrimo

18.0

(0.0)

P. Eugenia

19.0

(0.0)

1.7
(0.2)

2.0
(0.0)

2.0
(0.0)

1.9
(0.1)

1.9
(0.1)

14.3
7L4
100

57.1
71.4

0.0.'i4
(0.026)

0.1.^6
(0.037)

0.195
(0.045)

0.095
(0.023)

0.113
(0.29)

0.085
(0.041)

0.204
(0.044)

0.287
(0.036)

0.124
(0.024)

0.144
(0.036)

Cantina showed high gene flow (Njn > 1). Malarrimo and Punta
Eugenia, separated by nearly 40 i<m, showed relatively high levels
of gene flow {Njn 1 ). The gene flow between these populations
and Bahi'a Tortugas populations was variable (0.066 < Njn <
infinity) and no association between levels of gene flow and geo-
graphic distance was found.

Mean genetic distance among populations was low (Dnei =
0.0122, a = 0.009; Table 7). The phenogram developed to esti-
mate relationships among the five H. fulgens populations (cophe-
netic correlation = 0.795) yielded three clusters (Figure 2). The
first formed by La Bajada and Malarrimo at a distance of 0.0019.
The second formed by Los Morros and Punta Eugenia (Dm, i =
0.0037), which is linked to the first cluster at a distance of 0.01. At
the base of the phenogram. La Cantina is associated to both clus-
ters at a distance of 0.0201 .

Nei's D values derived from the paired comparison of indi-
vidual populations were not correlated with the geographical dis-
tance of the populations (Mantel test, /• = 0.0935, t = 0.2346, P
= 0.5927; Figure 3). The scatter plot did not show an island or
stepping stone model among populations.

DISCUSSION

Heterozygosity and the genetic distance are influenced by the
number individuals and the number of loci examined (Nei and
Roychoudhury 1974, Nei 1978). Likewise, particularly the het-
erozygosity has been negatively correlated with the number of loci
investigated (HartI et al. 1994). For these reasons, at a flrst exami-
nation of our data we thought that the average heterozygosity of W.
fiilf>en.s could has been overestimated because we studied only

seven polymorphic loci. However, in spite of the low number of
loci examined, the average heterozygosity we found among H.
fuli>ens populations (H^ = 0. 1 19) was similar to those reported for
H. discus hannai Ino (W^ = 0.123; Fujio et al. 1983), H. laevigala
(//a = 0.195), H. rubra (H^ = 0.140; Brown and Murray 1992),
and other mollusk (//^ = 0.129; Fujio et al. 1983). In addition, our
estimates of heterozygosity were also lower than those previously
reported for other benthic marine organisms (//^ = 0.285; Nevo
1978, Nevo et al. 1984), where a minimum of 15 loci were ana-
lyzed.

Another potentially problematic aspect of our study was the
sample size. Several authors claim that large sample must be ana-
lyzed to estimate adequate patterns of genetic diversification
(Lewontin 1974, Nei et al. 1983); however, Archie et al. (1989)
recommend that at least 20 individuals should be studied when it
is not possible to collect large samples, or particularly, for studying
rare or endangered species. We were limited to analyzing 20 in-
dividuals per population because the Mexican abalone is a col-
lapsed flshery. thus each abalone piece has a very high commercial
value and therefore it is very difficult to get large samples.

It has also been shown that the number of individuals to be
examined depends on the level of heterozygosity found (Nei
1978). That is to say. the sample size must be increased when
heterozygosity is high. This was not our case because the levels of
heterozygosity found for H. fulgens populations were low (<0.2).

Deviation from Hardy- Weinberg Equilibrium

While we observed fixed alleles in some loci and an apparent
heterozygosity deficit in the studied populations, the sequential

TABLE 3.

Prohahilities that the observed gene fre(|uencies in five populations of H. fulgena from Baja California, Mexico conform to tho.se expected
under Hardy-Weinberg equilibrium, u.sing the exact test (Weir 1996). Significant values are given in bold.

MDH-I

MDH-2

LDH

ME

LAP-1

LAP-2

GOT

La Bajada

1.000

_

0.172

1 .000

1.(100

0.024

-

Los Morros

1.000

1.000

(l.()21

1 .000

(l.(l.<(»

0.128

1 .000

La Cantina

0.536

0.177

1.000

0.223

1 .000

0.003

0.056

Malarrimo

-

1.000

().(»31

1 .000

1.000

1 .000

1 .000

P. Eugenia

-

0. 1 59

1 .()(K)

1.000

0.0.16

1.000

1.000

P < 0.05.

Genetic Analysis of Abalone Haliotis fvlgens

857

TABLE 4.

Locus-specific and overall F^-^ values across five populations of H.

fulgens from Baja California, Mexico. Significance of variation

among populations was determined by one tailed t test

of independence.

Locus

fsi

SE

MDH-1

MDH-2

LDH

ME

LAP-1

LAP-2

GOT

Overall

0.1355
0.0434
0.0064
0.0116
0.0663
0.0145
0.0450
0.0461

0.0812
0.0440
0.0233
0.0229
0.0669
0.0154
0.0325
0,0408

*** s
** ns
** ns
** ns
** ns
** ns
** ns
** ns

Significance level for / test ** p < 0.05,

P<0,OI.

BonfeiToni test suggested that none of the deviations from Hardy-
Weinberg equilibrium had biological significance. This result is
different from other abalone species, such as H. discus hannai
(Fujio et al, 1983) and H. rubra (Brown and Murray 1992), and
other bivalves such as Mytihts edulis L, (Kohen et al, 1976. Jo-
hannenson et al, 1990), Crassostrea virginica Gmelin (Singh and
Zouros 1978, Buroker 1983), and C. gigas Thunberg (Gosling
1981) that have shown departures from Hardy-Weinberg equilib-
rium by heterozygous deficiency.

Although f IS and f,T showed a positive sign that confirmed a
deficit of heterozygosity within populations, they were not statis-
tically different from zero in all the loci of H. fulgens. Brown and
Murray ( 1992) found a similar pattern of heterozygous deficiency
in H. rubra and H. laevigata with a positive overall for both F,^
and f,T-, but the heterozygous deficiency were not consistent
across all loci.

The estimated F^t across populations surveyed do not suggest
that H. fulgens populations are genetically isolated from one an-
other. In fact only 4% observed genetic variability is due to geo-
graphic subdivision and random genetic drift. Furthermore, the
five populations studied formed a homogeneous group with re-
spect to allele frequencies at six of the seven loci analyzed (Table
4). Therefore, we cannot consider these populations as different
stocks,

TABLE 5.

Weir and Cockerham (1984) estimates of Wright's F values

calculated separately for each locus for population of H. fulgens

from Baja California, Mexico. Means and standard errors were

obtained by jackknifing over loci. Confidence interval obtained by

bootstrapping over loci.

TABLE 6.

Number of migrants per generation for five populations of H.

fulgens from Baja California, Mexico. Pairwise estimates of gene

fiow (N,jn) based on Weir and Cockerham's (1984) Fst-

Los

La

P.

Morros

Cantina

Malarrimo

Eugenia

La Bajada

0,932

0,439

infinity

2.720

Lcs Morrcs

15,182

2.135

infinity

La Cantina

0.066

0.153

Malarrimo

10.435

Genetic Differentiation

The small genetic distance observed among populations (0,001
s D < 0,029) confirms that they are genetically homogeneous and
form part of an interconnected large population by gene flow.
Nevertheless, the phenogram and Mantel's test showed that the
differences among populations were small and not accumulative
geographically because none of them was joined to another by its
geographic proximity (Figures 2 and 3), A different pattern was
described by Brown and Murray (1992) for H. rubra, where a
broad-scale gene pool for this species was geographically homo-
geneous and accumulative.

Although all the analysis leads us to conclude that the five
populations surveyed from part of a large population, two impor-
tant issues that emerged from this study remain to be explained: ( 1 )
why we observed a high degree of homozygocity in H. fulgens
populations, despite the fact that F,s and F,t do not differ signifi-
cantly from zero, and (2) how can we explain the heterogeneous
flow observed?

In the first case, we hypothesize that inbreeding and genetic
drift could be provoking the heterozygous deficiency observed
within populations of H. fulgens in Baja California, although se-
lection against heterozygous could be another explanation. In fact
the inbreeding and genetic drive could be caused by a combination
of factors, such as the reduction of the adult population size ob-
served in the last 20 years as a result of a serial over-exploitation
of small population units within the larger fishery that the Mexican
abalone was in early times (Prince and Guzman del Proo 1993),
and the limited larval dispersal characteristic of these species to a
scale of tens to hundreds of meters (Prince et al, 1987, Prince et al,
1988) up to several kilometers (McShane et al, 1 988, Shepherd and
Brown 1993, Sasaki and Shepherd 1995),

Particularly, recent hydrodynamic experiments in Bahia Tortu-
gas have shown that the average cun'ents could potentially trans-
port larvae for 3 to 5 km along shore during the first 5 days of the

TABLE 7.

Locus

^IS

fiT

FsT

Nei's genetic distance (Nei 1972) above the diagonal and the

geographical distances (kilometers) below the diagonal among five

populations of H. fulgens from Baja California, Mexico.

MDH-I
MDH-2

0,0707
0.2360
0.3962
0.1602
0.2858
0.4941
0.2371
0.3180(0.051)

0. 1 289-0.5070

0.1540
0.2750
0.4000
0.1634
0.3264
0.5025
0.2704
0.3351 (0.044)

0. 1 868-0,4833

0.0897
0.0510
0.0063
0.0038
0.0568
0.0166
0.0437
0.0358(0.010)

0.0-0,0728

LDH

ME

LAP-1

LAP-2

GOT

Mean (SD)

99% Confidence

Interval

La Bajada
Los Morros
La Cantina
Malarrimo
P. Eugenia

La
Bajada

2,7

5,2

68.8

32.4

Los
Morros

0.0135

2.8
66.4
30.0

La
Cantina

0,0294
0.0100

63.6

37.2

Malarrimo

0.0019
0,0105
0,0277

.16.4

P.
Eugenia

0,0065
0,0038
0,0133
0.0052

858

ZUNIGA ET AL.

Genetic distance

003

002

001

-_ La Bajada

Malarrimo

Los Morros

Punta Eugenia

La Cantina

Figure. 2. Phenogram showing the genetic relationships of five H.
fulgens localities from Baja California Peninsula. Nei's genetic dis-
tance were clustered using UPGMA. The bars represent the standard
error. Cophenetic correlation was 0.799.

5 0 01 •

Geographic Distance (Km)

Figure. 3. Scatter plots demonstrating the no-relationship between ge-
netic distance and geographic distance.

pelagic cycle. (Guzman del Proo et al. in press). Thus a limited
larval dispersal in the neighborhood of the breeding population
increases the larvae survival, however, it also increases the in-
breeding and local competition within populations due to a re-
duced gene flow.

Regarding the heterogeneous gene flow observed among five
populations of H. fulgens analyzed, we also could explain it be-
cause of the limited larval dispersal. The hydrodynamic pattern in
Bahi'a Tortugas and neighboring coastal zone showed that larval
exchange could potentially take place between neighboring aba-
lone reefs at a limited distance under dominant swells coming from
the west. Nevertheless, it does not exclude the possibility of some
reduced interchange of larvae between more distant reefs, provid-
ing the dominant hydrodynamic conditions change, for instance,
when the direction of swells coming from the west change south-
erly, the intensity of the winds changes, or the tidal low changes
(Guzman del Prck) et al. in press). Thus we hypothesize that gene
flow along the distribution of abalone reefs could be variable de-
pending of the coastal topography, dominant swells, and driving
winds that determine the local coastal hydrodynamic and conse-
quently the level of larval interchange among reefs.

These types of findings have important implications in terms of
fishery management. Modern fisheries science demands a holistic
management of the fisheries; this concept includes the mainte-
nance of genetic diversity and the population structure, which are
critical for ensuring the long-term survival of any fishery (Shep-
herd and Brown 1993). Although this study was confined to a
limited area, the results are likely to be indicative of population
genetics of Mexican abalone and the factors influencing it, which
should be considered in future management policy.

ACKNOWLEDGMENTS

This research was supported by a grant from the Direccion de
Estudios de Posgrado e Investigacion-IPN (DEPI 942031). The
authors thank the support received for field work from the So-
ciedad Cooperativa de Prt)ducci6n Pesquera Bahi'a Tortugas and
they thank the Instituto Nacional de Pesca for using their facilities
in Bahi'a Tortugas. We thank also Jorge Carrillo and Jorge Belmar
for their help in the fieldwork. Jane L. Hayes kindly reviewed
English version of the manuscript.

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IRON METHIONINE (FeMET) AND IRON SULFATE {FeS04) AS SOURCES OF DIETARY IRON
FOR JUVENILE ABALONE, HALIOTIS DISCUS HANNAI INO.

KANGSEN MAI AND BEIPING TAN

Aqitacultiire Research Lahoratoiy
Fisheries College
Ocean University of Qingdao
Qingdao. P.R. China 266003

ABSTRACT The minimum dietary iron requirement and its bioavailability were determined for juvenile abalone (Haliotis discus
lumnai Ino.) using casein-gelatin-based diets supplemented with 0. 10, 20. 30, 60, 120, and 200 mg iron/kg from iron methionine
(FeMet) or iron sulfate heptahydrate (FeSOj ■ 7H,0). The experimental diets containing graded levels of dietary iron (24.9-212.7 mg
iron/kg) provided as either FeMet or FeSO^ were fed to juvenile abalone. Abalone juveniles of similar size were distributed in a
flow-through system using a completely randomized design with thirteen treatments and three replicates of each treatment. Abalone
fed the basal diet without iron supplementation exhibited significantly lower survival and carcass protein than the other groups. The
average weight gain rate (WGR, %). daily increment in shell length (DISL. |xm/day). and soft-body iron concentration (SB iron, p-g/g)
of the abalone were significantly affected by dietary treatment, and responded in broken-line models to increases in dietary iron levels
from the two iron sources. The optimal levels of dietary iron using FeMet and FeSOj as the supplemental iron sources, determined by
broken-line regression analysis, on the basis of maximum WGR. were 62.79 and 58.35 mg/kg. respectively. On maximum DISL, the
values were 66.12 and 62.1 1 mg/kg, respectively, and on maximum SB iron deposition were 66.78 and 64.55 mg/kg, respectively. The
content of iron and manganese in the shell, however, was maintained relatively constant regardless of dietary treatment. Significantly
reduced manganese deposition in soft-body parts was observed for the abalone fed diets containing high levels ( > 100 mg/kg) of iron
from FeSOj. However, this effect of excess iron on the utilization of manganese was not found when using FeMet as the iron source.
Based on these results, an optimal level of dietary iron was recommended to be 65-70 mg/kg with either FeMet or FeSOj as the iron
source. This experiment also showed that the bioavailability of dietary iron from FeSOj was as high as that from FeMet.

KEY WORDS: abalone, Huliolis discus Imnnai. iron requirement, bioavailability, nutrition, mollusks

INTRODUCTION

Iron is an essential trace element in all-higher animals, includ-
ing fish, because of its importance in cellular respiration and mi-
tosis (Robbins et al. 1972). Studies on dietary iron have been
conducted with some fish species. Iron deficiency in channel cat-
fish, Icralurus piinctatiis, has been characterized by suppressed
hematocrit, hemoglobin, plasma iron content and transferrin satu-
ration, and a dietary iron requirement of s30 mg/kg diet was
recommended for this species (Gatlin and Wilson 1986). A re-
quirement of 150 mg/kg diet has been reported for red sea bream,
Chrysophrus major, (Sakamoto and Yone 1978a). All these
recommended values were evaluated when iron sulfate
(FeSOj • IH2O) was the source of supplemental iron.

The nutritional value of dietary mineral sources depends not
only upon their contents in the feedstuff but also upon the bio-
availability of the element to animals (Paripatananont and Lovell
1997). Studies with mammals have shown that chelation of min-
erals to amino acids may increase their absorption rate in the
intestine (Ashmead 1992). Wedekind et al. (1992) reported that the
bioavailability of zinc from zinc methionine (ZnMet) in poultry
was greater than that from zinc sulfate (ZnS04). Paripananont and
Lovell (1995) also reported that using ZnMet as the dietary zinc
source reduced the optimal levels of dietary zinc for channel cat-
fish in both purified and practical diets as compared to the diets
containing ZnSOj. More recently, Paripananont and Lovell (1997)
reported that the coefficients of net absorption of chelated trace
minerals (Zn. Fe. Cu, Mn, and Se) were much higher than those of
inorganic forms of these minerals for channel catfish in both pu-
rified and practical diets. Similar results were observed by Li et al.
(1995) in shrimp (Penaeiis chinensis) in a practical diet. However,
Lim et al. (1996) reported that iron methionine and iron sulfate

were equally effective in improving growth and preventing anemia
in channel catfish.

Excess iron suppleinentation may, however, be detrimental be-
cause of the pro-oxidant nature of this mineral (Gatlin and Wilson
1986) and decreasing absorption of other ininerals such as man-
ganese (John.son and Korynta 1992). In addition, unnecessarily
high additions of iron and other micronutrients also increase the
price of feeds, as well as increase the input of minerals to the
aquatic environment.

There is no information on the requirement of dietary iron in
any mollusc species, including juvenile abalone, Haliotis discus
hannai Ino., one of the most widely cultured and commercially
important abalone. Also, no information is available on the bio-
availabilities of organic and inorganic .sources of dietary iron to
mollusc species. Therefore, the objectives of this study were to
determine the dietary iron requirement of juvenile abalone, H.
discus lumnai. with FeMet and FeSOj as the iron sources and to
compare the bioavailabilities of the two iron sources using a pre-
mium quality diet, based on casein-gelatin as the protein sources.
Determination of the iron requirement and bioavailabilities of this
species was not only based on the growth and survival of the
animal, but also on the tissue iron deposition.

MATERIALS AND METHODS

Feed Formulation and Manufacture

The basal diet formulation is given in Table 1. Ca.sein and
gelatin were used as protein sources. Crude protein level of the
experimental diets was about 30%, which is considered to be suf-
ficient to maintain optimum growth for H. discus hannai (Mai et
al. 1995b). Soybean oil and menhaden fish oil (1:1) was used as
the basal lipid source. Dietary lipid level was about 3.5%, which

861

862

Mai and Tan

TABLE 1.
Composition of the basal diet (dry weight basis).

Percent in Diet

Ingredient
Casein, vitamin-free (Sigma Chemical,

St. Louis, MO)
Gelatin (Sigma Chemical,

St. Louis. MO)
Dextrin (Shanghai Chemical Co.,

Shanghai, China)
Sodium alginate (Shanghai Chemical Co..

Shanghai, China)
SO/MFO (Food grade )^'
Choline chloride (Shanghai Chemical Co.,

Shanghai, China)
Fe-free mineral mix'"
Vitamin mix"^
Proximate analysis (means of triplicate)
Crude protein (%)
Crude lipid (%)
Ash (<7c)

Gross energy (kj/g)''
Iron (mg/kg)

25.00

6.00

38.50

20,00

3,50
,50

4,50
2,00

28,52
3,53
8,28

17,19

24.85

"Soybean oil and menhaden fish oil (1:1) with 0.001 "/f ethoxyquin.
''Fe-free mineral mix, each 1000 g of diet contained: NaCl, 0.4 g:
MgSOj • 7H,0, 6.0 g; NaH^POj • 2H,0, 10.0 g; KH,POj, 20.0 g;
Ca(H,POj)2 • H_,0. 8.0 g: ZnSO^ • 7H,6, 141.2 mg; MnSO^ • H,0, 64.8
mg; CuS04-5H,0, 12.4 mg: CoCl, • 6H,0, 0.4 mg: KIO„ 1.2 mg:
Na,SeO,. 0,4 mg,

"Vitamin mix, each 1000 g of diet contained: thiamin HCl, 120 mg:
riboflavin, 100 mg; folic acid, .W mg: PABA, 400 mg: pyridoxine HCl, 40
mg; niacin, 800 mg; Ca pantothenate, 200 mg: inositol, 4000 mg: ascorbic
acid, 4000 mg; biotin, 12 mg; vitamin E, 450 mg; menadione, 80 mg: B,,,
0,18 mg; vitamin A, 100 000 lU; vitamin D, 2000 lU: elhoxyqum, 400 mg,
''Estimated with an .\YR-1 bomb calorimeter.

was sufficient to support optimum growth utid provide enough
EFA for the abalone (Mai et al. 1995a). The compositions of
vitamin and mineral mixtures were modified from those used by
Uki et al. (1985). The casein-gelatin-based diet contained 24,85 ±
2,71 mg/kg of intrinsic iron. The basal diet was supplemented with
0, 10, 20, M. 60, 120, and 200 mg of iron/kg from either iron
methionine (FeMet) (Feed Additive Co,, Ministry of Chemistry
Industry of China, Jinan) or iron sulfate heptahydrate
(FeS04 • 7H2O) (Sigma, St. Louis. MO). Final iron concentrations
in the experimental diets (n = 3) were found to be: 24.85 ±2.71.
35.52 ± 1.96. 43.48 ± 2.15, 54,33 ± 2,56, 82.17 ± 3,04. 136.51 ±
3.78, and 210.13 ± 2.86 mg/kg dry diet from FeMet, and 24,85 ±
2,71, 34,13 ± 1,74, 41..59 ± 2,11, 53,28 ± 1,68, 82,46 ± 2.12,
135.39 ±3.17, and 212.65 ± 2.65 mg/kg dry diet from FeSO^ as
delermincd by ICP-AFS

Procedures for diet preparation were modified from those de-
scribed by Mai et al. (1995a. 1995b). Casein, gelatin and some
minerals that were in the form of small grains were ground indi-
vidually using a Pascal Mill and then passed through a mesh with
l25-(xm pore si/e. Dry ingredients were weighed on an electronic
balance and thoroughly mixed. After adding water (about 120%,
v/vv) to the mechanically mixed ingredients containing 2(Wr so-
dium alginate, a paste was made. The paste was shaped into 0.5-
tiim thick sheets, which were cut into 1 cm" flakes. The flakes
were dipped into an iicitieous sohition of CaCI, (5%. w/v) for 1

min. By this treatment, sodium alginate was converted to an in-
soluble calcium alginate gel, in which the nutrients were bound
(Uki and Watanabe 1992). The surplus solution was drained natu-
rally, then the flakes were sealed in a sample bag and stored at
-20 °C until use.

Animal Rearing

Juvenile abalone, H. discus hannai. used in this experiment
were derived from a spawning in June 1998, at Mashan Fisheries
Co. Shandong, China. Before the trial, shell length was measured
with calipers to the nearest 0.02 mm and the animals were weighed
to the nearest 0.01 g using an electronic balance.

Animals were kept in acrylic square cages (20 cm x 20 cm x 20
cm). Each rearing unit was stocked with 25 abalone juveniles.
Similar size juveniles (mean weight 0.702 ± 0.02 g; mean shell
length 16.110 ± 0.10 mm) were assigned to the rearing system
using a completely randomized design with thirteen treatments and
three replicates per treatment. The system was flow-through, with
water filtered to 30 |j.m by primary sand filters, then to 10 \i.m by
secondary composite sand filters. The flow rate was about 0.5 L
per min per cage. Cages were kept in dim light by screening with
black plastic drapes. During the experimental period, water tem-
perature ranged from 18.2-22.0 °C. salinity 30-34. pH 7.6-7.9.
Dissolved oxygen was not less than 7 mg/L, and there were neg-
ligible levels of free ammonia and nitrite (AOAC, 1995), The
rearing water contained 0.35 ± O.I mg Fe/L as determined by
ICP-AES (n = 3).

Prior to initiation of the experiment, the abalone underwent a
2-week conditioning period during which they readily adjusted to
an iron-depleted casein-gelatin-based diet (Table 1 ) and standard-
ized environmental conditions. The feeding trial was run for 16
wk. Abalone were hand-fed with the test diets at a rate equaling
5-10% of abalone wet weight per day. once daily at 17:00. Every
morning, uneaten feed and feces were removed to maintain the
water quality.

Sample Collection and Analysis

At the termination of the experiment, animals were not fed for
3 days, then all abalone were removed from the cage, weighed,
measured, and counted. Then, 15 abalone from each replicate were
frozen (-20 °C) for subsequent analysis. Growth was expressed as
weight gain rate (WGR, %) and daily increment in shell length
(DISL, (xm/day). The calculation formulae were as follows:

WGR(%) = |(Wt - Wi)/Vv'il X 100

DISL = [(SLt - SLi)/t| X 1000

Where Wt and Wi are final and initial mean weight (g), respec-
tively: SLt and SLi are final and initial mean shell length (mm),
respectively; and t is the feeding trial period (day).

Proximate analyses to determine protein, lipid, ash, and mois-
ture levels were conducted using con\enlional procedures (AOAC
1995),

Elemental analyses of shells and soft bodies of the abalone
were modified from the method described by Shearer ( 1984). The
shell samples were digested in a mixture of equal parts of hydro-
chloric acid (37%, ACS reagent) and nitrite acid (70%, ACS re-
agent) at a ratio of 1:20 (w/v). The soft body samples were di-
gested in perchloric acid (HCIO4, 70%, ACS reagent) at a ratio of
1:20 (w/v). Then, the digests were appropriately diluted with
Milli-0 water within the analytical capabilities of the ICP atomic

FeMet and FESO4 AS Dietary Iron

863

emission spectrophotometer (JY 70Plus. Jobin Yvon Co.). El-
emental concentrations of the samples are expressed on a wet-
weight basis as recommended by Shearer (1984).

Leaching

The leaching test of dietary iron was carried out according to
the method used by Coote et al. (1996). Pre-weighed feed was put
onto 100-micron mesh screens and allowed it to settle to the bot-
tom of experimental cages without abalone. Temperature and flow
rate were adjusted to match those of the experiment, the values
being 20 ± 0.8 °C, and about 0.5 L per min per cage, respectively.
At the end of the allotted time (0. 6. and 12 h, respectively), the
remaining feed was removed from the cages and dried overnight at
60 °C in an oven. Dried feed was submitted for analysis of total
iron with an ICP-atomic emission spectrophotometer.

Statistical Analysis

Data from each treatment were subjected to one-way ANOVA.
When overall differences were significant at less than 5% level.
Tukey test was used to compare the mean values between indi-
vidual treatments. Statistical analysis was performed using
STATISTICA^^^' package. Dietary iron requirement of juvenile
abalone was determined by the broken-line regression analysis
(Robbins et al. 1979. Robbins 1986). The linear segments of the
regression lines, below the breakpoints, were used to compare the
bioavailability of dietary iron mainly from FeMet with that mainly
from FeSOj by deriving the ratio of the slopes of the lines (Forbes
and Parker 1977, Paripatananont and Lovell 1995).

RESULTS

Leaching

The results of the 12-h leaching test are illustrated in Figure 1.
The iron content of the diets, supplemented with the two iron
sources, all declined within the whole test period. After 6 h in
seawater, the remaining iron content of the diets ranged from 20. 1 7
to 139.37 mg/kg for FeMet diet (Fig. lA), and from 20.17 to
137.17 mg/kg for FeS04 diet (Fig. IB). After 12 h of immersion
in seawater, the dietary iron content ranged from 18.66 to 77.97
mg/kg for FeMet diet (Fig. lA) and from 18.66 to 78.92 for FeSO^
diet (Fig. IB). There were similar leaching rates between the two
iron sources. After 6 h of immersion in seawater, the leached iron
accounted for approximately 20-35% of the total iron in the diets
for the two sources of iron, and this value increased to approxi-
mately 25-60% after 12 h of immersion in seawater.

Sunival and Growth

At the end of 16 wk, abalone fed the basal diet, without iron
supplementation, had significantly (ANOVA, P < .01) lower sur-
vival than abalone fed diets containing supplemental iron from
either FeMet or FeSOj. Abalone fed iron-supplemented diets did
not differ from each other in survival, regardless of iron level
(Table 2, Table 3). The average WGR (%) and DISL ((xm/day) of
the animals were significantly affected (P < .01 ) by the varying
levels of dietary iron from the two iron sources (Table 2 and Table
3). The mean weight gain rate ranged from 61.88 to 132.41% for
the abalone fed diets containing graded levels of iron from FeMet
(Table 2), and from 61.88 to 137.60% for FeSOj (Table 3). Daily
increment in shell length ranged from 63.99 to 85.46 (xm/day for
FeMet (Table 2), and from 63.96 to 86.54 |jim/day for FeSOj

A: FeMet

■in tir [Jh

2-J. 8 :t5. 5 ^ti. 5 5^. 3 S2. L' 136. 5 2iO. I

Dietary Iron (mg kg)

g 50

41.6 53.3 82.5 135.4 212.7

Dietary iron (mg kg)

Figure 1. Changes of iron content in the diets containing varying levels
of supplemental iron to the basal diet from either FeMet (A) or FeSOj
(B) with increasing immersion time (0, 6, and 12 h, respectively) in
seawater. Error bars are the SD, values significantly different
(ANOVA, Tuliey's test) from the controls (0 h) are indicated with
asterisks {*P < .05 and **P < .01).

(Table 3). Both WGR and DISL responded in broken-line models
to increases in dietary iron levels with the two iron sources (Fig. 2
and Fig. 3). The breakpoint in the regression line, which is con-
sidered to be the minimum dietary level for optimum response,
was 62.79 mg iron/kg diet for WGR, and that was 66.12 mg
iron/kg diet for DISL when using FeMet as the iron source (Fig. 2).
The breakpoints for WGR and DISL were similar when using
FeSOj as the iron source compared to FeMet, and the values were
58.35 and 62.1 1 mg iron/kg diet, respectively (Fig. 3). The ratios
of the slopes of the broken-line equations were 1.09 (2.1927/
2.0029) for WGR and 1.15 (0.5987/0.5196) for DISL with FeSOj
as iron source compared to FeMet (Fig. 2 and Fig. 3). Thus, The
bioavailability of dietary iron mainly from FeMet seems to be as
high as that mainly from FeSOj.

Carcass Composition

Data on percentage of soft body moisture, protein, and lipid are
shown in Table 4. There were no significant differences (P > .05)
in the contents of soft-body moisture and lipid of the abalone fed
varying levels of dietary iron from the two iron sources. The
content of soft-body protein, however, was significantly affected
by dietary treatment. The lowest protein level was obtained in the
abalone fed the basal diet, the value being 52.90%, and was ef-
fectively increased by supplementation of iron with the both
sources of iron.

864

Mai and Tan

TABLE 2.
WGR, DISL, und survival of abalone fed graded levels of dietary iron from FeMet for 16 wk (mean ± SD, n = 3).

Added Iron

Dietary Iron

Initial Shell

Initial Weight

Final Shell

Final Weight

WGR

DISL

Survival

(mg/kg)

(mg/kg)

Length (mm)

<g>

Length (mm)

Ig)

(%)

(pm/d)

(%)

0

24.9

16.052 ±.14

.695 ± .02

23.088 + .23"

1.1 24 ±.04"

60.88 ± 7.5"

63.96 ± 3.3"

88.00 ± 4.0"

10

35.5

16.111 ±.10

.704 ±.01

23.6 17 ±.22"

1.245 ±.04"

76.69 ± 4.3"

68.24 ± 1.1"

97.33 ± 4.6"

20

43.5

15.971 +.23

.686 ± .03

24.324 ±.12''

1.411 ±.02'

105.83 ±6.6'^

75.93 ± 1.2''

98.67 ± 2.3"

30

54.3

16.154±.10

.706 ±.01

24.760 ±.1 O*"-

1.527 ±.02''

1 16.38 ± l.O"'

78.24 ± .3"'"

1 00.00 ±.0''

60

82.2

16.195 ±.25

.708 + .04

25.595 + .17''

1.641 ±.03"

132.41 ± 11.8'

85.46 + 2.4"

1 00.00 ±.0"

120

136.5

16.093 ±.14

.700 + .02

25. 479 ±.14''

1.623±.04'''-'

131.71 + 1.0'

85.33 ± .3''

1 00.00 ±.0"

200

210.1

16.112 + .20

.707 ± .02

25.3 18 ±.46''

1.609 ±.06'*'=

129.87 ±5.4'

83.69 ± 2.6""

100.00 ± .0"

ANOVA

F value

.5206

.3276

57.3371

87.7834

57.9954

57.0539

9.5417

P value

.7835

.9114

.0000

.0000

.0000

.0000

.0002

' Means in the same column sharing a common superscript letter were not significantly different (P > .05) as determined by Tukey's test.

Elemental Concentration

The concentrations of shell ash. iron, and manganese are pre-
sented in Table 5. The contents of both the ash and the selected
elements of the shells were relatively constant {P > .05) regardless
of dietary treatment for the two iron sources.

The levels of soft-body ash, iron, and manganese are shown in
Table 6. After 16 weeks of feeding trial, no significant differences
(P > .05) were observed in the levels of soft-body ash among
dietary treatments. The concentrations of iron, however, were sig-
nificantly affected {P < .01 ) by the varying levels of dietary iron
from the two iron sources. The iron content ranged from 312 to
528 (o-g/g for FeSOj and from 312 to 530 (xg/g for FeMet. Iron
content also responded in a broken-line model to increases in
dietary iron levels with both iron sources (Fig. 4A and B). The
breakpoints in the regression lines were 66.78 mg iron/kg diet for
FeMet (Fig. 4A) and 64.55 mg iron/kg diet for FeSO^ (Fig. 4B).
The bioavailability of iron from FeMet in relative to that from
FeS04 was 101% (100 x 5.2225/5.1769). The manganese content
was strongly affected by the addition of iron from FeSOj, while
that was relatively constant irrespective of dietary iron levels from
FeMet (Table 6). Namely, the manganese content of abalone fed
diets containing 120 or 200 mg iron/kg was markedly lower than
that of abalone fed diets containing s60 mg iron/kg when using
FeS04 as the iron source.

DISCUSSION

The diets used in this experiment supported satisfactory aba-
lone growth. After 16 weeks of the feeding trial, all groups that
obtained sufficient dietary iron from the two iron sources grew
well both in mean weight gain rate and in mean daily increment in
shell length, in comparison to those reported by other authors (e.g.,
Uki et al. 1985, Uki and Watanabe 1992, Mai et al. 1995a, Mai et
al. 1995b, Mai 1998).

To our knowledge, there is only one previously published paper
pertaining to the dietary mineral nutrition of abalone, H. Iat'vai;ata.
(Coote et al. 1996). This is probably in part due to the problems
associated with leaching of minerals added to the soft tnoist diet.
In the present experiment, the special feed manufacture technology
was adopted so as to improve the water stability of the feed.
Leaching of dietary iron, however, still occurred (Fig. 1). We
observed the fact that the digestive tracts of the most abalone were
full of food within 2 h of feeding with the premium quality diets
(Mai et al. 1998). This, together with the fact that a series of
criteria responded in broken-line models to increases in dietary
iron levels with the two iron sources, makes the requirement of
dietary iron of juvenile abalone recommended in the present study
acceptable. If the water stability of dietary iron can be further
improved, the optimal level of dietary iron for this species may be
further reduced to a certain extent.

TABLE 3.
WGR, DISL, and survival of abalone fed graded levels of dietary iron from FeS04 for 16 wk (mean ± SI), n = 3).

Added Inm
(mg/kg)

Dietary Iron
(mg/kg)

Initial Shell
Length (mm)

Initial Weight

Ig)

Final Shell
Length (mm)

Final Weight

(g)

WGR

(%)

DISL

(Mm/d)

Survival

(%)

0

24.9

16.052+14

.695 ± .02

23.088 ± .23"

1.1 24 ±.04"

61.88 ±7..5"

63.96 ± 3.3"

SS.OO ± 4.0'

10

34.1

16.149±.I3

.709 ± .01

23.861 ±.25"

1.282 ±.0.3"

80.72 ± 1.6"

70. 1 1 ± 1 .6 '"

!()().()() + .0"

20

41.6

16.191 ±17

.7 13 ±.02

24..374±.10"

1 .433 ± .02'

101.08 ±7..5"

74.38 ±1.2"

1 00.00 ±.0"

30

53.3

16.1K8±.I0

.705 ±.01

25. 106 ±.21''

1.575 ±.08''

123.25 ±8.6'

81.07 ±3.5"^

97.33 ± 2.3"

60

82.5

16.228 ±.10

.7.^0 ± .02

25.686 ±.1 0'

1.7 19 ±. 0.5'-

135..16±4.6'

85.15 ± i.r

97.33 ± 2.3"

120

135.4

15.964 ±.20

.682 ± .03

25.483 ±.1.3'

1.6 19 ±.06''''

1.^7.60 ±5.8'

86.54 ± 1 .6'

1 00.00 ±.0"

200

212.7

15.999 ±.14

.681 ±.01

25.477 ± .20'

1 .672 ± .O.V'-

1.^7.55 ±7.2'

86. 17 ±3. 1'

1 ()().()() ± .0"

ANOVA

F value

2.2392

2.8451

(i0.2()94

66.1033

60.1299

41.0526

15.1333

P value

.1004

.0502

.()()()()

.0000

.0000

.0000

.OOOO

' Means in the same colum sharing a common superscript letter were no signiOcanlly different I/' > .05) as determined by Tukey's lest.

FeMet and FESO4 AS Dietary Iron

865

160
140
120

M 100

S 80

-i 60

40

20

0

♦ bl<ni=6J 79 mg/kg. V-136, 51-2 0029(63 79-Xt, R-=0. 9-1S6

blvpt = 66. 12 mg/kg. V=85. 33-0, 5196(66. 12-X). R'-O. 9403

• WGIi(»

■ DISKun/dl

100 150

Dietarv lion (lEg'kg)

160
140

120

w

1;

= 100

CO

a 80

u

.2 60

u

40

20

0

0

bkpt=58 .35 «g/kg, V=135. 36-2. 193T(58. 35-X), R' = 0.9955

bkpt=62. 11 mg/kg. V=86. 54-0. 5987(62. 1 1-X) , ll==0.9985

♦ WGR(»)

■ DISKua/d)

100 150

Dietary iron (mg.^'kg)

Figure 2. Regression of WGR (%) and DISL (fim/da.v) on dietary iron Figure 3. Regression of WGR (%) and DISL ()inVday) on dietary iron
levels and breakpoints (bkpt) in tlie lines for juvenile abalone fed the levels and breakpoints (bkpt) in the lines for juvenile abalone fed the
diets containing graded levels of iron methionine (FeMet) for 16 wk. diets containing graded levels of iron sulfate (FeSOj • THjOjfor 16 wk.

Significantly increased mortality was observed in the abalone
fed the basal diet without supplementation of iron, which con-
tained 24.85 mg iron/kg (Tables I and 2). This result is similar to
that reported by Sealey et al. (1997). They indicated that a defi-
ciency of dietary iron was found to increase mortality of channel
cattlfh due to enteric septicemia of catfish (ESC). They also re-
ported that the chemotactic migration by macrophages was de-
pres.sed in iron-deficient tlsh and a level of 60 mg/kg from either
FeMet or FeSOj provided the highest chemotactic indexes. The
mechanisms by which iron deficiency impairs immune responses
in mammalian species are not clearly understood (Sherman and
Morton 1984). Iron deficiency is responsible for the reduced ac-
tivity of several enzymes, including ribonuleotite reductase (Hoff-
brand et al. 1976) and myeloperoxidase (Baggs and Miller 1973).
Decreased protein synthesis due to the reduced activity of these
enzymes may be a factor in reduced immunocompetence through
decreased antibody production (Sherman and Helyar 1988). Rob-
bins et al. (1972) reported decreased protein synthesis in mammals
suffering from severe iron deficiency. In the present study, data
from the carcass composition showed that the lowest protein level
was obtained in the abalone fed the basal diet, and carcass protein
was effectively increased by supplementation of iron from the both
sources, while the content of carcass moisture and lipid was main-
tained relatively constant irrespective of dietary iron levels. More

studies should be conducted to understand the effects of dietary
iron on the immune responses and disease resistance in abalone.

Results of this study showed that WGR and DISL were the two
responsive parameters to dietary iron levels from both FeMet and
FeS04 and responded in broken-line models to increases in dietary
iron levels with the two iron sources. Significantly depressed
growth was noticed after a 16-wk feeding trial for the abalone fed
low-iron diets. Impaired growth has also been observed in fishes
fed low-iron diets (e.g., Gatlin and Wilson 1986, Lim et al. 1996).
However, studies with red sea bream (Sakamoto and Yone 1976),
yellow tail (Ikeda et al. 1973) and common carp (Sakamoto and
Yone 1978b) showed that the growth of these fish was not affected
by iron-deficient diets. In contrast, Suzuki et al. (1982) reported a
significant improvement in growth of eel fed a standard eel feed
supplemented with 250 mg iron/kg from iron amino acid chelate.
The present results indicate that supplementation of iron to the
basal diet is necessary to obtain normal growth of abalone. H.
discus haimai.

Many studies examining the dietary elemental requirements of
aquatic species have shown that depressed whole-body or tissue
levels of essential elements could result from insufficient dietary
intake (Lovell 1978, Ogino and Yang 1978, Ogino and Yang 1979,
Gatlin et al. 1982, Wilson et al. 1982, Paripatananont and Lovell
1995). Baker (1986) also indicated that studies on the mineral

TABLE 4.
Carcass composition in abalone fed various levels of supplemental iron from FeSOj or FeMet for 16 weeks (means ± SD, n = 3).

Supplemental
Iron*

Moisture ( % )

Protein

(%)

L

ipid ( % )

(mg/kg)

FeSOj

FeMet

FeSOj

FeMet

FeSOj

FeMet

0

77.06 ± .25

77.06 ± .25

52.90 ±.10"

52.90 ±. 10"

7.29 ±.16

7.17±.I3

10

77.20 ±

17

76.01 ±

20

53.60 ±.18"

53.37 ±.10"

7.42 ±.10

7.31 ±.10

20

77.10 ±

28

77.18 ±

15

53.37 ±.10"''

53.86 ±.14"

7.39 ±.10

7.34 ±.21

30

76.29 ±

18

76.93 ±

10

53.62 ±.11''

53.20 ±.13""

7.61 ±.10

7.49 ± .23

60

77.14 ±

28

77.28 ±

25

53.56 ± .20"

53.78 ±.11"

7.49 + .11

7.34 ±.10

120

77.33 ±

11

77.21 ±

18

53.59 ±.13"

53.42 ±.10"

7.43 ±.10

7.33 ± .23

200

77.04 ±

23

77.08 ±

17

53.33 ± .20""

53.63 ±.16"

7.41 ±.17

7.47 ± .20

ANOVA

F value

.4981

.8516

6.7800

17.6268

1.7016

.4072

P value

.7929

.5696

.0118

.0007

.2510

.8531

' The basal diet contained 24.9 mg of iron/kg diet, and the measured total dietary iron levels are the same as those in Tables 2 and 3. respectively.
"" Means in the same column sharing a common superscript letter were not significantly different (P > .05) as determined by Tukey's test.

866

Mai and Tan

TABLE 5.

Ash and iron, manganese content in the shell of abalone fed graded levels of dietary iron from FeSOj or FeMet for 16 wk (means ± SD,

n = 3).

Supplemental

Ash(%)"

Iron

(Mg/g)"

Mangane.se (Mg/gt''

(mg/kgl

FeSOj

FeMet

FeSOj

FeMet

FeSOj

FeMet

0

74.53 ±.10

74.53 ±.10

128 ±9.41

128 ±9.41

9.12 ±.78

9.12 ±.78

10

74.69 ±.16

74.56 ±.!0

119 ±7.94

130 ±4.82

8.74 ±.91

8.49 ± .73

20

74.56 ±.10

74.60 ±.10

135 ± 12.52

123 ±10.56

9.04 ± .46

9. 19 ±.95

30

74.60 ±.10

74.52 ±.16

128± 10.18

I18±12.18

8.86 ± .52

8.46 ± .78

60

74.57 ±.12

74.66 ±. 10

120 ±9.08

125 ±7.75

9.17 ±.54

9.02 ± .56

120

74.50 ±.13

74.55 ±.10

132 ±7.82

134 ±6.96

8.47 ± .90

8.77 ± .48

200

74.52 ± .20

74.46 + .12

126 + 5.65

129+11.03

8.68 ± .77

9.11 ±.79

anova

F value

.4811

.4210

.6730

.6584

.5782

.5956

P value

.8043

.8441

.6785

.68 1 8

.7144

.7008

* The basal diet contained 24.9 mg of iron/kg diet, and the meaured total dietary iron levels are the same as those in Tables 2 and 3. respectively.
" Dry weight basis.
'' Wet weight basis.

requirements of animals should include measurement of body
stores of the test element. Mineral analyses at the end of the
feeding trial indicated that soft-body iron concentrations of the
abalone increased linearly until the dietary iron reached 66.78
mg/kg for FeMet (Fig. 4A), and 64.55 mg/kg for FeSOj (Fig. 4B).
The reduced iron reserves were becoming depleted and deficiency
signs would most likely become apparent if these diets were fed
for an extended period of time. Therefore, the soft-body iron con-
centration was also the responsive criterion for estimating the di-
etary iron requirement of the abalone. However, similar responses
were not observed in shell iron concentrations of the abalone. This
suggests that shell iron deposition of the abalone was not a useful
criterion for determining the iron requirement of the abalone, es-
pecially when the experimental duration is not long enough.
Aquatic shellfish have special formation mechanisms for biomin-
erali/.ation of their hard tissue. Sakai (1980) found that the accu-
mulation of organic acids in the rearing water could lead to severe
shell erosion in the young abalone, and cause the shell to split
along the respiratory apertures. Chen (1989) reported that there

was a marked depression in calcium and zinc concentration in split
of H. (liversicolor siipertc.xui. These results, along with the data
obtained in the present study, implies that the rearing water qual-
ity, such as pH, perhaps play a more significant role than the
dietary mineral concentration in the diets in shell mineralization
and shell mineral deposition of abalone.

Interaction among minerals or nutrients can decrease intestinal
absorption of inorganic nutrients. Johnson and Korynta (1992)
indicated that excess iron decreased the absorption of manganese
in rats. Results of mineral analyses in the present study showed
significantly reduced manganese deposition in the soft-body of the
abalone fed diets containing high levels (>I00 mg/kg) of iron
from FeSOj. However, this effect of excess iron on utilization of
manganese was not found when using FeMet as the iron source.
The probable explanation is that when the mineral is bound in
chelated form, the interaction between the chelate and other min-
erals or other compounds is prevented to a certain extent.

The minimum level of dietary iron for juvenile abalone slightly
varied with iron sources and criteria. Data from growth, and soft-

TABLE 6.

Ash and iron, manganese content in the soft body of abalone fed graded levels of dietary iron from FeSOj or FeMet for 16 «k (means + SD,

n = 3).

Supplemental

Ash ( % )

Iron

iMg/g)'

Manganese

iMg/g)"

(mg/kg)

FeSOj

FeMet

FeSOj

FeMet

FeSOj

FeMet

0

11.51 ±.15

11.51 ±15

312 ± I2.86-'

312 ± 12.86-'

6.58 + .78"

6.58 ± .78

10

11.54±.12

11..59 + .10

376 ± 10.94"

359 ± 9.38''

6.46 ± .97"

6.28 ± .97

20

11.50±.10

11. .55 ±.10

426 ± 7.97"

419 ±8.58''

6.57 ± 1.02"

6.40 ±1.15

30

ll.57±.10

11 .49 ± . 1 8

458 ± I8.4I'-''

461 ±18.12-^^''

5.98 ± .76"

6.06 ± .86

60

11.49 ±.10

11.59 ±.14

519 ±9.74''

530 ± 1 7.74''

5.77 ± .94"

6.10 ±.81

120

1I.4S±.17

1I..57±.I0

528+ 12.66''

517 ± 10.88''

4.89 ± .72"

6.29 ± .66

200

11.58 ±.11

11. 56 ±.13

514± 18.2.5''

526 ±13.1 3''

4.92 ± .74"

6. 15 + .78

ANOVA

/•' value

.1428

.2089

.54.2785

56.3374

37.8865

.4186

P value

.9487

.9625

.0000

0.0000

.()()()()

.8627

* The basal diet contained 24.9 mg of iron/kg diet, and the measured total dietary iron levels are the same as those in Tables 2 and 3, respectively.

' Dry weight basis.

' Wet weight basis.

'' '' Means in the same column sharing a common superscript letter were not significantly different (P >.05) as determined by Tukcy's test.

FeMet and FESO4 AS Dietary Iron

867

600
500

3 400

0

- 300

0

t 200
o
100

0

bkpi=66-?8 mg/kg. Y=530- 5. 2225(66. 78-X). R-=0, 9852

50 100 150

Dietary iron (mg/kg)

bkp[ = 64, 55 mg/kg. V=538-5. 1769(54. 55-X). fi=0. 9541

100
Dietary

150

g.'kg)

Figure 4. Regression of soft-body iron concentration (pg/g, wet weight
basis) on dietary iron levels and breakpoints (bkpt) in the lines for
juvenile abalone fed the diets containing graded levels of iron either
from methionine (FeMet) (A) or from iron sulfate (FcSOj) (B) for
16 wk.

body iron concentration showed that about 63-67 mg/kg of dietary
iron from FeMet could maintain optimum growth and soft-body
iron deposition. When using FeSOj as supplemental iron source,
the minimum level of dietary iron was 60-65 mg/kg. We therefore
recommended that dietary iron requirement of juvenile abalone is
65-70 mg/kg. This estimated requirement is in agreement with
Lall and Hines (1987). who determined that the dietary iron re-
quirement of Atlantic salmon was 60 mg/kg. However, this re-
quirement is higher than those reported for channel catfish (30 mg

iron/kg; Gatlin and Wilson 1986. Lim et al. 1996). and lower than
those reported for eel ( 1 70 mg iron/kg; Nose and Aral 1987), and
sea bream (150 mg iron/kg; Sakamoto and Yone 1978a).

Studies with mammals have shown that chelation of minerals to
amino acids may increase their absorption rate in the intestine
(Ashmead 1992). He indicated that the higher bioavailability of
amino-acid-bound trace elements to animals is because chelation
protects the element from forming insoluble complexes in the di-
gestive tract and facilitates mineral transport across the intestinal
mucosa. He also suggested that the chelate could remain intact
until it reaches the site in the body where the element is needed.
Paripatananont and Lovell (1995) indicated that the relative bio-
availabilities of ZnMet. with ZnSOj as the standard, were 352%
for weight gain and 305% for bone zinc deposition in egg-white
diet in channel catfish. Our previous study also showed that the
bioavailability of ZnMet is approximately three times as high as
that of ZnSOj to juvenile abalone. H. discus hannai (Tan and Mai
2000). In the present study, however, we found that FeMet were
equally effective as FeSOj for improving growth and decreasing
mortality in abalone. This is in agreement with Lim et al. (1996),
who repotted that iron methionine and iron sulfate were equally
effective in preventing anemia in channel catfish. The reason for
the difference between the efficacy of chelated zinc and chelated
iron as mineral sources for animals is that calcium and/or phos-
phorus bind dietary zinc and decrease its absorption through the
intestinal mucosa (Lewis et al. 1994), but iron is not similarly
inhibited; therefore, chelation may be not as effective for iron as
for zinc (Lim et al. 1996).

Results of the present study indicated that a level of 65-70 mg
of iron/kg either from FeMet or from FeS04 was sufficient for
growth and tissue iron deposition in the abalone. Both iron methi-
onine complex and iron sulfate heptahydrate were equally utilized
by abalone, H. discus hannai. Further studies should focus on the
responses of the abalone to dietary iron from the two sources of
iron, using other criteria, such as immune parameters and hema-
tological values.

ACKNOWLEDGMENT

The authors are grateful for financial support by grant Nos.
39670572 and 39770589 from the National Natural Science Foun-
dation of China (NNSFC). We also thank Mr. Fulong Liu of Shan-
dong Commodity Inspection Bureau for assistance in elemental
analyses.

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Sherman, A. R. & L. Helyer 1988. Iron deficiency, immunity, and disease
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Sherman, A. R. & P. E. Morton 1984. Copper metabolism in the iron-
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Suzuki. K., Y. Iwahasi & T. Takatsuka 1982. The effects of iron amino
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roles of amino acid chelates in animal nutrition. Noyes Publication.
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Tan, B. & K. Mai. 2000. Zinc methionine and zinc sulfate as sources of
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Uki, N. & T. Watanabe. 1992. Review of the nutritional requirements of
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test diets for abalone. Bull. Jpn. Soc. Sci. Fish. 51: 1825-1833.

Wedekind, K. J., A. E. Hortin & D. H. Baker 1992. Methodology for
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phosphorus requirement of channel catfish. J. Nutr. 112: 1197-1202.

7<)H/7i<;/ oj Shellfish Research. Vol. 19, No. 2, 869-881, 2000.

POTENTIAL DISPERSION OF REPRODUCTIVE PRODUCTS AND LARVAL STAGES OF
ABALONE {HALIOTIS SPP.) AS A FUNCTION OF THE HYDRODYNAMICS OF BAHIA

TORTUGAS, MEXICO

SERGIO A. GUZMAN-DEL PROO,' ' FELIPE SALINAS,^
OLEG ZAYTSEVr ' JORGE BELMAR-PEREZ,' AND
JORGE CARRILLO-LAGUNA' '

Ecology Laboratory, Department of Zoology. National School of
Biological Sciences. National Polytechnic Institute. Prol. de Carpio y Plan
de Ayala. Mexico. D.F.

^Oceanology Department. Interdisciplinary Center of Marine Sciences.
National Polytechnic Institute. Playa el Conchalito s/n. La Paz. B.C.S..
Mexico
^Becario COFAA

ABSTRACT Field ob.servation.s of currents and water mixing were made in autumn 1996. at four coastal sites close to Bahia Tortugas,
on the central part of the Baja California Pacific coast, to evaluate the influence of the hydrodynamics on the transport of abalone larvae
[Haliotis spp.). Current measurements and full-scale Lagrangean experiments on .surface-water transport were carried out during the
main spawning season of this genus in the area. Tidal currents seem not to be the dominant factor, but instead both wind- and
wave-driven currents appear to be the most important factors for larval transport in this coastal area. Additional echo sound and aerial
surveys confirmed that the reef topography and kelp beds attenuate current velocity. The hypothesis of larval dispersion is that during
a typical 3- to 5-day pelagic period, larval and postlarval stages might be retained primarily in areas close to parental reefs. Flushing
time in Bahia Tortugas was evaluated as five semidiurnal tidal periods. Sufficiently intensive currents at the mouth of the bay (up to
25 cm/sec) may complicate larval interchange between northern and southern vicinities of the bay.

KEY WORDS: abalone, Huliolis. dispersal, larvae, hydrodynamics, transport

INTRODUCTION

Currently, abalone (Haliotis spp.) catch off the Baja California
Penninsula coastal zone is so limited that the stability of the fish-
eries is threatened. Catch has dropped frotn 6.000 tons annually in
1950 to around 600 tons (meat weight) in 1997 (Semamap 1997).
Some authors attribute this scarcity to oveifishing and consequent
low levels of recruitment (Guzman del Proo 1992. Prince and
Guzman del Proo 1993), which in turn has led to strict manage-
ment measures based on a quota system for each fishing zone
(Ramade-Villanueva et al. 1998).

A crucial consideration in the management and administration
of these fisheries is a clear understanding of the concept of a unit
stock (Shepherd and Brown 1993), and this requires knowledge of
larval dispersal. Two different hypotheses on larval transport have
been proposed for abalone. The first postulates that the larvae can
be transported for long distances from their parent reef and supply
other distant reefs (Forster et al. 1982, Tegner and Butler 1985).
The second establishes that larval interchange between reefs is
limited and that each reef is a small independent stock that replen-
ishes itself, with very limited larval mixing between neighboring
reefs (Prince et al. 1988, McShane and Smith 1991). The first
hypothesis implies a management policy that would cover long
stretches of coastline, whereas the second would require individual
management of each reef.

Information that could help to solve this question is a knowl-
edge of the type of circulatiiin and hydrodynamic patterns in the
coastal areas, where abalone are found on locally isolated rocky
reefs parallel to the coast from the intertidal zone down to 20-m
depths (average). Large abalone concentrations are found among
giant kelp beds {Macrocystis pyrifera) and abundant algal vegeta-
tion that serves as their habitat and food.

The reproductive period oi Haliotis fulgcns and H. corrugata is
from late summer, during fall and the onset of winter (Sevilla

1972, Belmar-Perez and Guzman del Proo 1992, Garcia and Ortiz
1992). Spawning for boths species in Tortugas begin in summer
(August) and last trough the autumn, ending in December or Janu-
ary (Andrade 1971, Guzman del Proo unpublished data); however,
the peak spawning ocurs over a more restricted period (October
through November). Larval metamorphosis, trocophore to veliger
stage, takes place in the water column, where larvae float for some
3 to 5 days and for a maximum of 1 5 days before the settling larvae
attach to the bottom and begin their benthic life (Leigthon 1974).
Recruitment into the fishery depends to a large degree on high
settling success on a suitable rocky substrate (McShane 1996).

Larval dispersal depends on the coastal topography and coastal
hydrodynamics adjacent to abalone habitat (Shepherd et al. 1992).
Some authors propose larval dispersion is limited to tens or hun-
dreds of meters, with almost immediate settlement taking place in
the vicinity of the parent stock (Prince et al. 1988). but others
suggest that larvae are transported for kilometers, depending on the
current regime, wave action, and storms (Sasaki and Shepherd
1995). Since juveniles and adults are practically sedentary organ-
isms whose displacements do not exceed tens of meters (Shepherd

1973, 1986), larval supply and postlarval settlement turns out to be
a critical phase for recruitment and subsequent abundance of aba-
lone adults. Although there are uncertainty of the relationship be-
tween larval supply and recruitment in abalone (McShane 1995), it
is only in this manner that adult abalone populations can replace
themselves and exchange genetic material. A larval exchange on
large distances or confined areas, has important implications for
the management of this fishery.

In this article, we analyze the results of hydrodynamic studies
made in autumn 1996 at selected abalone reefs close to Bahia

869

870

Guzman-del Proo et al.

Tortugas, where current velocities and wave action were measured
by Eulerian and Lagrangean methods. The objective was to evalu-
ate, during the peak spawning season, the influence of the hydro-
dynamics on the transport of abalone larvae, and the potential
larval conectedness between the abalone reefs located at north and
south of Bahia Tortugas. This allowed us to formulate preliminary
hypotheses regarding the potential larval dispersion of Haliotis
fulgens and H. corrugata. which inhabit this zone.

AREA OF STUDY

The zone under study covered the area where abalone reefs are
located, to the north and south of Bahia Tortugas, including the
bay proper. This coastal zone forms part of the area which exploits
the Bahia Tortugas Fishing Cooperative (Fig. I ).

Four sites were selected for study: La Colitloruda (Station I. 12
m depth) to the north of the bay; the mouth of the bay (Station II,
16 m depth); La Pinta (Station III. 10 ni depth), to the south of the

bay; and La Bajada (Station IV. 6 m depth), inside the bay (Fig. 1 ).
La Colitloruda and La Pinta are highly productive abalone reefs,
with irregular bottom topography and are normally covered by
extensive giant kelp (Mucracystis pyrifera) beds (Fig. 4, above,
and Fig. 9). La Bajada (Fig. 1 ) is a shallow and protected bed of
boulders with no kelp and where juvenile abalone are abundant.
The mouth of the bay is a strait, where most of the water is
exchanged between the bay and the ocean.

Given that the main channel of the mouth of Bahia Tortugas is
normal to the NW-SE line, waves that manage to get into the bay
come from the SW. Main wave energy entering the bay comes
from this direction, and significant waves heights coming from the
SW and W are in the range from 1.40 to 2.75 m (Secretari'a de
Pesca 1981). Between August to December, where is the entire
spawnig season for H. fulgens and H. cornigata. winds are from
the W. In November, winds blow from both the north and the west.
In Bahia Tortugas the average annual winds are from the NW, and
these are the dominant winds (Secretan'a de Pesca 1981).

Figure I. Study area. Sample site locations and main kelp l)eds (dashed I in the area ol abalone reels close lo Bahia lorlugas. November 1996.

Potential Dispersion of Abalone Larvae

871

Tides in Bahia Tortugas are mixed, semidiurnal, ranging from
1.40 m to 2.00 m with a time lag to tidal measurements made with
the mareograph at Guerrero Negro (30 km to the north) but is an
almost perfect match with the wave-tide gauge at Isla de Cedros,
located 120 km to the northwest (Secretaria de Pesca 1981).

MATERIALS AND METHODS

The field observations were made during the fall (September
through November) using three methods: ( I ) measurements of sea
level variations and horizontal current components by "Inter-
Ocean" S4. current meter of electromagnetic induction placed 40
cm from the bottom at four selected stations (Fig. 1): (2) vertical
profiling of currents by acoustic Doppler profiler "Sontek"
(ADCP) at the same sites and at the mouth of the bay; and (3)
Lagrangean experiments using fluorescent dye (Uranine) to esti-
mate advection and diffusion. The measurements were made at
tidal ebb and flood. Depth profiles were made with "Furuno"
echo-sounder.

Current measurements at the fixed stations were made between
November 26-30, 1996. At station I (La Colitloruda. Fig. 1), we
recorded horizontal current components and sea level variations
(to define the variables of tides and waves) with a 2-Hz sampling
frequency. At station III (La Pinta. Fig. 1 ), the equipment was
programmed to measure variations in the sea level for 10 min
every hour with the same frequency. Currents and tides at stations
II and IV (Fig. 1) were recorded with a sampling interval of I min.

At stations I and III devices were in.stalled close to giant kelp
beds, but not inside them, and additional ADCP profiles were
made both close to the current meters as well as inside adjoining
kelp beds. Water exchange between the bay and the ocean was
determined by means of a series of current profiles at the mouth of
the bay.

To record surface currents, dye-tracer experiments were done
on September 13 and 14, 1996 and repeated on November 27,
1996 at the same four sites. The displacement and position of each
one of the dye patches were determined by a boat with a "Magellan
5000" GPS. These position records were combined with aerial
photos, which allowed us to determine the size of the dye patches
by using the boat size as a scale reference. The boat was always
located at the same point of the spot.

RESULTS

During field observations, typical wind patterns were observed
in the area. Normally, the breeze pattern was recorded at the wind
intensity up to 4—5 m/s, but on November 27, the wind increased
over a 6-h period up to 8-10 m/s, and on the 29th, there were
winds up to 12 m/s from the SE coupled with rain. This was
possibly caused by the influence of hurricane Fausto. which passed
at the extreme south of the Baja California peninsula

Bottom Currents and Tides at Fixed Stations

During the measurements the tide was mixed with a range of
1.9 m (Fig. 2a). Water movement at each of the stations is de-
scribed below.

Station I, La Colinoruda

During all measurements, the average near-bed current was
about 13 cm/sec and the direction of the current was consistently
around 350° (NNW), which indicates a net transport in that direc-
tion (Fig. 2b). At this station there is no notable influence of tidal
currents. Possibly, this effect can be explained by both local to-

pography and the fact that we placed the equipment close to kelp
beds where tidal currents are attenuated by the presence of the
kelp. Short-period local waves and swells, in comparison to tides,
cross the kelp losing little energy and develop a type of mass
transport known as Stokes transport (Phillips 1980), which masks
the tidal current. The direction of the transport, or at least its
average value, is the same as the direction of incidental waves. The
spectral density and direction diagrams of sea-level variations at
the site during the experiment showed a swell of up to 2 meters
coming from the NWW (Fig. 3).

Station II (mouth of the bay)

This is the only station where data obtained showed the pres-
ence of significant tidal currents. The near-bed currents at the
mouth are reversible and changed direction from 130°- 1 50° (the
ebb) to 350°-360° (the flood) with a current speed of 1-2 cm/s
during the high and low water and about 10 cm/s during the flood
and ebb (Fig. 2c). Disturbance caused by strong west winds on
November 28 led to intense wind- and wave-driven along-shore
currents, masking tidal currents until November 30, when tidal
influence became evident once again (Fig. 2c). Intensive winds can
interrupt the "pump-style" tidal currents at the mouth of the bay
and cause a current along the coast.

Station III (La Pinta)

Current velocities vary from 12 to 24 cm/sec in a constant
direction of 180° (toward the south). They were not generated by
tides, but were caused by another dynamic process; wind-driven
flow and a current that compensates for the permanent wave trans-
port (Fig. 2d). Aerial photography showed the existence of small-
scale topographic eddies at the site (Fig. 4, bottom panel).

Station IV (La Bajada)

Here, the current meter was in operation for 43 h. We observed
that the near-bottom current was not influenced by the tide. Shown
in Figure 5 are four different hydrodynamic situations: ( 1 ) from
12:30 p.m. on November 26, the current had an average speed of
4 cm/s, wind was 3^ m/s; (2) with no wind, we ob.served that the
average currents were of low intensity and flow in all directions;
(3) here, the drift currents were between 5-6 cm/s, but local winds
increased their intensity up to 6-7 m/s, 200°-230° NE; and (4)
here, the currents were very similar to (2), This pattern was caused
to the combined effect of wind-driven currents and bottom friction,
because the average depth was about 5 m.

ADCP Profiling at Fixed Stations

Figure 6 shows typical vertical profiles of currents measured on
November 30 close to autonomous devices at four fixed stations in
the abalone reefs. Wave action was filtered from current profiles
for periods of up to 20 s.

The La Colifloruda profile (Fig. 6a) was a good match with
data recorded by the current meter. Current intensification up to 30
cm/s was registered in sub-surface layer. At depth, the current
speed varied from 1 0 to 20 cm/s. Although the current speed in the
water body around the kelp beds was significant, inside them it
dropped considerably.

Current profiles measured close to Station 11 at the mouth of the
bay (Fig. 6b) show the tidal current, measured during the flood,
had two-layer structure. Current speed was attenuated deeper than
8-10 m from 30-33 cm/s to 20 cm/s. Similar profiles in the
opposite direction were recorded during the ebb. The profile in

872

Guzman-del Proo et al.

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12 24 12 24 12 24 12 24 12
Time (h)

FiRure 2. Tidal level variations and velocity of currents al the llxed stations: 1 (La COlillonidal, 11 (!-a Boca) and III (La I'intal on November

2ft, I'Wft. i

Fig. 6c, taken inside the bay close to La Bujada (.Slalion IV). ehorcJ at this station. However, at the siirlaee, we note a counler-

represent.s the vertical distribution of" the current with a wind thai current caused by a SW wind (Fig. 6d). 1

blew at 10-12 m/s (November 30), with a gradient typical for '*

currents induced by wind. The current profile at La Pinta (Station Waltr Exchange Between (he Ray and Pacific Ocean

III) presents a more complicated structure. At depths greater than

three meters speed and ilireclion (to the S) were a good match with The series of current profiles were made during the ebb to

current recording provided by ihc autonomous instrumeni an- evaluate the v\ater exchange between the bay and ocean. Data

*

Potential Dispersion of Abalone Larvae

873

28 Nov. 7:00-7:30

0.01

1E-05

0.001 r

0.0001 T

0.01

0.1
Frecuency, Hz

28 Nov. 7.00-7300

90 180 270

Direction, grad

360

29 Nov 19:00-19:30

1E-05

*£ 0.01 f - -

? 0.001 i"

0.0001

0.01

0.1
Frecuency, Hz

29 Nov. 19.00-19.30

90 180 270

Direction, grad

360

30 Nov. 7:00-7:30

30 Nov, 7.00-7.30

1

E

10

1

0.1

0.01

0.0001

1E-05

: : : i/v;:: ; ;;::;;:

i 1 \ - r Tirii r\~ N" i — 1 — i -i-i-i ri-

1 i--i-r-rri-in i — rV' ~i~i"i ri-

160

0.01

0.1
Frecuency, Hz

90 180 270

Direction, grad

360

Figure 3. Typical spectral deasities and directional distribution of the wave action at La Colifloruda at three different wind speed: November
28, 6-8 m/s (signiflcant wave heights were from 0.9 to 1.0 m) and November 29 and 30, up to 15 m/sec (significant wave heights were up to 2.0
m). Main wave direction was from the West in the course of experiment time.

obtained showed most of the profiles have the shape portrayed in
Figure 6b. which is typical for a channel. In addition to this, they
are vertically uniform because of the tidal pressure. Near-bottom
speed was slower because of bottom friction. The width of the
main mouth of the bay is 3,000 m. with a depth ranging from
10-19 m. The total area of the cross-section of the mouth is 41.400
m" at an average tidal level (Fig. 7b). By using the vertical current

profiles at the mouth of the bay (Fig. 7a). we calculated the water
exchanged during a tidal cycle (November 26. 1996) was about 29
X 10" m-.

The bay tidal prism for the same tidal cycle was 37 x 10''m''
(the surface area of the bay is 24 xlO^m", and tidal variation during
this cycle was 1.58 m). The approximate volume of the bay at an
average sea level is 145 x 10''m\ and the approximate time the

874

Guzman-del Proo et al.

Figure 4. Kelp litd lahovu) and sniull-sculi' uddy Ihi'low) in the vicinity ol' La I'inta.

water renniins witliiii R;ilii;i Tiirliiiias is llvf scniidiiiriial liil;il
cycles.

A comparison ot the tidal prism with the water exchange cal-
culated from current profiles shows that 77% of the water was
exchanged through the main channel and only 23'/r through the
straits between the islands at the southern part of the hav. where
the depth was 2 to 3 m.

Lagraiif'eaii Kxperimenls Willi Fluorescent Dye

The aqueous solution of Uranina was released instantly on the
surface and. becau.se of vertical mixing, the coloring agent was
dispersed to depths of 5 to 6 m during the experiment, which lasted
between 2 and 3 h. The results recorded with aerial photography
show the Lagrangean trajectories of dye spots and enable calcu-

Potential Dispersion of Abalone Larvae

875

St.lV(LaBajada)

10

E
o

o
o

>

0

12

0 12

Time (h)

3 ^

E

■a

5cm/s

Figure 5. Currents recorded at Station IV inside Bahia Tortugas, at different times of recording (one sample per minute), November 26 through
28, 1996. Tides did not affect current pattern at this site, current velocity growth at intervals (a) and (c) was forced by wind.

lation of the average current velocity of the surface layer (Fig. 8
and Table 1 ). Figure 8a shows the location of the experiments. In
September (Fig. 8c. ebb. and Fig. 8d. tloodl. inside the bay cur-
rents varied between 4 and 9 cm/sec. and trajectories of dye
patches were changed according to the tidal cycle, but not signifi-
cantly (patches 2, 3, and 4 in Fig. 8c: 3 and 4 in Fig. 8d). During
the experiments made in November (Fig. 8b, spots 4 and 5, the
ebb), the trajectories were similar to those of September. We also

observed that the transport of surface water in the narrows between
islands was always out of the bay, with speeds from 4 to 7 cm/s,
during both the ebb tide (Fig. 8d. spots 1 and 2) and the flood (Fig.
8b. spot 3). Thus, the tides do not change circulatory patterns (in
clockwise direction) in the southern part of the bay and larvae are
transported into the bay through the main channel can possibly be
carried out the bay through the narrows between the islands.
Experiments at the main mouth (patch 1. Fig. 8c; spots 1 and 2,

876

Guzman-del Proo et al.

a

u
Q

Speed (cm/s) o 90 i80 270 360 90

10

12

La Colifloruda

0 10 20 30 40 50 Direction (degree)

a
u
Q

0

2

4

6

8

10
12
14 I

Speed (cm/s) 0 90 180 270

16 t.

La Boca

0 10 20 30 40 50 Direction (degree)

Speed (cm/s) 0 90 1 so 270 360 90

Speed (cm/s)

0 90 ISO 270 360

0
1

^ 2

B

i" 3
a

C

T — • — rr^ — I — ' — r-

La Bajada

0
2

1 4

•S

e- ^

10 t

La Pinta

0 10 20 30 40 50 Direction

0 10 20 30 40 50 Direction (degree)

Figure 6. Typical vertical current profiles at fixed stations. Waves were filtered through these profiles with periods under 20 s (Novemher 30,
1996).

Fig. 8b) confirm that tidal currents are between 17 and 25 cm/s and
supply almost 80% of the exchange of water between the bay and
the ocean.

Figure 8e shows the trajectories of the patches recorded in the
northern section of the area inside the Macrocyslis forest. In both
September and November, we observed the dye transport in the
kelp beds was toward the coast (to the NE), because of the wave
action. This result does not match well with the data recorded by
the current meter located close to the algal forest, which always
detected currents toward the N (Fig. 2b). The trajectories of the
patches at Playita Brava (at the extreme north of the mouth of the
bay. Fig. Be) were toward the south for both tides (flood and ebb).
This effect can be explained by means of compensating currents
along the coast forced by the swell. It appears the same process
occurs in the southern part of the area (Fig. 8f), where the dis-
placement of the patches is also toward the south with low speed
(4-6 cm/s).

DI.SCUSSION

Hydrodynamic Features of (he '/.one

On the whole, we found thai al noilheni and southern vicinities
of the Bahia Tortugas tidal currents were not dominant, and there

is a slow average water transport along the coast from the NNW to

the SSE with average velocity of 4-5 cm/s. This transport was
related to currents forced by wind and wave actions, but inside
kelp beds along-shore water movements were attenuated and wave
transport to the coast by swells prevailed, as at the sites La Coli-
lloruda and La Pinta.

The water exchange between the bay and the Pacific Ocean, as
measured by a series of vertical current profiles at the mouth (Fig.
6b), allowed us to estimate the bay Hushing lime as 5-semidiurnal
tidal cycles, that is, 2.5 days of residence inside the bay. Therefore,
the tide acts as a pump that transports water in and out of the bay
at a speed up to 25 cm/s (Figs. 2c and 6b). The existence of this
reversing flow is very important because its presence can interrupt
the along-shore current from the north, which has a slower speed
and leads to limited communication belueen the northern and
southern groups of abalone reefs. The study shows the effects of
swell (periods between 10-25 s) detected at stations 1 and 111 (La
Colitloruda and La Pinta). On these external reefs, the currents
were caused mainly by wind and by waves rather than by tides,
especially inside kelp beds. The maximum tidal range was 1.9 m
over the 4 days we examined, and the assemblage of the wave
energy spectra shows that average wave heights were between 1 .5
and 1.8 m, with an average period about 17 s (Fig. 3). From this,
we conclude that the wave energy flux outside the bay dominated
the tidal energy and that nonlinear water transport (Stokes trans-

Potential Dispersion of Abalone Larvae

877

Bahia Tortugas

Pacific Ocean

Q)

20 cm/s

9

8

7

6

5

4

3

2

1

0

T

1

»

T

»

t

I

»

»

\ 302m

320m

357m

373m

396m

357m

325m

304m

276m/

5

10

\2564

4315

5376
m2

6240
m2

6718
m2

5739
m2

4867
m2

3707

190§/

15

in

" b

^--- ,

-^

^--^

Figure 7. Scheme of water exchange calculations during the ebb: (a) Average velocity of the current at the main mouth of Bahia Tortugas on
November 28, 1996; (b) vertical section of the mouth and partial area of each profile.

port) was significant because of wave action. This explains the
relative constancy of direction of the current at these sites. At
station IV, the origin of the currents was mostly caused by wind
and bottom friction. Speed was slow, between 3 and 8 cm/s in the
bottom layer, and significant only during the maximum flood and
ebb. This became evident with the observed displacements of the
dye patches (Figs. 8c. 8d).

At La Colifloruda, the near-bed current recorded by the au-
tonomous current meter was persistently toward the coast, as was
shown in the experiment with the dye spots (Fig. 8e). but current
profiles recorded outside of the kelp forests show along-shore
water movements to the SSE. Possibly this disagreement may be

explained by the influence of reef topography and the presence of
extensive kelp beds. It is true that inside kelp beds we found a slow
water movement toward the coast caused by wave action, whereas
outside of them, along-shore flow to the south was recorded. It was
obvious that the presence of massive kelp beds was a key envi-
ronmental element in this zone. In the water column down up to a
depth of 20 m. we can see the influence of algae formations, such
as kelp {Macrocyslis pyrifera) and other groups of laminarians.
such as Eisenia, Egref>ici or Cystoceira. Together, they fomi a
physical barrier that can attenuate the current sometimes to one-fifth
of the speed (Jackson and Winant 1983, Bernstein and Jung 1979).
Seabed topography composed of different slopes and channels

878

Guzman-del Proo et al.

Bahia
Tortugas

OA

■ - 1 3 Sep (flood)
* -13 Sep (ebb)
A -14 Sep (flood)
o - 27 Nov (ebb)

Bahfa
Tortugas

A - 14 Sep (flood)
o -27 Nov (ebb)

J]

Bahfa Tortugas

^ Morros
7^2

ada

1 3 Sep (flood) ^

Finuru 8. I,agran|>eun Irajotlork's of the displaccmcnl of I'raniiie spots in the area of Bahia TortiiRas, Septemher 13 and 14 and Novemher 27,
I9'>6. Average velocities are shown in 1'ahle I.

Potential Dispersion of Abalone Larvae

879

TABLE 1.

Average velocities of tlie surface transport of Uranine spots.

Lagrangean experiments in Bahia Tortugas, September and

November 1996.

Average

Average

Date

Spot

speed

Date

Spot

speed

(figure)

number

(cm/s)

(figure)

number

cm/s

27 Nov (8b)

1

17.2

13 Sep(8d)

1

5.0

ibid.

T

24.7

ibid.

-)

4.5

ibid.

3

4.7

ibid.

3

8.3

ibid.

4

7.1

ibid.

4

5.9

ibid.

5

6.3

14Sep(8e)

1

1.4

13 Sep (8c)

I

20.7

ibid.

-)

7.6

ibid.

2

8.2

27 Nov (8e)

1

2 2

ibid.

3

4.8

ibid.

2

5.8

ibid.

4

5.7

14 Sep (80

1

6.2

ibid.

T

4.6

.

and exposure to the coastline to swell are other influential factors
(Fig. 9). Kelp beds, taken together with bottom topography and the
degree of exposure, function as plankton traps (Jackson and
Stratham 1981 cited in McShane et al. 1988) and explain the slow
diffusion and advection of Uranine observed at reefs at La Coli-
floruda and La Pinta (Figs. 8e and 8f).

Within these coastal circulation processes, the on and offshore
transport by internal waves or tidal bores, which is linked to the
lunar cycle and the composition of diurnal and semidiurnal tidal
components needs mention. Pineda (1994) has reported these
waves advect water from hundreds of meters to a few kilometers
from the shore, and, in spite of a limited scope of action, the
importance of this transport can be critical in moving nutrients,
food, and planktonic larvae in coastal waters. This type of circu-
lation, in combination with the transport capacity of floating ma-
terial and neuston through slicks and serial waves, may have a
significant influence on larval recruitment in benthic systems and
pelagic communities in coastal waters (Pineda 1991). We do not
believe this dynamic process is continuous in time nor that it
affects coastal waters shallower than 20 m, because internal waves
are usually associated with the main picnocline, which was 45-50
m depth in Bahia Tortugas. So. at this site, internal waves must be
reflected due to a sharp, bottom morphology. Therefore, we did not
measure these types of waves, although in the coastal zone with a
gentle slope internal waves must participate in the regional hydro-
dynamics.

Larval Dispersion Caused by Local Hydrodynamics

The main questions needed to explain the distribution of aba-
lone banks are where and how far can larvae be transported from
their point of origin? To study of the larval-transport problem, it is
necessary to determine first, if some measurements of currents at
certain points are sufficient to trace the trajectory of small sections
of water and abalone larvae? This is far from being true. The larvae
trajectories are complicated because coastal currents are not uni-
form and any extrapolation to assign values for distance traveled
by the larvae will be only a rough estimate and will depend on the
prevalence and alternation of tidal currents, wind drift, local
waves, and swell at each site.

In this study, we are obviously assuming that Hciliotis larvae or
gametes are passively transported just like the inert fluorescent dye

used in our experiments. Although the veliger larvae and postlar-
vae, before settling, have a certain ability to move in a given
direction and to choose an attractive substrate for settling at the
bottom (Strathmann 1974), the available evidence suggests that the
veligers of abalone behave as passively transported particles (Mc-
Shane 1992) and that eddies in coastal waters concentrate larvae,
an observation consistent with passive transport of larvae (Tanaka
et al. 1986, cited in McShane 1992).

Leighton (1974) notes that the duration of larval life is con-
trolled primarily by temperature and varies from 4 to 15 days. He
observed that larvae of H. fulgens, at 22-23 °C settled by the
fourth day. and H. corrugata at 21-22 °C settling was observed as
early as 3.5 days. In the reefs, the reproductive success (spawning
and settling) depends on a set of hydrological conditions, since
transport of larvae away from reef habitat would cause high mor-
tality, particularly because abalone larvae have only a few days
during which they are competent to settle (McShane 1992). Then,
we assume the results presented here are applicable to the earliest
stages of larval life of Haliotis before settling, i.e., the first 4—5
days when most larvae are settling. Our assumption is also sup-
ported on studies carried out al Bahia Tortugas abalone farm,
where have been shown that the trocophore stage occurs after

La Colfloruda ^ ^T -titTv \ \

\ \>^'
Los H/brros \ \ * >

^ \-

X •

Pacific (Dcean , _. ^ ~ ,, ,

La Pinta ^i-.

Oisbnce (m)

Figure 9. Bathymetric profiles of the sampling stations: (a) La Colif-
loruda; (b) Los Morros-La Boca; and (c) La Pinta.

880

Guzman-del Proo et al.

12-15 h after fertilization at 16-20 °C and most settlement of
veliger stages takes place 4-6 days later (Mason-Suastegui et al.
1992).

Abalone larval dispersal distances and settlement are critical
components of abalone population dynamics, which continue to be
a concern and for which there is not much agreement. In a broad
sense, abalone are considered short-distance dispersers (Allison et
al. 1998). Nevertheless, some authors state that Haliotis larvae can
be transported .several kilometers (Forster et al. 1982. Tegner and
Butler 1985), in some cases up to 10 km (McShane et al. 1988).
Other authors believe that the larvae settle very quickly in the
neighborhood of the parent stock on a small spatial scale of tens to
hundreds of meters (Prince et al. 1988). McShane et al. (1988,
1991) and McShane and Smith (1991) have supported too the
hypothesis of that settlement is highly variable on small spatial and
temporal scales. Some models propose that larvae may be dis-
persed over short or large distances depending on whether they are
released near high relief reefs or in open waters respectively (Mc-
Shane et al. 1988). Recently, Sasaki and Shepherd (1995) have
suggested a model of larvae dispersal for Haliotis discus wherein
the scale of dispersal is related to the intensity of the inducing
storm event. Whatever the case, there is little dispute that abalone
larvae are passively dispersed as considered in this study.

Our calculations of the Lagrangean transport velocities (Table
1 ) show that in some areas in and near Bahia Tortugas average
cuiTents can potentially transport larvae for 3 to 5 km along-shore
during their pelagic cycle. The northern reefs group (La Colitlo-
ruda-Playita Brava) has 10 exploited abalone reefs, while the
southern one (La Pinta-Morros) contains 12 spots (Soc. Coopera-
tiva Bahia Tortugas 1996). The average distance between two reefs
is about 1-2 km. That means that potentially larval exchange could
take place between neighboring reefs. However, extensive kelp
beds, small-scale topographic eddies, and onshore-wave mass
transport significantly attenuate this along-shore larval flux. To be
transported along the coast, larvae must leave their parent reef, but
only a small number of them have a chance to do this because of
small water movements inside the kelp beds. This limited number
must be sufficient to maintain the larval exchange between neigh-
boring abalone reefs of northern or southern sites near Bahia Tor-
tugas, but it is not obvious that larvae leave their spawning area to
be directly transported from the northern reefs group (La Colitlo-
ruda) to the southern one (La Pinia) or the reverse. Under normal
conditions, the most probable way for larval exchange between
northern and southern reefs is through Bahia Tortugas. During the
Hooding, larvae arc transported into the bay in the northern part of
the main strait by tidal tlow; during the ebb, they come out through
the southern straits. This is possible because the flushing time is
less than the larval pelagic cycle. This scheme is valid under the

hydrodynamic conditions observed, when the swells coming from
the W induce long-shore average transport to the SE and tidal
currents are not dominant. Thus, northern reefs donate larvae to
southern ones. Under a different direction of swell, for example
from the S, this scheme will be reversed. It is clear that only a
small number of larvae can be transported this way. It may be that
storms can only improve the larvae exchange between the northern
(La Colifioruda) and southern (La Pinta) groups of abalone reefs.

CONCLUSIONS

Larval dispersion are limited and restricted to short distances in
reefs whose coastal morphology and sublittoral relief are complex
and covered by extensive kelp beds. This hypothesis is similar to
Prince et al. ( 1988) and McShane and Smith ( 1991 ), who state that
the larvae remain in the clo.se vicinity of the parent stock. The
results of our experiment point more to this type of dispersion than
any other kind. Nevertheless, we cannot reject the possibility that
more distant transport, including through Bahia Tortugas, might
occur along those areas of the coastline with no massive algae
formations, with gently sloping bottoms, significant tidal currents
at the mouth of the bay, and with currents induced by wind and
waves. Storms and strong swells that occur during certain times of
the year in this area, especially winter, could lead to more distant
transport such as tho.se postulated by Sasaki and Shepherd (1995).
During this time of the year, density and coverage of kelp reefs
become less dense, and the current velocity should be more intense
with the lesser foliage. This could also be true that for El Nifio
years when in Baja California all Macrucystis beds disappear, as
occurred with the last El Nifio in 1997 (Guzman del Proo unpub-
lished data).

We believe fisheries management should take a new approach
and abalone reefs should be managed as small local and indepen-
dent stock units, whose larval repopulation depends on coastal
hydrodynamics, which varies from place to place. We do not be-
lieve it is advisable to continue to manage them under a policy of
long stretches of coastline, as has been done to date.

ACKNOWLEDGMENTS

We are grateful lo the Consejo Nacional de Ciencia y Tecno-
logia (projects 2598PN. ()47PN-I297) and Instituto Politecnico
Nacional (project 980801) for funding of this study. We thank
Lucio Godinez-Orta and Manuel Saldivar-Reyes for echo-
sounding assistance, abalone fishermen of the Cooperativa de Pro-
duccion Pesquera Bahia Tortugas for help in the field studies.
Thanks to Drs. Ellis Gla/icr, Georganne Weller and Barbara Lucas
who edited the English-language text. Thanks also to Dr. Scoresby
Shepherd as well other anonymous referee for usctui critical com-
ments on ihe manuscript.

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519-530 In: S. A. Shepherd. R. W. Day & A. J. Butler AJ (eds.).
Progress in Abalone Fisheries Research. Mar. Freshwater Res.

Secretari'a de Pesca. 1981. Estudios del Medio Fi'sico e Investigacion de
Campo en la Costa Oe.ste de la Peninsula de Baja California. Sec. Pesca
Mexico. 41 pp.

Secretari'a de Pesca. 1985. Estudios del Medio Fi'sico para una Ubicacion
Alternativa del Puerto Pesquero de Bahi'a Tortugas, B.C.S. Sec. Pesca
Mexico. -30 pp.

Semarnap. 1997. Anuario Estadi'stico de Pesca 1997. Secretari'a del Medio
Ambiente Recursos Naturales y Pesca Mexico. 241 pp.

Sevilla. M. L. 1971. Desarrollo gonadico del abulon azul Haliotis fulgens
Phillipi. Revista de la Sociedad Mexicana de Historia Natural. Mexico
XXXI:129-139.

Shepherd, S. A. 1973. Studies on southern Australian abalone. Australian
/ Mar. Freshwater Res. 24:217-257.

Shepherd, S. A. 1986. Movement of the southern australian abalone Hali-
otis laevigata in relation to crevice abundance. Austr. J. Ecol. 1 1 :295-
302.

Shepherd. S. A. & Brown. 1993. What is an abalone stock? Implications
for the role of refugia in conservation. Can. J. Fisheries Aquatic Sci.
50:2001-2009.

Shepherd, S. A., D. Lowe & D. Partington. 1992. Studies on southern
Australian abalone (genus Haliotis ) XIII. larval dispersal and recruit-
ment J.Exp. Mar Biol. Ecol. 164:247-260.

Sociedad Cooperativa Bahi'a Tortugas. 1996. Calalogo de bitacoras de
captura de abulon. Soc. Coop. B. Tortugas. Mex. Mecanografiado. 3 pp.

Strathmann. R.R. 1974. The spread of sibling larvae of sedentary marine
invertebrate. Am. Nat. 108:29—14.

Strathmann, R.R. 1986. What controls the type of larval development?
Summary statement for the evolution session. Bull. Marine Sci. 39:
616-622.

Tegner, M. J. & R. A. Butler. 1985. Drift tube study of the dispersal
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Progr Ser. 26:73-84.

Journal of Shellfish Research. Vol. 19, No. 2, 883-889. 2000.

THE COMBINED EFFECTS OF TEMPERATURE AND SALINITY ON GROWTH,
DEVELOPMENT, AND SURVIVAL FOR TROPICAL GASTROPOD VELIGERS OF

STROMBUS GIG AS

MEGAN DAVIS

Harbor Branch Oceanographic Institution

5600 US 1 North

Ft. Pierce. Florida 34946

ABSTRACT The precompetent period for many planktotrophic larvae of marine invertebrates is influenced by environmental factors
such as food supply, temperature, and salinity. In thi.s laboratory study veligers of the tropical gastropod Strombus gigas were grown
in 16 temperature (20-32 °C) and salinity (30-45 ppt) combinations to e.xamine growth, development, and survival to metamorphic
competence. These environmental conditions are typical of the waters where veligers of this species naturally disperse. Temperature,
and to a lesser extent salinity, can be used to estimate length of the precompetent period during the reproductive season. Veligers grown
in 24 to 32 °C and 30 to 40 ppt survived well and the estimated precompetent period was 16 to 24 days long. Development was arrested
and mortality was high at 20 °C regardless of salinity, and at 45 ppt regardless of temperature. To estimate dispersal potential and
supply of lar\'ae to local and distant settlement sites it is important to understand how variation in environmental conditions affects the
length of lar\al life.

KEY WORDS: Larvae, temperature, salinity, spawning season. Strombus gigas, queen conch

INTRODUCTION

Larvae develop over a range of environmental conditions char-
acterizing the locations they inhabit. In the field, the spawning
season and release of larvae is usually synchronized with favorable
conditions to maximize larval growth and survival (Sastry 1986).
Temperature and salinity conditions affect larval growth and sur-
vival of many marine invertebrates (Kinne 1963. Kinne 1964)
including crustaceans (Mene et al. 1991, Brown et al. 1992), echi-
noderms (Watts et al. 1982), and mollusks (Tettelbach and Rhodes
1981. Zimmerman and Pechenik 1991). Typically, temperature
influences survival and growth and salinity affects survival more
than growth (Tettelbach and Rhodes 1981. Nagaraj 1988, His et al.
1989, Lemos et al. 1994).

Little is known about the combined effects of temperature and
salinity on growth and survival of larvae that disperse in tropical
oligotrophic waters. The veliger larva of the commercially fished
gastropod Strombus gigas (Linnaeus) disperses horizontally and
vertically over a wide geographic range in the tropical coastal and
open ocean oligotrophic waters of the Caribbean region (Mitton et
al. 1989. Posada and Appeldoom 1994. Stoner and Davis 1997a,
Stoner and Davis 1997b, Stoner et al. 1997). The juveniles and
adults of this species are found in waters that range from 17 to 32
°C, and salinities that range from 30 to 40 ppt (Davis pers. obs.).
However, optimal culturing conditions for veligers of 5. gigas are
28 to 30 °C and 35 to 37 ppt (Davis 1994a). These conditions are
found in the Caribbean waters during the peak reproductive
months (July to September), but each hatch of veligers may expe-
rience variations in temperature (19-31 °C) and salinity (35-50
ppt) during the 6- to 8-mo egg-laying season (Davis et al. 1984.
Stoner et al. 1992. Pitts and Smith 1993, Glazer pers. comm.).

The combined effects of temperature and salinity on growth,
development, and survival to metamorphic competence for ve-
ligers of S. gigas were tested in the laboratory. Veligers were
exposed to 16 temperature (20-32 °C) and salinity (30-45 ppt)
combinations found during the spawning season. These data pro-
vide insight into the upper and lower lethal limits for survival and
the conditions that affect growth of 5. gigas veligers. The results
also assist in determining how temperature and salinity influence

the precompetent period and supply of larvae to settlement sites
throughout the spawning season.

MATERIALS AND METHODS

This laboratory experiment was designed to test the combined
effects of four temperatures (20, 24, 28, and 32 °C) and four
salinities (30, 35, 40. and 45 ppt) on growth rates, development,
and survivorship to metamorphic competence for veligers of S.
gigas. The experiment was a 4 x 4 factorial design, with all 16
temperature and salinity combinations tested.

The study was conducted from June to September, 1994 at the
Caribbean Marine Research Center. Vero Beach Laboratory in
Florida. Newly laid egg masses were collected from a spawning
population near Lee Stocking Island, Bahamas (Stoner et al. 1992)
and shipped to the Vero Beach Laboratory. The egg masses were
incubated in a flow-through system for 4 days at ambient tempera-
ture (28 "O and salinity (35 ppt) (Davis 1994a). On the day of
hatching, several strands of the egg mass were placed in 3-L glass
hatching containers.

All temperature and salinity combination treatments were not
run simultaneously. Each hatch of veligers was used with one
temperature treatment and all four salinity treatments (32 °C treat-
ment was conducted in June, 28 °C in August, 24 °C the beginning
of September, and 20 °C the end of September). A control treat-
ment using optimum temperature (28 °C) and salinity (35 ppt)
(Davis 1994a) was run with each hatch of veligers. The control
treatment for the 20 °C-treatment was 24 °C and 35 ppt because it
was difficult to maintain water at 28 °C due to heater failure. The
control treatments were used to test for differences among each
hatch of veligers and/or time periods. By day 20. controls from
each experiment had mean shell lengths that were not statistically
different (F,,^ = 1.927, P = 0.1791; see "Results"). This allowed
for treatments to be compared statistically.

Water temperature was maintained using an incubator for the
20 and 24 °C treatments and a heated water bath for the 28 and 32
°C treatments. Seawater used in all treatments was filtered ( 10 |xm)
and sterilized with ultraviolet light. Depending on salinity of am-
bient seawater, salinity was lowered to 30 ppt by mixing 1 12 to
162 mL distilled water L"' ambient seawater. To increa.se salin-

883

884

Davis

ity to 40 and 45 ppt, 3.8 and 7.5 g Instant Ocean L"' ambient
seawater was mixed together, respectively. A refractometer was
used to measure salinity. Treatment water was made in the con-
tainers 24 hr in advance to allow the Instant Ocean to dissolve
completely and the temperatures to adjust to treatment conditions.
To assure that the use of Instant Ocean did not have a negative
effect on larval growth and survival, veligers were cultured in
100% Instant Ocean made to a concentration of 35 ppt (41 g
Instant Ocean L^' distilled water) at a temperature of 28 °C. There
were no negative effects on growth and survival for veligers grown
exclusively in Instant Ocean (see "Results"). Salinity and tempera-
ture were monitored daily. Temperatures were maintained within
±2 °C and salinities were maintained within ±1 ppt.

A sample of 30 newly hatched veligers was measured to de-
termine initial size. A dissecting microscope equipped with an
ocular micrometer was used to measure shell length from apex to
siphonal canal at 20x magnification. Veligers were initially
stocked at 125 L"' in an 800-mL transparent, polypropelene con-
tainer. This concentration is similar to that used in standard aqua-
culture practices for this species (Davis 1994a). There were four
replicate containers for each treatment. Initially, the veligers were
acclimated to the temperature and salinity treatment over a period
of 2 hr by gradually lowering or increasing temperature and/or
salinity. Every 48 hr the veligers were placed in new water and
containers. The veligers and treatment water were removed by
pouring them through a submerged sieve with the appropriate size
mesh (120-300 ixm). A wash bottle filled with water of the cor-
responding temperature and salinity treatment was used to move
the veligers from the sieve into the new container.

Daily veligers were fed cultured phytoplankton to satiation.
They were fed exclusively Isochrysis galbana from day 0 to 10 and
a mixture of /. i^alhana and Chaetoceros gracilis at a 3: 1 ratio from
day 10 to metamorphic competence (Davis 1994a). The final con-
centration of phytoplankton in each container was 5.000 to 10.000
cells/mL (Davis 1994a).

As the veligers grew, the number of veligers in the Ireatmenl
containers was gradually reduced to 62 veligers L"' on day 7, 31
veligers L"' on day 13, and 12 veligers L'' on day 20. This
reduction in concentration was based on standard aquaculture pro-
cedures for this species (Davis 1994a). Every other day 5 veligers
were removed from each replicate for measurements and develop-
mental observations. To avoid damaging veligers during observa-
tions, they were removed carefully with a pipet and placed in a
.seawater-filled Petri dish. Based on velar lobe development, seven
developmental stages were identified and recorded: ( I ) hatching;
(2) two lobes; (3) beginning four lobes (4a); (4) four lobes (4b); (5)
beginning six lobes (6a); (6) six lobes (6b); and (7) elongated six
lobes (6c). Developnienlal stage at a given age was based on when
5()7f or more of Ihc veligers were at thai stage. The veligers re-
moved for measurements and observations were only returned
when the concentration was below the designed concentration for
that day. On day 7, 13, and 20 all veligers in each replicate were
observed, concentration was reduced, and dead veligers were re-
moved and recorded lo deteriniiie mortality. The Irealments were
run until the veligers showed the documented signs of competence
such as green pigmentation on the propodium, six elongated lobes,
buccal mass development, and swim-crawl behavior (Brownell
1977, Davis 1994b. Noyes 1996, Davis 2()()()| or imlil all the
veligers were approaching death or had died.

ANOVA following the guidelines of Day and Quinn (1989)
was used to determine if shell lengths and mortalilv were sii;nifi-

cantly different for veligers grown in different temperature and
salinity combinations. Cochran's test was used to test for homo-
geneity of variances. Tukey's multiple comparison test of means
was used to compare shell length and mortality data. The statistical
program JMP, developed by SAS Institute, Inc. for Macintosh, was
used for the statistical analy.ses.

RESULTS

Growth

Even though the temperature and salinity treatments were not
run simultaneously and were conducted with four hatches of ve-
ligers, the mean shell length of control veligers including Instant
Ocean veligers were not significantly different by day 20 (F, ,5 =
1.927, P = 0.1791; Fig. I). Only on day 12 was there a difference.
The mean shell length was statistically smaller for the 20 °C con-
trol veligers (F-, ,, = 8.431, P < 0.05, Tukey's te.st, P < 0.05)
compared to the other control veligers. Average growth rates for
veligers in the control treatments ranged from 26 to 3 1 p,m d~' and
the first morphological signs of competence were observed be-
tween 24 and 26 days.

Temperature had a stronger intluence on the growth rates of
veligers of 5. gigas than salinity (Figs. 2 and 3). However, veligers
grown in the extreme high salinity treatment (45 ppt) grew slowly
(2-19 |jLm d"') at all temperatures and did not show morphological
signs of metamorphic competence (Figs, 2 and 3). On days 16 and
20, veligers grown al 24 and 32 °C and salinity 45 ppt had shell
lengths that were not significantly different (Day 16: F, , =
6.7279, P = 0.050; Day 20: F, , = 5.2517, P = 0.0705; Fig. 2).
On day 16, shell lengths of veligers grown at 28 °C and 45 ppt
were not different from those of veligers grown at 24 "C and 45
ppt, but their shells were smaller than those of veligers cultured at
32 "C and 45 ppt (F. 7 = 7.5294, P < 0.05, Tukey's test, P =
0.05; Fig. 2).

Highest overall growth rates (44-52 |jim d"' ) were achieved for
veligers grown al 32 °C and salinities 30, 35, and 40 ppl (Fig. 3).

■S

60

zuu

■

-•— Control 20 1

-■— Control 24 °C

-A- Control 28 "C - 10

ly

tA

-•— Control .12 °C

C-,

^

900

5

^Jx/^

^

r r ^

^

'rj^

^

■

«xt

" ^

^

<

.100

^

0 2 4 6 8 lU 1: 14 16 18 20 22 24 lb
Age (days)

Fi("urt' I. (hohIIi of .V. ,i,'(^'fl.v vulijjiTs jirown in aintnil conditions. The
control Irt-atnicnl for 24 and .^2 (' Irealmcnls was 28 C and 35 ppt.
Control lor 20 C was 24 (' and 35 ppt and control for 28 ' C was 28
(' and Instant Ocean (lO) mixed to .15 ppl. Data points represent
means and standard deviations (;i = 4 replicate containers, 5 veligers
were measured from each replicate for each data point).

1200

E
a.

c
u

.J

C/5

1200

900 -

on
g

s 600

Temperature and Salinity of Veliger Growth
1200

885

2 4 6 8 10 12 14 16 18 20 22 24 26
Age (days)

0 2 4 6 8 10 12 14 16 18 20 22 24 26
Age (days)

1200

900

60

c
1)

= 600

-^30 ppt

-^35 ppt

-A- 40 ppt

-•—45 ppt

—o— Control

0 2 4 6 8 10 12 14 16 18 20 22 24 26
Age (days)

4 6 8 10 12 14 16 18 20 22 24 26
Age (days)

Figure 2. Growth of S. gigas veligers cultured in 16 temperature and salinity treatments. The control treatment for 24 and 32 C treatments was
28 ' C and 35 ppt. Control for 20 °C was 24 °C and 35 ppt and control for 28 C was 28 C and Instant Ocean mixed to 35 ppt. Data points
represent means and standard deviations (h = 4 replicate containers, 5 veligers were measured from each replicate for each data point).

Although shell lengths on day 16 differed for veligers grown at 32
°C and 30. 35. and 40 ppt (F,,^ = 5.0989, P < 0.05), veligers in
the three treatments all showed morphological signs of metamor-
phic competence on day 16 (Fig. 2). Shell lengths were not dif-
ferent for veligers cultured at 32 °C and salinities 30 and 35 ppt,
and 32 °C and salinities 30 and 40 ppt (Tukey's test. P > 0.05).
Shell lengths were larger for veligers grown at 32 °C and 40 ppt

than veligers grown at 32 °C and 35 ppt (Tukey's test. P < 0.05;
Fig. 2).

Veligers grown at 24 and 28 °C and salinities 30. 35, and 40 ppt
had similar growth rates (29-34 jjlui d"' ) and showed morphologi-
cal signs of metamorphic competence by day 22 and 24, respec-
tively (Figs. 2 and 3). Overall growth patterns for veligers grown
in these conditions were similar; however, there were statistically
significant differences in shell lengths on day 20 (F, ,g = 4.7709,
P < 0.05; Fig. 2). On this day, shell lengths were larger for veligers
grown at 24 °C and 35 ppt and 28 °C and salinities 30 and 35 ppt
(Tukey's test, P < 0.05), but all other shell lengths were not sta-
tistically different (Tukey's test, P > 0.05; Fig. 2).

The lowest growth rates (1-8 |xm d~') occurred for veligers
grown at 20 °C at all salinities (Fig. 3). and none of these veligers
showed morphological signs of metamorphic competence. Shell
lengths for veligers grown at 20 °C and all salinities were not
different on day 16 (F, y = 3.8884, P = 0.0607; Fig. 2). However,
on day 20 shell lengths for veligers cultured at 20 °C and salinities
40 and 45 ppt were smaller than veligers grown at 20 °C and
salinities 30 and 35 ppt, which had shell lengths that did not differ

Salinity (ppt) from each other (F, « = 8.4120. P < 0.05, Tukey's test. P = 0.05;

Fis;. 2).

Developmental Stages

Temperature ( C)

Figure 3. Overall mean growth rates of S. gigas veligers cultured in 16
temperature and salinity combinations (n = 4 replicate containers, 5
veligers were measured from each replicate for each data point).

Veligers of 5. gigas grown in control conditions (24 and 28 °C,
35 ppt) developed through all velar lobe stages and showed mor-
phological signs of metamorphic competence (Fig. 4). Veligers
grown at temperatures 24, 28, and 32 °C and salinities 30, 35, and

886

Davis

2H
24
20

S- If'

OJ IT
Clfl '-

20 °C

11

30 35 40 45

Salinity (ppt)

Control

28
24
20

lA

•a

(L> 19
00 '■^

<

8
4
0

24 °C

■ fx-

H6b

Qfxi

a4h

D4a

30 35 40 45

Salinity (ppt)

Control

28
24
20

a

GO '-
<
8

4

0

28 °C

I

40 45

Salinity (ppt)

Control

30 35 40 45

Salinity (ppt)

Control

Figure 4. Developmental stages of S. gigas veligers cultured at 16 temperature and salinity combinations. The control treatment for 24 and 32
°C treatments was 28 "C and 35 ppt. Control for 20 °C was 24 C and 35 ppt and control for 28 "C was 28 "C and Instant Ocean mixed to 35
ppt. Veligers hatch on day 0 with 2 lobes. The legend represents the following stages of velar lobe development: 4a, beginning four lobes; 4b,
four lobes; 6a, beginning six lobes; 6b, six lobes; and 6c, elongated six lobes. The data were calculated as a percentage of veligers at each stage.
The data points changed when 50% or more of the veligers developed to the next stage (n = 4 replicate containers, 5 veligers were examined from
each replicate for each data point).

40 ppl developed six elongated lobes (stage 6c) and showed mor-
phological signs of metamorphic competence (Fig. 4). Fifty per-
cent or more of the veligers in these treatments were in the fol-
lowing stages: 4a, beginning four lobes for 1 to 2 days; 4b, four
lobes for 2 to 3 days; 6a, beginning six lobes for 2 to 4 days; 6b,
six lobes for 9 to 15 days; and 6c, elongated six lobes for 2 to 6
days prior to showing morphological signs of competence.

Development was arrested for veligers grown at all low tem-
perature (20 C) treatments and at all high salinity (45 ppt) treat-
ments (Fig. 4). Veligers grown at 20 °C did not develop past 4
lobes (stage 4b) at 40 and 45 ppt and only started six lobes (stage
6a) at 30 and 35 ppt. The veligers grown at high salinity 45 ppt and
temperatures 24, 2<S, and 32 "C did not develop beyond the begin-
ning of six lobes (stage 6a).

Mortality

Percent moitality (mean ± .SD) was low (tiays 0-7: 6.6% ±
2.39^, days 7-13: 3.4'/< ±5.1'/^. and days 1 .V2(): 1 .5'/, ± 2.9'/f 1 for
veligers grown exclusively in the Instant Ocean control treatment
(28 "C and 35 ppl). Therefore, high mortality for veligers grown at
45 ppt was not attributed to the addition of Instant Ocean salts.
Overall mortality was highest in all low temperature (20 "C) and
all high salinity (45 ppt) treatments (Fig. 5).

At the end of the 0 to 7 day interval, percent mortality was not

different and was highest for veligers grown at 20 and 24 "C and
at salinity 45 ppt (F,^ = 0.6962, P = 0.4360; Fig. 5). Percent
mortality was low and not different for veligers grown at 24 °C and
salinities 30, 35, and 40 ppt (F,,, = 1.2521, P = 0.3313; Fig. 5).
For veligers grown at 28 °C mortality was not different for veligers
in 30 and 35 ppt treatments, but mortality was lower in salinity 40
ppt (F,., = 6.62.54, P < 0.05, Tukey's test P = 0.05; Fig. 5).
Mortality for veligers grown in 32 °C and all salinities was high
and not different among salinity treatments (F, j, = 1.4286, P =
0.2870; Fig. 5).

Mortality in the middle interval. 7 to 13 days, increased for
veligers grown at 20 "'C and all salinities and veligers grown at 45
ppt and all temperatures (Fig. 5). Percent mortality was not dif-
ferent for veligers grown at 20 °C and 30 and 35 ppt and tempera-
tures 24 and 32 °C and 45 ppt (F, ,, = 2.2068. P = 0.1447: Fig.
5). Veligers in the treatments 20 ' C and salinities 40 and 45 ppt
and 28 °C and 45 ppt had the highest mortality for this interval and
percent mortality was marginally different (Fii, = 3.966, P =
0.0573; Fig. 5).

This general trend coiuiiuicd into the last interval. 13 to 20
days, at which time mortality was highest and not different for
veligers grown at 20 °C at all salinities and 28 "C at 45 ppl (F, ,,
= 1.8573, P = 0.1953; Fig. 5). Mortality was also high and not
different for veligers grown at 24 and 32 °C and 45 ppl (F, , =
1.5010. /' = 0.2751; Fig. 5). At the end of the 7 to 13 day and 13

Temperature and Salinity of Veliger Growth

887

Days 0-7

3
o

u~

o

"a

C

o

o

"a

C
o

Salinity ppt

Temperature C

Days 7-13

45
40
35 Salinity ppt

Temperature C

Days 13-20

100

3 80

o

o

"a
C

I

60

40

20

0

45
40
35 Salinity ppt

24

28

32

Temperature C

Figure 5. Percentage of mortality of S. gigas veligers cultured in 16
temperature and salinity combinations. Mortality was calculated as
the percentage of veligers that died during each interval. Data points
represent mean of 4 replicate containers and 5 veligers were measured
from each replicate.

to 20 day interval, mortality was lowest and not different for
veligers grown at 24. 28, and 32 °C and salinities 30, 35, and 40
ppt (days 7-13: F-,.^ = 1.5680, P = 0.1929: days 13-20: F, ,^ =
0.7976. P = 0.5672: Fia. 5).

DISCUSSION

In the natural environment, timing of the reproductive period
and release of larvae is usually synchronized with conditions that
are most favorable for maximization of larval survival and conti-
nuity of the species (Sastry 1986). Veligers of S. gii;as showed
morphological signs of metamorphic competence and maintained
high survival (71%-93%) at temperatures 24 to 32 °C and salini-
ties 30 to 40 ppt. It is not surprising that veligers grew and survived
well in these conditions because these values are typically found
during the reproductive season and within the geographic region
where veligers naturally disperse. During the non-reproductive
season, salinity stays within a narrow range, but temperature can
drop as low as 17 °C in some waters where juvenile and adult S.
gigas are found.

S. gigas veliger growth, development, and survival was influ-
enced primarily by temperature, and salinity had a lesser effect as
has been shown with other crustacean and molluscan larvae
(Tettelbach and Rhodes 1982. Nagaraj 1988. His et al. 1989. Le-
mos et al. 1994). Therefore, temperature rather than salinity can be
used to predict time to metamorphic competence during the egg-
laying season. For instance, at the beginning and end of the season
when temperatures are 24 to 28 °C, the precompetent period will
be 22 to 24 days long. During the peak months of the season when
temperatures are as high as 3 1 °C. the precompetent period can be
just 16 days. Veliger abundance is high in the peak months not
only because copulation frequency and number of egg masses laid
increases 2-fold compared to the beginning and the end of the
season (Davis et al. 1984. Weil and Laughlin 1984, Stoner et al.
1992, Stoner and Davis 1997a), but also because temperatures are
optimal for larval growth and survival.

Other studies have indicated that veligers of S. gigas and
Stroinbus costaliis (Gmelin) develop normally at 28 °C, decrease
growth at 24 °C and have 100% mortality at 32 °C (Aldana Aranda
and Torrentera 1987, Aldana Aranda et al. 1989, Glazer pers.
comm.). However, in this study a temperature of 32 °C provided
conditions for fast growth and high survival. This is likely the
highest temperature veligers encounter in most waters where they
disperse. This temperature is probably near the upper physiological
tolerance for the.se veligers, especially since most tropical marine
organisms cannot survive and actively grow at temperatures higher
than 35 °C (Kinne 1963). There are several examples of molluscan
(Lucas and Costlow 1979. Tettelbach and Rhodes 1981, Robert et
al. 1988, His et al. 1989) and crustacean (Brown et al. 1992) larvae
rapidly decreasing survival and reaching a growth plateau between
30 and 35 °C. If veligers of 5. gigas are growing at near-maximum
rate at 32 °C, elevated food conditions may be the only factor
capable of sustaining an increased growth rate at this high tem-
perature (Boidron-Metairon 1995, Hoegh-Guldberg and Pearse
1995).

At a temperature of 20 °C, veliger development was arrested
prior to showing any morphological signs of competence and sur-
vival rate was as low as 10% regardless of salinity. In isolated
cases embryo development and larval release may occur at low
temperatures (Rodriguez et al. 1991). This may be the case for
embryos developing in egg masses laid in February in the Florida
Keys when temperatures were as low as 19 °C (Glazer pers.
comm.). However, based on the results from this study even with
a successful hatching, it is unlikely that veligers would survive and
develop to metamorphic competence at these low temperatures.
Low larval abundance at the beginning and end of the spawning
season may be due to low number of egg masses (Davis et al.

888

Davis

1984, Stoner et al. 1992) and decreased larval survival at low
temperatures, especially in the beginning of the season.

Environmental conditions such as light are known to trigger
veligers of S. gigas to migrate vertically (Barile et al. 1994, Stoner
and Davis 1997b). Temperature may also influence migratory be-
havior, and the depth at which veligers migrate will depend upon
their acclimation and tolerance abilities (Young and Chia 1987).
The majority of veligers in the Exuma Sound, Bahamas were
located above the thermocline (30 m) where temperatures of 28 to
29 °C are optimal for growth, and only on occasion veligers were
found as deep as 100 m where temperature was 25 °C (Stoner and
Davis 1997b). Salinity probably has little influence on vertical
distribution of veligers in these same waters because salinity above
the halocline (30 m) was 38 ppt and decreased only to 37 ppt at
100 m (Stoner and Davis 1997b).

In this experiment veligers of S. gigcis showed morphological
signs of metamorphic competence when salinity levels were 30 to
40 ppt and temperatures were 24 to 32 °C. However, at high
salinities, such as 45 ppt, larval growth was reduced and mortality
was high. It is likely that S. gigas veligers disperse in relatively
stable salinity conditions because they are found in coastal and
open ocean waters of the Florida Keys, Bahamas, and Caribbean
Sea (Posada and Appeldoorn 1994, Stoner et al. 1997, Stoner and
Davis 1997a, Stoner and Davis 1997b). However, on occasion
veligers have been collected in shallow bank areas in the Bahamas
where salinity can be as high as 43 ppt (Pitts and Smith 1993,
Jones 1996) and in nearshore waters of the Florida Keys where
salinity can peak at 50 ppt due to influx of hypersaline Bay water
(Lapointe and Clark 1992, Fourqurean et al. 1992). It is possible
that short-term exposure to high salinity in shallow waters may
slow veliger growth temporarily, but long-term exposure would
severely limit survival and growth to metamorphic competence. In
this study the growth rate of veligers cultured at 45 ppt ranged
from 2 to 19 (xm day'' with temperatures 20 to 32 °C, respec-
tively. Based on these growth rates, it is possible that if veligers
survived being cultured at 24 to 32 °C and 45 ppt over a long
period of time, metamorphic competence could be achieved in
approximately 40 to 50 days after hatching. High salinity and
temperature conditions are known to cause developmental stress
due to reduction of dissolved oxygen (Kinne 1964). Therefore,
growth and survival of veligers to metamorphic competence could
be inhibited in locations where evaporation processes are highest
with elevated temperatures.

Future studies need to determine what effects short- and long-

term exposure to fluctuations in temperature and salinity have on
growth and survival rates of veligers of S. gigas at different de-
velopmental stages. Kinne (1963) suggested, for example, that a
constant temperature of 20 °C and temperatures fluctuating be-
tween 15 and 25 °C with an average of 20 °C do not necessarily
have the same biological effects. Veligers of Crepiditla fornicata
(Linnaeus) grown under cyclic temperature regimes showed im-
mediate changes in shell growth and carbon content in relationship
to each cyclic change in temperature (Lucas and Costlow 1979).
Additional studies .should also focus on how temperature and sa-
linity may change the documented metamorphic competence cri-
teria. For instance, a recent investigation showed that heat shock
(35-37 °C) may induce metamorphosis at a younger developmen-
tal stage when no green pigmentation is present (A. Boettcher
pers. comm.).

In summary, temperature appears to be the ecological param-
eter controlling onset and completion of the typical 6- to 8-month
spawning season and the geographical distribution of S. gigas.
Therefore, it is likely that this species has adapted egg production
to correspond with the most favorable environmental conditions
for larval growth and survival. Egg laying, larval abundance, and
temperature are highest during the peak reproductive months. July
to September (Stoner et al. 1992). Therefore, recruitment success
in these months should be highest based on high number of ve-
ligers in the plankton, and the probability that veliger predation
decreases with increasing growth and developmental rate (Rumrill
1990). At the beginning and end of the reproductive period larval
abundance is low due to lower number of egg inasses (Davis et al.
1984. Stoner et al. 1992) and temperatures are below optima for
growth and survival. These veligers are likely to disperse to distant
populations due to an increase in length of larval life, but settle-
ment success may decrease due to longer exposure to predators
and advection from settlement sites (Rumrill 1990). The advantage
of variations in larval growth and development during the repro-
ductive season is that this benthic species disperses and recruits to
both local and distant settlement habitats which in turn maintains
genetic continuity over a wide geographic range.

ACKNOWLEDGMENTS

The author wishes to thank Dr. Allan Stoner for guidance and
editorial support and Chris Metzger for assistance in the labora-
tory. This research was supported by a grant to the Caribbean
Marine Research Center from the National L'ndersea Research
Program. Harbor Branch Oceanographic Institution number 1364.

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DISTRIBUTION AND ABUNDANCE OF STROMBUS GIGAS VELIGERS AT SIX FISHING SITES
ON BANCO CHINCHORRO, QUINTANA ROO, MEXICO

ALBERTO DE JESUS-NAVARRETE''^ AND
DALILA ALDANA-ARANDA-

Lahoratorio de Pesqiten'as Artesanales.
El Colegio de la Frontera Sur,
Unidad Chetumal. A. P. 424 Chetumal,
Q. Roo. Mexico C.P. 77000
'Centra de Iiivestigacion y de Estiidios
Avanzados Unidad Merida. A. P. 73 Cordemex,
Merida Yucatan Mexico

ABSTRACT In order to study the distribution and abundance of Strombus gigas veligers, duplicate plankton samples were collected
every 2 mo from August 1997 to July 1998 at six sites on Banco Chinchorro. Plankton tows were made with a conical net that had
a 0.5-m diameter opening and 202n,m mesh size. During the sample period, 798 veligers were collected. Larvae were more abundant
during the rainy season (July through August), (58.62%", 467 larvae) and "nortes" season (October through December) (35.46%, 283
larvae), while only 5.76% (48 larvae) were counted in the dry season (March through May). Larval density varied from 0.00093
veligers • 10 m~' in May to 7.42 veligers • 10 m"' in August. A high percentage of larvae were stage I (89.08%^), with lower abundance
of stage II, III, and IV veligers (3.76%, 0.25%, and 6.52%, respectively) and only 0.38%f of the larvae were competent. High abundance
of early stages suggests that Banco Chinchorro is an important source of veligers. Considering that surface current trajectory in this
region is northwestward, the presence of competent larvae in the Southern part suggests an origin in areas outside of Banco Chinchorro,
or in places situated downstream. It is possible that Banco Chinchorro supplies larvae to its shelf, the Quinlana Roo coast and Florida.

KEY WORDS: Banco Chinchorro, Caribbean, distribution, larvae, queen conch, SiiDinhus gigas

INTRODUCTION

The queen conch, Strombus gigas (Linne 1758), is a gastropod
widely distributed in the Caribbean (Stoner 1997). It has been
fished since Arawak Indians inhabited the Caribbean region (Ran-
dall 1964. Keegan 1992).

Queen conch stocks have declined throughout the region over
the past 10 y, and various regulations have been implemented
independently in most Caribbean nations (Berg and Olsen 1989.
Appeldoorn 1994). International trade of conch is now monitored
by the Convention on International Trade of Endangered Species
(CITES) in order to ensure the species" survival.

The biology and ecology of queen conch is relatively well
studied (Randall 1964, Brownell and Stevely 1981, Appeldoorn
and Ballantine 1982. Stoner et al. 1996. de Jesijs-Navarrete and
Oliva-Rivera 1997). However, detailed larval descriptions (Davis
et al. 1993) and larval distribution and abundance have been sur-
veyed only recently (Stoner et al. 1992. Posada and Appeldoorn
1994, Stoner and Davis 1997).

In Quintana Roo, Mexico the culture of queen conch (Cruz
1984), larval diets and feeding behavior (Aldana-Aranda and Pa-
tino-Suarez 1998), growth of juveniles in pens (de Jesiis-
Navarrete et al. 1994) and juvenile and adult ecology (de Jesiis-
Navarrete and Oliva-Rivera 1997). have been studied. Fishery
biology investigations have determined that this resource is over-
exploited in Banco Chinchorro (Chavez and Arregufn 1994).

Recruitment in commercial species with a planktonic phase is
complex and is further complicated by the fact that larvae may
drift hundreds of kilometers from their site of origin before settling
to the benthos. As a result, many local populations depend on
distant sources for larvae. Thus, stock management of the species
is a multinational problem (Berg and Olsen 1989).

This research represents the first investigation of queen conch

larvae in the western Caribbean. The .study was designed to deter-
mine the abundance and distribution of 5. gigas veligers and to test
the hypothesis that Banco Chinchorro is an important larval pro-
duction site.

MATERIALS AND METHODS

Study Area

Banco Chinchorro is a false atoll situated offshore from south-
em Quintana Roo within the Mexican Exclusive Economic Zone
( 18°23'-18°47'N. 87°14'-87°27'W) (Fig. 1). The bank is geologi-
cally similar to the Belize reefs. Turneffe. Glovers and Lighthouse
(Jordan and Martin 1987). Chinchorro is 46 km long. 19 km in the
widest part, and has an area of 561 km". Depth inside the lagoon
reef decreases from 12 m in the south region to 7 to 3 m in the
central part to 2 m in the north. Chinchorro has four keys, two
small keys known as Cayo Norte. Cayo Centro. which is the larg-
est, and Cayo Lobos, the most southerly and smallest. Surface
current pattern in the reef lagoon is poorly known. The principal
transport is towards the northwest, and the current arrives to Chin-
chorro from the south.

Sampling

Samples were collected every 2 mo. from August 1997 to July
1998. at six sites in the reef lagoon: Cayo Lobos (18°23'N,
87°23'W), Isla Che (18°29'N. 87°26'W). Cayo Centro (18°35'N.
87°2rW), Cayo Centro West (18°36'N. 87°2rW). Penelope
(18°42'N, 87°15'W). and Cayo Norte (18°46'N, 87°20'W)
(Fig. 1 ). The dry season is from March to June, and the rainy .season
is from July to October. The cold season, characterized by strong
winds from the north, known locally as "nortes," is from Novem-
ber to February.

Duplicate surface plankton tows were made at each site using

891

892

DE JESUS-NAVARRETE AND ALDANA-ARANDA

/'

/

}l Cayo Centre

f West

y •18-36'N, g7'«W

y/ Norte ^k

X I8"<6' N.87-20'W ^^

/ ^

Penelope # 4

18° 4rN,87° Ib'W i

-t

18* 21 N, 87-23'W

Figure 1. Map of Banco Chinchorro showing the area of sampling
locations within the reef lagoon.

a conical net, that had a 0.50-m diameter opening and 202-|jim
mesh size. Tows were made from a boat traveling in circles, ap-
proximately 200 m in diameter, for 15 min at a mean velocity of
1 m-s"'. The tow volume was measured using a 2035 MK4 flow-
meter display connected to a calibrated General Oceanic®
2031HR2 flowmeter suspended in the mouth of the net. Plankton
samples were preserved in a 5% neutral formaldehyde-seawater
mixture (Stoner and Davis 1997). In all sites, tows were diurnal
and additional night collections were made only at Cayo Centro
and Cayo Lobos.

In the laboratory, the entire volume of each plankton sample
was sorted for Stromhits veligers using a dissecting microscope
(20x). Positive identifications for S. f;itf<is were made following
the descriptions of Davis et al. (1993). Veligers were counted and
.shells were measured for total length with a calibrated ocular mi-
crometer. Veligcr density was siandardi/ed to 10 m"\ Larvae were

divided into four size classes for analysis of abundance patterns:
stage I ( 150 to 450 p.m shell length (SL), Stage II, (451 to 650 \xm
SL), stage III, (651 to 950 jxm SL), stage IV (950 to 1200 p-m SL)
and competent larvae >1200 |jim SL) (Davis et al. 1993). Abun-
dance data were analyzed for spatial and temporal variation using
a two-way ANOVA. Data were transformed to Log (x-i-1) prior to
analysis to improve homogeneity of variance.

Temperature (°C) and dissolved oxygen (mg/L) were recorded
simultaneously, at the water surface at each site using an oxygen
meter (YSI model 58). Salinity (%c) was measured with a tem-
perature-conductivity meter (OHAUS model 50).

RESULTS

Plivsical Measurements

Temperature ranged from 26.3 ± 0.9 (n = 6) in December to
29.3 ± 0.3 (n = 6) in October. Dissolved oxygen varied between
5.9 ± 0.37 mg/L in July to 7.0 ± 0.56 mg/L (n = 6) in August.
Salinity varied from 35.9 ± O.I2%r in October to 37.0 ± 0.9%c
(n = 6) in March (Table 1).

Distribution and Larval Abundance by Site

A total of 798 Slroinbiis gigcis veligers were collected from
August 1997 to July 1998. Most were collected at Penelope (376
larvae; 47.1%). Cayo Centro followed in importance with 41.0%,
which included both the day samples (30.9%) and night samples
(10.1%). The remainder were collected at Cayo Norte (4.3%),
Cayo Centro West (3.8%), Isla Che (2.9%), and Cayo Lobos
(0.9%).

Considering a 4-nio long period for each climatic season, larvae
were distributed in the following manner: 58.5% of the veligers
were captured in rainy season (July to October), 35.4% was col-
lected in "nortes" (November to February), and 6.0% were col-
lected in the dry season (March to June). On the whole the rainy
season and the season of "nortes" contributed 94.0%' of the ve-
ligers collected.

Larval Density

Density varied from 0.00093 larvae- 10 m ' to 7.42 larvae- 10
m \ The greatest density of larvae occurred at Penelope in August
with 7.42 larvae- 10 m \ followed by Cayo Centro with 4.95 lar-
vae- 10 m ' in October. Cayo Centro (night) with a density of 1.81
larvae- 10 m ' in July and Penelope had 1.05 larvae- 10 m"' in
May. The remainder of the collection sites had densities less
than I larvae- 10 m~' (Fig. 2). There were no significant differ-

TABI.K 1.
Temperature (C), salinity (%r.), and dissolved oxygen (mg/I,) al Banco Chinchorro Quintana Roo, Mexico, Augu.st 1997 to July 1998.

Cayo Lobos

Isla Che

Cayo Centro
T °C %, 0,

Centro West

'enelopc

Cayo Norte

Month

TX

%c

O2

T°C

%f,

Oj

T°C

%o

Oj

T C

9r(

0,

T°C

%, O2

August

28.2

36.2

7.7

28.6

36.2

7.5

29.0

36.4

6.5

29.8

.36.2

6.3

29.3

36.4

6.8

29.0

36.4 7.3

October

29.6

3.5.8

6.7

29.3

36. 1

6.2

29.5

35.9

6.6

28.9

.36.1

6.3

29.3

35.9

6.x

29.7

35.9 7.3

December

25.2

36.0

6.1

26.9

36.0

.5.8

27.1

37.6

6.5

25.0

36.2

6.2

27.0

36.0

6.0

26.5

36.0 6.2

March

2.'i.9

37.6

6.3

27.1

37..';

6.5

27.5

36

6.2

27.2

37.8

64

27.5

37.8

6.0

27.6

37.7 6.3

May

27.6

35.0

f).5

28.2

36..";

6.3

28.4

35.0

6.8

284

36.0

5.9

26.5

.36.5

6.2

26.5

35.6 6.6

July

29.1

.36.2

5.8

29.8

.^6.2

5.5

29.3

36.4

64

29.8

36.0

5.8

—

—

—

—

— —

STROMBUS GIGAS VELIGERS at QUINTANA Roo

893

u
>

vx

u

August

n=366

L

October

n=280

-1

December

n=3

htarch

n=2

May

n-46

JJy

n=101

Cayo LobQS IslaChe Cap Cayo Orto F^mope Cayo
Lotos M^ Cef«o Cenh) Nigrt Nnte

V\fe9t

Figure 2. S. gi%as larval density in Banco Chinchorro by month and
site.

ences in the abundance of veligers between sites or montiis (two-
way ANOVA. Table 2).

TABLE 2.

Results of two-way ANOVAs for veligers abundance of S. gigas,
August 1997 to July 1998.

Source of

Sum of

Mean

Signiflcance

Variation

Squares

d.f.

Square

F-ratio

Level

Site

14.114

7

2.016

0.943

0.486

Month

19.382

5

3.876

1.814

0.135

Residual

74.797

35

2.137

Total

108.294

47

Site refers to sites of sample in Banco Chinchorro.

Penelope. Finally, three competent larvae (0.4%) were collected at
Cayo Lobos (Fig. 3).

DISCUSSION

Despite abundant evidence that temperature influences repro-
duction in 5. gigas (Randall 1964, Weil and Laughlin 1984, Stoner
et al. 1996), Corral and Ogawa (1987) noted that reproduction
occurs year round in Banco Chinchorro regardless of temperature.
Our results also support the occurrence of year-round reproduction

1(X)
50
0

100

^^

50

^

"^— '

<u

u

C

c«

-a

1U0

fi

s

50

.Q

«

0

a>

>►

-4^

C8

inn

a>

^

50

Stage I

n=711

stage II

n=3()

stage III

n=2

stage IV

1

1=52

Larval Distribution for Size Class

Stage I larvae were most abundant (89.1%, 71 1 larvae) and 30
larvae (3.8%) were stage II. These sizes were distributed over all
sample sites, but with greatest abundance at Penelope and Cayo
Centro. Two larvae (0.2% ) were stage III (701-950 |jim), and were
collected at Isia Che and Cayo Centro. There were 52 stage IV
larvae (6.5%); these were more abundant in Cayo Centro and

100

50

I

Competents

n=3

Cayo
Lobos

Loboa

B Oie CCentro
West

Cayo
Centro

Centro

Cayo
Mode

Figure 3. S. gigas larval size class distribution in Banco Chinchorro.

894

DE Jesus-Navarrete and Aldana-Aranda

because larvae were collected throughout the year. However, lar-
val abundance peaked in August and October, during the wanner
months. Stoner et al. (1992) reported high Strombiis veliger abun-
dances in the Bahamas during the wanner season (June to Sep-
tember) and Posada and Appeldoom (1994) also found greater
abundance of larvae in July, during the period of reproductive
activity at Los Roques National Park, Venezuela.

In Chinchono, larvae were more abundant at Penelope and
Cayo Centre. The.se sites have tidal channels between the inner
lagoon and offshore reef, where the water flow is intense. It is
possible that larvae spawned in deep waters, can be transported to
reef lagoon by the tidal flow, or action of internal bores, as has
been reported for other invertebrate species (Pineda 1995, Shanks
1998, Stoner and Smith 1998).

The larval density found at Chinchorro (0.00093 to 7.42 lar-
vae-10 m~^) is very similar to densities reported in other parts of
the Caribbean. In the Bahamas, a maximal density of 4.16 lar-
vae-10 m"' and a minimal of .04 larvae- 10 m"' was reported
(Stoner et al. 1992). In the eastern Caribbean veligers density in
oceanic waters was low (0.20 ± 0.251 larvae- 10 m"^) compared
with protected zones (0.51 ± 0.45 larvae- 10 m~"'). The maximal
abundance occurred in waters off Los Roques National Park, Ven-
ezuela, with a density of 1.22 larvae- 10 m""" (Posada and Appel-
doom 1994). In Florida the larval density varied 0.36 to 0.91
larvae- 10 m"'. The greatest abundance was found in June and was
related with temperature and wind variations (Stoner et al. 1996).
The presence of a greater quantity of larvae near competence size
than newly hatched larvae (<500 jjim) was attributed to a process
of transport of larvae from Mexico, Belize or Cuba, and to meso-
scale oceanographic processes like eddies in the current from
Florida (Stoner et al. 1996).

In Chinchono, we found a high percentage (89%) of larvae of
early stages (1 and II; 244 to 780 (xm) and this indicates that
Chinchorro is an important source of veligers. Stages III and IV
stages were not abundant, hut they were present, which suggest a
continuous local recruitment. In the Bahamas, the presence of
intermediate size to 900 (jim shell length suggested total develop-
ment in the Bahamas Bank (Stoner et al. 1996).

There was a variation in the size of larvae from south lo north
in Banco Chinchono: the few competent larvae were collected at

Cayo Lobos and the vast majority of newly hatched larvae were
found in Cayo Centre and Penelope. The presence of competent
larvae in Lobos Key, in the south of Chinchono, indicates the
anival of larvae from the exterior of the bank and possibly from
other parts of the Caribbean. Speeds of 0.8 m-s"' to 1.2 m-s"'
(Kinder 1983), would permit larvae to cross distances of approxi-
mately 900 km between Chinchono and the eastern Caribbean.
Drifters released in Jamaica have passed near Chinchono and were
picked up on the coast of Quintana Roo (Metcalf et al. 1977, Grant
and Wyatt 1980).

Even though Chinchono has a diminished abundance of adults,
larval density was high (7.42 larvae- 10 m"'). This may reinforce
the perception that healthy population of adults is associated with
high larval abundance (Stoner and Davis 1997).

It is thought that the Caribbean insular arches located down
stream are important sources of larvae within the scheme of
metapopulations (Stoner 1997). Banco Chinchono, is definitively
not a source site, since Chinchono receives competent larvae from
the down stream populations and is able to produce larvae from its
own shelf. It is likely that larvae disperse from Chinchono to sites
of the Quintana Roo coast and possibly to Florida, since it has been
demonstrated that drift cards liberated in Chinchono anived in
Florida (Merino-Ibarra 1986).

It is possible that Mexico, and particularly Chinchono, does not
receive larvae from Belize, due to the coastal circulation pattern
(Merino-Ibana 1986). and the fact that larvae were not found in
Hol-Chan Marine Reserve (de Jesus-Navarrete. unpubl. data). Fur-
thermore, larvae density in the south coast of Quintana Roo was
low ( 1 .4 larvae- 10 m ') (Oliva-Rivera and de Jesiis-Navanete, in
press). Larvae were present year round in Chinchono, a charac-
teristic shared only by Florida, and this represents the longest
reproductive season in the Caribbean.

ACKNOWLEDGMENTS

El Consejo Nacional de Ciencia y Tecnologi'a (CONACyT)
grant 420P-N9506 supported this research. Comments on the
manuscript by Allan W. Stoner, Megan Davis. Scott Monks and
one anonymous reviewer were greatly appreciated. AJN sincerely
thanks to Megan Davis for training in the identification of Strom-
hiis liificis larvae.

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iarvicullure (it three Caribbean Cimchs: Queen Conch Stnmihus nifias.

Milk Conch Slrnmhiis tii.\luliis and I-ightlng Conch Sirnnihiis /ih,i,'(//.v.

Aqiimiilliirc 167: 16.1-178.
Appeldoorn, R. S. 1994. Spatial variability in the morphology of niieen

conch and its implications for management regulations, pp. 1 4.")- 1 57.

In: R. S. Appeldoorn y Q. Rodriguez (eds.). Queen Conch Biology,

Fisheries and Marlculiure. fundacion Cientifica Los Roques. Caracas.

Venezuela. .1.56 pp.
Appeldoorn. R. S. & I). L. Ballanlnie I9S2. Field release oteullured queen

conch in Puerto Rico: implications for slock resioralion. /'loi . Gulf aiul

Ctirihh. l-ish. Iiisl. .15: X9-9S.
Berg. C. J. Jr. & D. A. Olsen. 1989. Conservation .ind nian:igenienl of

queen conch tSlioiiihii.', f;if'<i.\) fisheries in the Caribbean, pp. 422—442.

In: J. F. Caddy. (Ed.). Marine Invertebiale Fisheries; Their Assessment

and Management. Wiley. Nev^' York.
Brownell. W. N. & J. M. .Slevely 1981. The biology, fisheries, and man-
agement of the queen conch, Sinimbus gigas. Mar. Fish. Rev. 4.1: 1-12.
Chivez. E. A. & F. Arregui'n-Sdnchez. 1994. Simulation modelling for

conch fishery management, pp. 169-189. In: R. S. Appeldoorn y Q.

Rodri'gue/ (eds.). Queen Conch Biology. Fisheries and Mariculture.

Fundacion Cientifica Los Roques. Caracas. Venezuela. .156 pp.
Cruz. R. S. 1984. Avances en la expermientaciiin de la produccion masiva

de caracol en Q.Roo. Me,\ico. Pkh: Uiilf unil Ccirihh. Fish. Ins:. .17:

1 2-20.
Corral, J. L. & J. Ogawa. 1987. Cullivo masivo del caracol Stromlnis gifiiis

en eslanques de concrelo. Pmc. Giilfaiul Citiihh. Fi\h. Insl. .18: 144-

.151.
Davis. M., C. Bolton & A. W. Stoner, 199.1. A comparison of larval

development growth, and shell morphology in three Caribbean Slrom-

bus species. The Veliger. 36: 236-244.
de Jesus-Navarrete, A. & J. J. Oliva-Rivera. 1997. Densidad. crecimiento

y reclulamienlo del caracol rosado Stromhus gigas L. en Punla Gavilan,

Quintana Roo. Mexico. Rev. Biol. Trof>. 45: 797-801.
de Jesiis Navarrele. A.. J. Oliva-Rivera. A. Pelayo. M. Gongora. A. Me-
dina & M. Dominguez-Viveros. 1994. Desarrollo Cientffico y Tecno-

Idgico para el cultivo del caracol. Bolelin informativo. SEPESCA/

CIQRO. 45. pp.
Oram. C. J. & J. R. Wyatt. 1980. Surface currents in the eastern Cayman

and weslem Caribbean seas. Bull. Mar. Sti. 30: 613-622.

Strombus gigas Veligers at Quintana Roo

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Jordan. D. E. & E. Marlin. 1987. Chinchorro: morphology and composition

of a Caribbean atoll. Atoll. Res. Bull. 310.
Keegan, W. F. 1992. The people who discovered Columbus: the prehistory

of the Bahamas. Inv. Press Florida. Gainesville. FL. 279 pp.
Kinder. T. H. 1983. Shallow currents in the Caribbean sea and Gulf of

Mexico as observed with satellite tracked drifters. Bull. Mar. Sci. 33:

239-246.
Merino-Ibarra, M. 1986. Aspectos de la circulacion costera superficial del

caribe mexicano. con base en observaciones utilizando tarjetas de de-

riva. Ann. Cienc. del Mar y Limnol. UNAM. 13: 31^6.
Metcalf. W. G., M. C. Statculp & D. K. Atwood. 1977. Mona passage drift

bottle study. Bull. Mar. Sci. 27: 586-591.
Oliva-Rivera. J. J. & A. de Jesiis-Navarrete. En prensa. Composicion.

distribuciiin y abundancia de larvas de moluscos gastropodo.s en el sur

de Quintana Roo, Mexico y norte de Belice. Rev. Biol. Trap. 48.
Pineda, J. 1995. Internal bores in the nearshore: warm-water fronts, seward

gravity currents and the onshore transport of neustonic larvae. J. Mar.

Res. 52: 427-i58.
Posada, J. & R. S. Appeldoorn, 1994. Preliminary observations on the

distribution of Strombus larvae in the eastern Caribbean, pp. 191-199.

In: R. S. Appeldoorn & B. Rodriguez, (eds.). Queen Conch, Biology,

Fisheries and Mariculture. Fundacion CientiTica, Los Roques, Caracas

Venezuela. 356 pp.

Randall. J. E. 1964. Contributions to the biology of the queen conch,
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Shanks, A. L. 1998. Abundance of post-larval Callinectes sapidus. Pe-
luieus spp., ilea spp.. and Libinia spp. collected at an outer coastal site
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Stoner, A. W.. V. J. Sandt & I. F. Boidron-Metairon. 1992. Seasonality in
reproductive activity and larval abundance of queen conch Strombus
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Stoner, A. W., P, A. Pitts & R. A. Amgstrong, 1996. Interaction of physical
and biological large scale distribution of juvenile queen conch in sea-
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Stoner, A. W. 1997. The status of Queen Conch, Strombus gigas. Research
in the Caribbean. Murine Fish. Rev. 59: 14—22.

Stoner, A. W. & M. Davis. 1997. Abundance and distribution of Queen
Conch veligers {Strombus gigas Linne) in Central Bahamas: I. Hori-
zontal patterns in relation to reproductive and nursery grounds. J. Shell-
fi.sh Res. 16: 7-18.

Stoner, A. W. & N. P. Smith. 1998. Across-.shelf transport of gastropod
larvae in the Central Bahamas: rapid responses to local wind condi-
tions. J. Plank. Res. 20: 1-16.

Weil, M.E. & Laughlin, G.R. 1984. Biology, population dynamics, and
reproduction of the queen conch Strombus gigas Linne in the Archi-
pelago de Los Roques National Park. J. Shellfish Res. 4: 45-62.

Joiinuil ofShfllfish Resfurch. Vol. IQ. No. 2. 8y7-SW, 2000.

RAPANA VENOSA (VALENCIENNES, 1846) (MOLLUSCA: MURICIDAE): A NEW GASTROPOD

IN SOUTH ATLANTIC WATERS

GUIDO PASTORINO,' - PABLO E. PENCHASZADEH,'^'
LAURA SCHEJTER/ AND CLAUDIA BREMEC" "^

Museo Argentina de Ciencias Naturales

Ay. Angel Gcdhirdo 470 3° piso lab 57

C1405DJR Buenos Aires, Argentina
-CONICET
^FCEyN-UBA

Facultad de Ciencias Exactas y Naturales. UNMdP

Dean Funes 3350. 7600 Mar del Plata. Argentina
' Instituto Nacional de Investigacion y Desarrollo Pesquero

Casilla de Correo 175

7600 Mar del Plata. Argentina

ABSTRACT Rapaiui veiiosa (Valenciennes 1846) (Gastropoda: Muricidae). a mollusk native to Eastern Asia, is reported for the first
time in Argentine waters in the north of Bahi'a Saniborombdn. During a routine bottom sampling, a female specimen of 97.1 and 76.3
mm shell length and width, respectively, and egg capsules were found approximately on 35.3°S-56.4'W in 13 m of water. The possible
way of entrance is discussed. The finding of egg capsules permits the supposition that the introduced population is sexually mature and
actively breeding. The presence of banks of mussels {Myriliis edidi.s plalensis) and oysters iOsirea puelchana). probable prey, together
with the occurrence of the egg capsules point out that the development of R. veiiosii in .Argentina could be ecologically and
economically important.

KEY WORDS: Rapana venosa. Muricidae, South Atlantic, mollusca, invasions. Argentina

INTRODUCTION

Rapana venosa (Valenciennes 1846) is a marine gastropod na-
tive of Eastern Asia where it is used as a food resource (Hasegawa
1996). Harding and Mann (1999) mentioned the Sea of Japan, the
Yellow Sea, the East China Sea, and the Gulf of Bohai as the
precise places of origin. Since the description of this species in
1846 it was reported in several countries in Europe and Asia,
sometimes as R. thoinasiana. which is mostly used as a synonym.
Powell (1972) recorded pagurized shells of R. venosa in New
Zealand waters. However, these shells were considered as a food
item thrown off an Asian fishing boat (Marshall and Crosby 1998).
Drapchin (1953) points out the Black Sea as the first place of
penetration out of Rapana' s traditional geographic distribution.
Cesari and Mizzan (1993) mentioned several authors who reported
the expansion of this gastropod along the Mediterranean Sea. In
1998, R. venosa was recorded in the Chesapeake Bay, U.S.A.
(Harding and Mann 1999). This was the first mention of the spe-
cies in America. Scarabino recorded the same species in Uru-
guayan waters in April to May of 1998 (pers. comm.).

In this paper we follow the systematic arrangement proposed
by Kool (1993). After a phylogenetic study of the family Muri-
cidae he concluded that the genus Rapana belongs to the family
Muricidae and to the subfamily Rapaninae.

After a routine bottom sampling off Bahi'a Samborombon. Bue-
nos Aires province, Argentina we found egg-capsules and one
adult specimen that belong to R. venosa. This constitutes the first
written mention of the species in South America.

RESULTS AND DISCUSSION

Egg Capsules and Eggs

Egg capsules of R. venosa were collected November 18, 1999
in 13 m of water with a bottom trawl of 120 mm mesh size from

35.436°S-56,373°W (trawl 74, INIDEP EH-09-99) (Figure 1).
The whole egg mass (Figure 2) has 208 capsules, which is within
the average number (115-220) cited by D'Assaro (1991). Each
capsule has an average number of eggs per capsule of 840. ranging
from 790 to 890 (;; = 20). The egg capsules contained embryos at
a morula-gastrula stage. 240 microns in diameter. Collected cap-
sules measured 20 to 30 mm in length, including the curved tip.
and 3.5 mm in width at the smallest diameter at the base. The
general form and measurements are in agreement with Chung el al.
(1993) and the detailed illustrations shown by D'Assaro (1991).

Figure 1. Map showing the collection localities of R. venosa (Valenci-
ennes) and the egg capsules (O).

897

898

Pastorino et al.

Figure 2. Egg capsules ot R. venosa (Valenciennes). Scale bar = 1 cm.

Adult Specimen

One female specimen of R. venosa (Figure 3, A-C) was also
collected on November 18. 1999 in 13 m of water with a bottom
trawl from 35.53 1°S-56.532°W (INIDEP trawl 75 EH-09-99).
The specimen was large, reaching 97. 1 mm in length and 76.3 mm
in width. It has epibiosis of polychaetes Serpulidae and the Cirri-
pedia Bulaiiits venustiis Darwin. Bottom sediments on both trawls
were composed of fine sand.

Together with Corhiciila fliiininea (Mijller), C. UirgiUieiti
(Philippi) (Ituarte 1981. Ituarte 1994), and Liinnupenui fortiinei
(Dunker) (Pastorino et al. 1993; Darrigran and Pastorino 1995) this
is the forth species of recently invading mollusks into Argentine
waters. However, it is the first gastropod and the first from typical
marine environment. All the other invading species of mollusks
(all bivalves) are freshwater {Corhiciila spp.) oreurihaline species
{Limnoperna fortiinei).

Both species of Corliicida were apparently introduced as food
lor crew consumption on Asiatic ships. R. venosa is a common and
esteemed delicacy in Japan which is called "Akanishi" (Ha.segawa

1996). However, this way of entrance seems less probable. Be-
cause Limnoperna fortiinei specimens are not used as food in their
native countries it is supposed that the introduction was produced
as larvae carried in untreated ballast water from commercial or
military ships (Pastorino et al. 1993, Darrigran and Pastorino
1995). This is probably the same way that R. venosa used to enter
North America (Harding and Mann 1999) and Argentine waters as
well.

In several papers Carlton (1992 and refs. therein) reviewed
probable mechanisms of introduction of non-indigenous marine
organisiris to North American waters. He mentioned the movement
of oysters and the concomitant movement of organisms on the
oyster shells or in associated sediments and detritus as one of the
most important of these mechanisms. Ceratostoma inornatum (Re-
cluz) (Muricidae) was introduced in U.S.A. apparently because of
the commercial oyster industry. Another species, Urosalpin.x ci-
nerea (Say) (Muricidae), was introduced to the northeast Pacific in
the same way.

The finding of egg capsules allows us to think that the intro-
duced population is sexually mature and actively breeding. Mussel
banks of Mytiliis edulis platensis d'Orbigny and the local oyster
Ostrea piielchana d'Orbigny are distributed all along the Argen-
tine northern coast at depth of 35 to 45 m and both are suitable
prey for R. venosa. The presence of these possible prey together
with the occurrence of the egg capsules points out the development
of R. venosa in Argentina as ecologically and economically im-
portant.

ACKNOWLEDGEMENTS

We appreciate the bibliography sent by T. Kimura (Mie Uni-
versity, Japan) and H. Saito (National Science Museum, Japan).
Thoughtful reviews were provided by Roger Mann and an anony-
mous reviewer. This study was supported in part by a research
grant from Fundacion Antorchas, Argentina and the project BID
802/OC-AR-PICT No. 01 -0432 1 from the National Agency for
Scientific and Technical Promotion, Argentina to P.P. This is
INIDEP contribution No. 1155.

Figuru 3. R. vcnma (Valenciennes). I'liree views of llie t'emulc lndi\idual collected (A-C). Scale bar = 3 cm.

Rapana venosa in Argentina

899

LITERATURE CITED

Carlton. J. 1992. Introduced marine and e.stuarine mollusks of North
America: an end-of-the-20th century perspective. J. Shellfish Res.
ll(2):489-505.

Cesari, P. & L. Mizzan. 1993. Observazioni su Rapiiim venosu (Valenci-
ennes 1846) in cattivita (Gastropoda. Muricidae. Thaidinae). Bollenino
del Miiseo civico di Sim in miliinde di Venezia 42:9-21.

Chung. E.. S. Kiin & Y. Kim. 1993. Reproductive ecology of the purple
shell. Rapciiui venosii (Gastropoda: Muricidae). with special reference
to the reproductive cycle, depositions of egg capsules and hatchings of
larvae. Korean J. Malacol. 9(2):1-I5.

Darrigran, G. & G. Pastorino. 1995. The recent introduction of a freshwater
asiatic bivalve. Limnnperna fnrnmci (Mytiliade) into South America.
V<'«?f/-38(2):I71-175.

D"Assaro, C. N. 1991. Gunnar Thorson's world wide collection of proso-
branch egg capsules: Muricidae. Ophelia 351:1-101.

Drapchin. E.J. 1953. Novii molliusc v Cernoni more. Priioda 8:92-95.

Harding. J. M. & R. Mann. 1999. Observations on the biology of the veined
rapa whelk. Rapana venosa (Valenciennes. 1846) in the Chesapeake
Bay. / Shellfish Res. 18(1):9-17.

Hasegawa. K. 1996. Rapana venosa: Database of Endargered Marine and

Freshwater Animals in Japan III. pp. 50-55. Japan Fishery Resource
Conservation Association (ed.).
Ituarte. C. F. 1981. Primera noticia acerca de la introduccion de peleci'po-
dos asiaticos en el area rioplatense (Mollusca. Corbiculidae). Neotro-

pica naiy.i9-$3.

Ituarte. C. F. 1994. Corbicnia and Neoeorbicula (Bivahia: Corbiculidae)
in the Parana. Uruguay, and Ri'o de La Plata Basins. Nautilus 107(4):
129-135.

Kool. S. P. 1993. Phylogenetic analysis of the Rapaninae (Neogastropoda:
Muricidae). Malaeologia 35(2): 155-260.

Marshall, B. & D. D. Crosby. 1998. Occurrence of the tropical and sub-
tropical gastropod Stromhus vomer vomer (Roding 1798) (Mollusca:
Strombidae) off northeastern Northland. New Zealand. New Zealand J.
Mar. Freshwater Res. 32:135-137.

Pastorino. G.. G. Darrigran. S. M. Marti'n. & L. Lunaschi. 1993. Linmo-
perna fortunei (Dunker 1857) (Mytilidae). nueve bivalvo invasor en
aguas del Ri'o de la Plata. Neotropica 39( 102-102):34.

Powell, A. W. B. 1979. New Zealand Mollusca. Marine, Land and Fresh-
water Shells. William Collins Publishers LTD. Auckland. 500 pp.

Journal of Shellfish Rescurch. Vol. 19. No. 2. 901-904, 2000.

SHOREBIRD FEEDING ON STRANDED GIANT GASTROPOD EGG CAPSULES OF
ADELOMELON BRASILIANA (VOLUTIDAE) IN COASTAL ARGENTINA

PABLO PENCHASZADEH,' FLORENCIA BOTTO,^ AND
OSCAR IRIBARNE- '

^Museo Argentino de Ciencias Natitrales "Bernardino Rivadavia"
CONICET-UBA. Angel Gallardo 470. (1405) Buenos Aires. Argentina

Ecologia. Departamento de Biologia (FCEyN)
CONICET-Universidad Nacional de Mar del Plata
(7600) Mar del Plata. Argentina

ABSTRACT We report predation by shorebirds on large egg capsules of the gastropod Ailelomelon brasiliana. This gastropod is a
common inhabitant of sandy substrates in the northern coastal area of Argentina and is the only South American volutid known to have
free, unattached egg capsules. These capsules are commonly found strewn on beaches, especially after storms. Two shorebirds were
observed feeding on embryos inside capsules, the American oystercatcher (Haematopus palliauis) and the South American stilt
(Himantopus melanurus). During periods when egg capsules were stranded both birds switched to prey on embryos inside capsules
stranded on the sandy beach. Field observations and a field experiment showed that birds prefer to prey on capsules that have embryos
in advanced developmental stages. It is interesting to note that of all the shorebirds observed at the study site, only the local
non-migratory species preyed on capsules.

KEY WORDS: Gasteropods. egg capsules, predation. shorebirds. southwest Atlantic

INTRODUCTION

The large marine gastropod Adelomelon brasiliana (Lamarck
1811) is a common inhabitant of sandy substrates (5-10 m in
depth) in the Province of Buenos Aires, Argentina (36°S to41°S).
This is the only South American volutid known to have free,
unattached egg capsules (Penchaszadeh et al. 2000). The first
known description of its spawn was provided by d'Orbigny (1846)
who recorded large number of egg capsules stranded on the beach
in San Bias Bay (40°00'S, 62°.30'W). The capsules are oblate-
spheroid in shape, yellowish, but nearly transparent, thin with a
smooth polished surface like wet gelatin, and possess considerable
rigidity (Dall 1889). They measure 40 to 80 mm maximum diam-
eter with a volume up to 140 mL (Penchaszadeh and De Mahieu
1976). Newly laid eggs have an average diameter of 240 microns.
Nine to 3.3 embryos per capsule (Penchaszadeh and De Mahieu
1976) develop, ingesting proteins, amino acids, and sugars con-
tained in the intracapsular liquid and in the inner wall of the
capsule (De Mahieu et al. 1974). Capsules do not carry nurse eggs
(Penchaszadeh and De Mahieu 1976).

These capsules are commonly found stranded on beaches along
the northern Argentinean coast, especially after storms. Eggs and
embryos can remain alive on the beach for a period of at least 7
days in winter (P. Penchaszadeh pers. obs.). Once in the intertidal
it is unlikely that capsules return to the sea and remain intact
because wave motion is too heavy and they would be readily
broken. Death of the embryos is mainly from desiccation and
increases in temperature. Field observations suggest that some
shorebird species utilize this food source.

The southwest Atlantic coastal and estuarine environments, in-
cluding the coast of the Buenos Aires province are inhabited by
several resident shorebirds (i.e. American Oystercatcher //aemar«-

'Corresponding author: CC 573 Correo Central (7600). Mar del Plata.
Argentina. E-mail osiriba^'mdp.edu.ar

pus palliatus and South American Stilt Himantopus melanurus)
and are important stopover and wintering sites for several North
American migratory shorebirds (Botto et al. 1998. Iribame and
Martinez 1999). Frequent beach strandings of gastropod egg cap-
sules have been reported in this area (Penchaszadeh and De
Mahieu 1976) and shorebirds have been observed feeding on them
(O. Iribame and F. Botto pers. obs.) Given that they may provide
a previously unidentified food source, our purpose was to docu-
ment their use by birds and describe any preference for capsules
with embryos in different stages of development.

MATERIAL AND METHODS

The study was conducted near the mouth of one of the east-
ernmost tidal channels (Arroyo San Clemente: 36°22'S, 56°45'W)
of Samborombom Bay (a coastal basin 100 km long within the La
Plata River estuary. Argentina). The area is characterized by large
and dense populations of the fiddler crab ilea uruguayensis and
the burrowing crab Chasmagnathus granulata (Boschi 1964). The
littoral zone extends into a large 5/>((m/;o-dominated salt marsh
(Bortolus and Iribame 1999).

During two periods of stranded capsules (December 2, 1998
and January 17, 1999) we sampled the shoreline to obtain an
estimate of the density of capsules accumulated per meter of shore.
One hundred random samples of 1-m tran.sect were established
from the high tide line to the water. Then all capsules were counted
in this area at high tide (the width varies from a few centimeters up
to 2 m). Given that the intertidal has a variable width, we will
report density as numbers of capsules per meter of coastline.

Three study periods (spring through autumn of 1995 and 1996.
1996 and 1997. and 1997 and 1998) were used to generate infor-
mation on the interaction between birds and capsules. During these
periods, monthly or bimonthly field observations were taken in the
area observing bird species present and species that fed on egg
capsules. A beach (500 m long and 60 m wide at low tide) with

901

902

Penchaszadeh et al.

large patches of Uca was used to evaluate bird activity in relation
to egg capsules. Observations of bird behavior were performed
from mid-morning to late afternoon, and bird species were iden-
tified following the field guide of Narosky and Yzurieta (1987).
Observers hiding in two different sites recorded bird behavior
using 10 X 50 binoculars and an 18 to 36x spotting scope (usually
at less than 40 m from the bird activity arena), and data were
recorded with a portable tape recorder. Each individual bird was
observed for a maximum of 15 min.

To determine if shorebirds prey differentially on capsules with
embryos in different stages of development we used two different
methods, a comparative field sampling experiment and a field
experiment, described as follows in the next two sections.

Comparison of Stages of Embryonic Development of Egg Capsules
Stranded on the Beach and from the Subtidal Area

To evaluate possible food preference we compared stranded
capsules versus capsules collected by a trawl boat (5-10 m depth)
from nearshore areas and classified them into four categories of
embryonic development: category I includes embryos not visible
to the naked eye, category II includes small, newly formed snails
< 5 mm, category III includes embryos 5 to 8 mm in length, and
category IV includes pigmented, fully developed snails with cal-
cified shells of about 8 to 1 1 mm in length. The assumption was
that transport of capsules to the beach does not interfere with the
embryo's stage of development. We also added two categories of
capsules: Pecked which are those capsules that were empty with a
clear mark of stabbing by birds, and Broken which are those cap-
sules empty with some kind of rupture, but not clearly identified as
bird stabbing. These two categories were added to evaluate the
incidence of bird predation on gastropod embryos. A relatively
large number of capsules was found stranded on the beach on
December 2, 1998, probably produced the previous day. Thus
these capsules had been exposed to predation presumably for one
day. One hundred-eight randomly (obtained by randomly allocat-
ing a l-nr quadrate) collected capsules were obtained to identify
the proportion of capsules with embryos at different developmen-
tal stages. A sample of 210 capsules was also obtained from the
nearshore 5 to 8 m in depth the same day. and embryonic devel-
opmental stages were recorded. The null hypothesis of no differ-
ence between the frequency distribution of capsules with embryos
in different stages, between sites, was evaluated using Kolmog-
orov-Smirnov test (Zar 1984).

Delerininalion of Fcedina Preferences of Shorebirds on Egg Capsules

To evaluate il shorebirds prefer lo feed on capsules wilh em-
bryos al different tiexelopmental stages, egg capsules were col-
lected by a trawling boat 5 lo 10 in in water depth off ihe coasi of
Mar del Plata (.38"S. 58"W, Argentina) and classified into the same
categories as above. Then 400 of these egg capsules, 100 of each
developmental stage, were distributed in the middle intertidal area
tluring a period of low tide. They were lefi alive stranded on the
sand for 4 h during which time shorebirds were observed feeding
on them. After this period we counted the number of egg capsules
of each developmental stage that were eaten by shorebirds. Then,
an index of food selection Chesson's alpha (Chesson 1978) was
iisetl to evaluale preference on these food items. The index mea-

sures an invariant degree of preference on the part of the predator
(Pearre 1982). The index is: Chesson's alpha = (r/p,) / S, (r,/p,),
where r, and p, are the proportion of prey item i in the diet and the
environment, respectively (Strauss 1979).

RESULTS AND DISCUSSION I

i

i

During the three study periods we spent a total ot 45 days j

making field observations and we observed seven major strandings
of egg capsules. During most strandings the entire field sampling
area was covered by a band of capsules approximately I m in
width. In two of these strandings we sampled the density of cap-
sules and obtained similar values. In the first stranding, density of
capsules per meter of coastline was 12 capsules m~' (SD = 21. /;
= 100), while it was 21 capsules m"' (SD = 16,/; = 100) during
the second stranding. This amount represents between six and ten
thousand capsules accumulated in our study area (500 m of coast-
line). We have no information on density of capsules stranded
during other events, bul our impression is that they were similar in i
magnitude. Given a range of nine to 33 embryos per capsule and
a wet weight of 0.15 g per embryo (0.0125 g dry weight), a
predator could obtain a total of 1 .35 to 4.95 g wel weight (0. 1 125-
0.4125 g dry weight) of food per capsule. This is a significant
amount of food that can be readily utilized. !

Several species of shorebirds, terns, and gull species were ob-
served feeding in this area during periods of egg capsule stranding.
These species were American golden-plover {Pliivialis dominica:
observed for 260 min). black-bellied plover (P. sc/Kotarola: ob-
served for 320 min), ruddy turnstone (Arenaria interpres; observed |
for 180 min), whimbrels {Numenius phaeopiis; observed for 140
min), gull billed tern (Sterna nilotica: which dived picking up
crabs from the intertidal; observed for 67 min). Brown Hooded
Gull {Lunis macidipennis: observed for 45 min), and the two-
banded plover {Cluuadriiis fidklaiuUcits: observed for 210 min).
Most of them (P. dominica. P. sipiatarola. A. interpres. and N.
pluwopus) are long range (from Canada and the U.S.A. lo central-
southern Argentina) migratory shorebirds (e.g., Myers and Myers
1979, Morrison and Ross 1989) and in this area, they were found
always preying on the fiddler crab Uca urugiiayensis (see Iribame
and Martinez 1999). The two-banded Plover migrates from south-
ern Patagonia (Argentina) to the northern Argenlineaii coast during
winter (Myers and Myers 1979). None of these species were ob-
served preying on egg capsules. The kelp gull Lams dominicaniis
was seen attempting to feed on capsules (three different days, five
indi\ iduals). Capsules were picked up in the bill and dropped on
ihe beach from several meters in height. However, this method was
not successful, the capsules were never broken, and kelp gulls
were not seen eating them.

Two other species of birds were observed feeding in this area.
the American oystercatcher [Haenialnpiis pidlianis: observed for
197 min) and ihe South American slill {Hinianlojms nielaniinis:
observed for 235 min ). In this area oystercatchers spent most of the J
lime preying on Ihe stout razor clam Tagehis pleheiiis. (Iribame et i
al. 1998), while the South American stilt fed on small items in-
cluding small gastropods (Lilloridina aiistralis) and newly re-
eruiled crabs (F. Botio pers. obs.). However, during periods of
capsule stranding they switched lo prey on the embryos inside
stranded capsules thai were lying on the sandy beach. Shorebirds
walked along the beach inspecting capsules, strongly pecking
some o\ ihem lo break Ihe capsule and eat the developing gastro-

I

Shorebird Feeding on Stranded Giant Gastropod Egg Capsules

903

pods found inside. Pec]<ing by Oystercatchers left a triangular
rupture in the capsule wall approximately 20 mm on each side
folded into the capsule, while the third side of the triangle re-
mained attached to the wall.

We have no evidence that birds used the walls of the capsule or
the liquid inside as food, even though this would be nutritive
(Miloslavich 1996. De Mahieu et al, 1974). All evidence suggests
that they fed only on the embryos. Egg capsules of gastropods are
structurally and chemically complex and very difficult to digest
(Miloslavich 1996). Indeed, they can remain on the shore for a
long time without degradation. They seem to have strong protec-
tion against bacteria, predation, and physical stress, which may be
an evolutionary response for egg protection (Pechenik 1986).

Observations on the oystercatcher and the South American stilt
behavior were performed while they were feeding exclusively on
capsules. Capture of sinall developing snails from inside the cap-
sule was identified by swallowing action observed in the throat of
the bird. Focal sampling of 13 American oystercatchers was per-
formed for a total of 64 min while they were feeding on capsules.
The feeding strategy of this shorebird was to "stab" the capsule
with its bill, perforating it and introducing their long bill into the
capsule. Once inside they probed for snails. These shorebirds
needed 43 sec (SD = 23, n = 84) to empty one capsule, eating on
average nine embryos per capsule (SD = 5. n = 84). A focal
sampling of nine South American stilts was performed for a total
of 31 min while they were feeding on capsules. The feeding strat-
egy was similar to the one seen for oystercatchers. These shore-
birds needed 25 sec (SD = 18. « = 38) to stop eating in one capsule,
eating on average five embryos per capsule (SD = 6, « = 38).

Comparison between the abundance of different stages of de-
velopment of embryos in stranded capsules (observed after the
predation event) with those from subtidal samples showed a dis-
tinct pattern. While most subtidal capsules had embryos in an
advanced developmental stage, the ones left stranded on the sea-
shore were mainly at the first developmental stages (Koiniogorov-
Smirnov test; P < 0.05) with a large proportion of eggs broken:
most of them were cleariy identified as being pecked by birds (see
Fig. lA). The field experiment showed that this difference might
be due to differential shorebird predation (Fig. IB). Only the
American oystercatcher was seen feeding in this area during the
experiment and the Chesson Index cleariy shows they preferred
capsules with embryos in advanced developmental stage (III and
IV) (Fig. IB).

Embryos in early developmental stages are very small; they
cannot be seen through the capsule walls and are clustered in the
lower section of the capsule. However, capsules with embryos in
advanced developmental stages (HI and IV) are transparent, em-
bryos are larger, (more than 5 mm in length), and in stage IV, they
have well-developed feet and usually are seen crawling on the
interior side of the capsule wall (Penchaszadeh et al. 2000). These
embryos can easily be seen from the exterior through the capsule
wall, which may allow visual predators such as the American
Oystercatcher and the South American Stilt to select their prey.

There is abundant literature showing predation on eggs by
shorebirds (i.e. Crossin and Ruber 1970. Farraway et al. 1986).
However, we know of no other previous report of predation on egg
capsules of prosobranchs. Moreover, of all shorebirds observed in
this study, the only two species that took advantage of these cap-
sules were both local non-migratory species (Narosky and Di Gia-
como 1993. Martinez, and Bachmann 1997. Bachmann and Mar-

LU
O
<

Z
LJJ

o

tr

LU
Q_

70

60

50

40

30

20

10

0

0.6

□ SUBTIDAL
^ INTERTIDAL

IV PECKED BROKEN

UJ °'
Q

0.2

B

1 1

IV

EMBRYONIC STAGE OF DEVELOPMENT

Figure 1. (A) Proportion of embryonic stages found inside egg cap-
sules of Adelomeloii brasiliana at intertidal and subtidal levels of Sam-
borombom Bay in summer 1998. The categories also include the pro-
portion of damaged egg capsules (BROKEN) and those that had clear
marks of bird pecking (PECKED). (B) Chceson Index of food selection
by shorebirds for egg capsules containing different embryonic stages
resulting from the intertidal predation experiment.

tinez 2000). The other local non-migratory species was the kelp
gull, which attempted to feed on capsules without success. There
is no evidence that the two-banded Plover, a short-range migratory
shorebird (Narosky and Yzurieta 1987). or any of the long-range
migratory shorebirds used capsules as food. Even species like the
ruddy turnstone. which feed on eggs of Terns at other sites
(Crossin and Huber 1970, Farraway et al. 1986) were not seen
feeding on capsules. This pattern may be the result of a food source
that is spatially restricted, highly sporadic, and unpredictable,
which make them prone to be used by resident species.

ACKNOWLEDGMENTS

This project was supported by the Universidad Nacional de
Mar del Plata (051/94). by the Fundacion Antorchas (grant no.
13016/1-00012). by the International Foundation for Science
(grant no. A/2501-1), and by the National Geographic Society
(grant no. 6487-99). F. Botto was supported by a .scholarship from
the Consejo Nacional de Investigaciones CientiTicas y Tecnicas.
The review of two anonymous referees largely improved the
manuscript.

904

Penchaszadeh et al.

LITERATURE CITED

Bachmann. S. & M. M. Martinez. 1999. Feeding tactics of the American
oystercatcher {Haemutopus palluitiis) on Mar Chiquita coastal lagoon.
Ornilol. Neolrop. 10: (in press).

Bortolus, A. & O. Iribarne. 1999. Effects of the SW Atlantic burrowing
crab Chasmagnathus granulala on a Sparlinu salt marsh. Mm: Ecoi
Progr. Ser. 178:79-88.

Botto. F.. O. Iiibame. M. Martinez, K. Delhey & M. Carrete. 1998. The
effect of migratory shorebirds on the benthic fauna of three SW At-
lantic estuaries. Esuiaries 21:700-709.

Boschi, E. E. 1964. Los cruslaceos Decapodos Brachyura del litoral bo-
naerense. Bol. Inst. Biol. Mar. 6:1-99.

Chesson, J. 1978. Measuring preference in selective predation. Ecology
59:211-215.

Crossin, R. S. & L. N. Huber. 1970. Sooty tern egg predation by ruddy
turnstones. Condor 88:521-522.

Dall. W. H. 1889. Scientific results of the explorations by U.S. fish com-
mission steamer Albatross. 7: preliminary report on the collections of
Mollusca and Brachiopoda obtained in 1887-1888. Proc. U. S. Nail.
Museum 12(77.^):2!9-362.

De Mahieu, G.. P. E. Penchaszadeh & A. Casal. 1974. Algunos aspectos de
las variaciones de proteinas y aminoacidos libres totales del liquido
intracapsular en relacion al desarrollo embrionario en Adelomelon bra-
siliana (Lamarck. 1811) (Gastropoda. Prosobranchiata, Volutidae).
Cah. Biol. Mar. XV(228):215-227.

d'Orbigny. A. 1846. Voyage dans ramerlque meridionale. Molliisques v.
Paris. 1-758.

Farraway, A.. K. Thomas & H. Blokpoel. 1986. Common tern predation by
ruddy turnstones. Condor 88:521-522.

Iribarne, O. & M. Martinez. 1999. Predation on the southwestern Atlantic
fiddler crab (Uca uruguayensis) by migratory shorebirds (Pluvialis
dominica. P. sqiiatarola, Arenaria inlerpres and Niimeniiis pluieopus).
E.siuaries 22:47-54.

Iribarne, O., J. Valero, M. Martinez. L. Lucifora & S. Bachman. 1998.

Shorebird predation may explain the origin of Holocene beds of stout
razor clams in life position. Mar. Ecol. Prog. Ser. 167:301-306.

Martinez. M. M. & S. Bachman. 1997. Kleptoparasilism of the American
oystercatcher Haematopus palliahis by gull Lanis spp in Mar Chiquita
lagoon, Buenos Aires, Argentina. Mar. Oniiol. 25:65-68.

Miloslavich. P. 1996. Biochemical composition of prosobranch egg cap-
sules. J. Moll. Stud. 62:133-135.

Morri.son. R. I. G. & R. K. Ross. 1989. Atlas of neartic shorebirds on the
coast of South America. Can. Wild. Sen: Spec. Puhl. 2:131-323.

Myers, J. P. & L. P. Myers. 1979. Shorebirds of coastal Buenos Aires
province. Argentina. Ibis 121:186-200.

Narosky. T„ & A. G. Di Giacomo, 1993. Las Aves de la Provincia de
Buenos Aires. Literature of Latin America, Buenos Aires, Argentina.
127 pp.

Narosky, T. & D. Yzurieta. 1987. Guia para la identificacion de aves de
Argentina y Uruguay. Asociacion Omitologica del Plata. Buenos Aires.
Argentina.

Pearre, Jr., S. 1982. Estimating prey preference by predators: uses of vari-
ous indices, and a proposal on another based on x" Can. J. Fish. .Aqual.
Sci. 39:914-923.

Pechenik. J, A. 1986. The encapsulation of eggs and embryos by mollusks:
an overview. Am. Malacol. Bull. 4:165-172.

Pencha.szadeh. P, E. & G. de Mahieu. 1976. Reproduccion de gasteropodos
prosobranquios del Atlantico suroccidental. volutidae. Physis 35:145-
153.

Penchaszadeh, P. E., P. Miloslavich, M. Lasta & P. M. S. Costa. 2000. Egg
capsules in the genus Adelomelon (Caenogastropoda; Volutidae) from
the Atlantic coast of South America. Nautilus 113(3): (in press).

Strauss, R. E. 1979. Reliability estimates for Ivlev's electivity indisex. the
forage ratio, and a proposed linear indisex of food selection. Trans. .Am.
FmA. Soc. 108:344-352,

Zar. J. H. 1984, Biostatistical Analysis. Prentice-Hall, Englewood Cliffs.
NJ.

♦

I

J<ninh:l of Slwlljl^^h Research. Vol. 14. No. 2. 905-91 1, 2000.

NEURAL EXTRACT INDUCTION OF EGG-LAYING AND SUBSEQUENT EMBRYOLOGICAL
DEVELOPMENT IN HARD AND SOFT EGG CAPSULES OF THE MARINE SNAIL,

CHORUS GIGANTEUS

JEFFREY L. RAM,' CARLOS GALLARDO,'

C. RODRIGO MERINO,' MICHAL L. RAM," AND

JORGE NAVARRO^

Department of Physiology. Wayne State University,

Detroit. Michigan 48201
~Deparment of Cardiology. Wayne State University,

Detroit. Michigan 48201
' Institute of Zoology. Universidad Austral de Chile, Valdivia. Chile

Institute of Marine Biology, Universidad Austral de Chile. Valdivia. Chile

ABSTRACT The great abundance, long evolutionary history, and diversity of gastropods make this class of mollusks interesting
for studies of the evolution and mechanisms of reproductive adaptations. This study investigated induction of laying of egg cap-
sules and subsequent intracapsular embryonic development of the Chilean muricid snail Chorus giganieiis. whose natural population
has suffered a serious decline during the last 20 years. Central nervous system (CNS) extracts ( supernatants of homogenates of
combined circumesophageal. pedal, and buccal ganglia that were boiled and centrifuged) caused the laying of both soft bulb-shaped
capsules and hard well-formed capsules. Latency between injection and capsule laying was 3-6 h. Neither control injections of vehicle
(filtered sea water) nor injection of e.\tracts containing less than half of a CNS per recipient caused laying. All but two of 36 capsules
laid in response to extract injection contained eggs. Eggs in soft capsules developed normally initially but were subject to infec-
tions; although eggs in some hard capsules showed arrested development, in others development appeared to proceed normally,
and veliger larvae developed in one hard capsule that was maintained for 49 days. The capsule walls of induced hard capsules had
a similar multilayered microscopic structure to spontaneously laid capsules. Soft capsules had a less compact middle lamina with
missing or much less compact outer lamina. These experiments demonstrate the presence of a putative hormone activating egg laying
in nervous system extracts of C. giganleus and demonstrate that normal intracapsular developinent can occur in some of the resultant
capsules.

KEY WORDS: Chorus giganleus. development, egg capsule, egg-laying, muricid snail, neurohormone, reproduction

INTRODUCTION

Declines of natural fisheries of some species have prompted
research to enhance natural reproduction or to develop aquacul-
ture. For example, in Chile the population of the economically
important muricid snail Chorus gigaiueiis Lesson 1829 has re-
cently undergone a large reduction. Despite increasing effort, the
catch of C. giganteiis decreased from 2.800 metric tons in 1980 to
less than 100 tons annually in 1995 and 1997 (Pinto-Aciiero and
Soto 1997. SERNAPESCA 1998). Development of aquaculture of
C giganleus is hampered by lack of basic knowledge about this
species, including about its reproduction. Spawning of C. gigan-
leus in the field has been reported in January, March, April, and
September (Jaramillo and Garrido 1990); however, the occurrence
of egg capsules in the field varies considerably from year to year,
making a secure means of obtaining egg capsules a priority in
establishing aquaculture for this species. The present study focuses
on methods of regulating the reproduction of C. giganleus by
means of laboratory control of egg-laying and subsequent intrac-
apsular development, with the eventual goal of applying this
knowledge to aquaculture.

Marine gastropods employ a variety of methods for packaging
and protecting their eggs (Solem 1991 ). Mechanisms for releasing
gametes vary from broadcast spawning followed by external fer-
tilization, as in most vetigastropods, to more highly evolved pat-
terns, found in caenogastropods, that include internal fertilization
with deposition of eggs in specialized capsules (gastropod classi-
fication according to Ponder and Lindberg 1997). Among neo-

gastropod species, including muricids such as C. giganleus. egg
capsules are hardened into definitive shapes by the ventral pedal
gland (VPG). As described for several species (Na.'ssarius, Ankel
1929. Concholepas concholepas. Castilla and Cancino 1976, Busy-
con. Ram 1977, Eupleura caudata etlerae. Gruber 1982). during
oviposition, a soft bulb-shaped capsule containing eggs is passed
from the female gonopore through a groove in the side of the
foot into the VPG, from which it sub.sequently emerges as a hard-
ened, well-formed capsule. The walls of the egg capsules usually
consist of multiple layers. In muricids, these layers typically in-
clude an outer protective lamina, a thick middle lamina com-
posed of multiple fibrous layers, and one or two thin inner laminae
encompassing the albumen with the suspended eggs (D'Asaro
1988).

Egg-laying behavior and formation of egg capsules in gastro-
pods is controlled by neuropeptides. Induction of egg laying by
nervous system extracts (Kupfermann 1967, Strumwasser et al.
1969, Geraerls and Bohlken 1976. Ram 1977) or the purified or
synthetic neurohormone (Chiu et al. 1979, Ebberink et al. 1985.
Ram and Ram 1989) has been shown in several species, but not
previously in any muricid snail. In Busycon, the caenogastropod
that previously has been most intensively studied, only egg-less
capsules were laid unless multiple injections (every 2-3 h for 24 h)
were made (Ram 1977. Ram et al. 1982). The present study in-
vestigated induction of capsule-laying by nervous system extracts
in C. giganleus. the presence of eggs in induced capsules, and the
effect of the hardening process on capsule wall structure and pro-
tection of embryos.

905

906

Ram et al.

MATERIALS AND METHODS

Animals

C. gigaiUeus for most experiments had been cultured for ap-
proximately one year in perforated plastic trays tied to long float-
ing cables anchored in Bahi'a Metri, adjacent to the Centro de
Acuicultura y Ciencias de Mar (CEACIMA-METRI) de Univer-
sidad de Los Lagos in Metri, Chile (41°36'S, 72°42"W). Large
(>11 cm shell length) females were used as both donors and re-
cipients of nervous system extracts. Recipients were selected from
a small number of animals available from trays in which recently
laid egg-capsules were present. For experiments, snails were
placed individually in 40 cm x 40 cm x 40 cm plastic tanks with
constantly flowing 50-(j.m filtered sea water at a temperature of
15-17 °C.

Recipients in other experiments (pilot experiments and experi-
ments on dosage and sensitivity to ambient temperature) were
female C. f^iganteus that had been cultured by J. Navarro in a
laboratory culture system in which animals had been observed to
lay egg capsules several days prior to injection experiments. Ani-
mals in these experiments were held at 15 °C in small aerated
aquaria with no flow-through sea water. Experiments were con-
ducted during February and March of 1998 and 1999.

Nervous System Extracts and Injections

Nervous systems were dissected from large female C. gigan-
leiis. For example, one series of 12 animals had shell lengths of
12.4 ± 0.2 cm (mean ± SEM): wet weight without shell of 60 ± 4
g; gonad weight of 1 .5 ± 0.3 g; and gonad index ( = gonad weight/
wet weight without shell) of 0.023 ± 0.004. Central nervous system
(CNS) extracts were made from combined circumesophageal gan-
glia, pedal ganglia, and buccal ganglia, dissected along with a short
piece of esophagus, as described previously for Busycon (Ram
1977). CNS and esophagus anatomy of C. giganletis is similar to
that previously illustrated for the muricid snail Concholepus c»n-
cholepas (Ram et al. 1998). Dissected CNSs were immediately
placed on ice and frozen in liquid nitrogen in groups of four.
Typically, four frozen CNSs were homogenized in 0.6 mL of
ice-cold filtered sea water in a motor-driven glass-teflon honiog-
eni/er, then placed on a boiling water bath for 10 min, cooled on
ice, centrifuged for 25 min, and diluted to 2 mL to give a concen-
tration of 2 CNS/mL. Extracts were either frozen in liquid nitrogen
until needed or injected immediately into recipients.

To test the minimal dosage of extract to elicit egg-laying, an
extract containing 2 CNS/mL was diluted 2-fold. 4-fold, 8-fold,
and 16-fold in filtered sea water. Diluted extracts were fro/en in
liquid nitrogen until thawed for injection.

The typical procedure utilized boiled extracts because in Bk.vv-
(■{III the substance eliciting egg laying was known to be stable to
boiling but was sensitive to proteolytic or other enzymes in un-
boiled extracts exposed to ambient temperatures for short periods
of lime. To lest the sensitivity of C. gigdiilciis extracts to ambient
icmpcratures. half of an unboiled CNS extract was placed at room
lemperature for 10 min and then boiled as usual; to the other half
of the extract, 0.3 mg bcnzamil (a protca.se inhibitor )/niL of extract
was added after which the extract was held at room temperature for
10 min followed by boiling. Extracts were frozen in liquid nitrogen
until injection into recipients. Animals were injected through the
side of the foot with 0.25 mL extract/recipient or, for control
injections, 0.25 mL filtered sea water. Similar injections into an

isolated foot showed this to be an effective route for injecting into
the pedal sinus. The specific timing of injections will be described
in the Results. The general sequence of injections was to inject
recipients once or twice with extracts, then to inject with control
solutions at similar intervals, and finally, to inject again with ex-
tracts. Thus, each animal acted as its own control. The rationale for
doing two extract injections prior to the control injections in one
experiment was that in another species (Busycon canaliculatiiin)
multiple injections at a 2- to 3-h interval had been necessary to get
insertion of eggs into capsules. Following injection, animals were
examined approximately once per hour to look for capsules thai
had been laid.

Intracapsular Development and Egg Capsule Micromorphology

Egg-containing capsules were placed in aerated vials in filtered
sea water at 15 °C, as used previously to study the complete
intracapsular development of C. giganteus (Gallardo 1981, Gonza-
lez and Gallardo 1 999). Normally, intracapsular development of C.
giganteus embryos into veliger larvae and release of the larvae
from the capsules takes approximately 70 days at 15 °C (Gonzalez
and Gallardo 1999). The size of the capsules, physical properties
and shape (hard or soft, with or without peduncle), and numbers of
eggs were noted, and selected capsules were fixed and/or photo-
graphed at various developmental stages.

Egg capsules were fixed for 2 h in a mixture containing 2.5%
glutaraldehyde, 10% para-formaldehyde, 2% acrolein, and 0.2 M
phosphate, pH 7.2 (Rodriguez, 1969). After washing with 0.2 M
phosphate buffer, capsules were postfixed for 2 h in buffered 1%
OsOj and embedded in epon-aruldite (Richardson et al. 1960).
Semi-thin ( I \x.m) sections were cut with a glass knife, stained with
toluidine blue borax, and mounted for observation under optical
microscope. Capsule wall laminae are described with reference to
previous studies in other muricacean snails (D'Asaro 1988. Gar-
rido and Gallardo 1993, Rawlings 1995).

Histology

To assess gonadal maturity in both responding and non-
responding females, gonads were dissected, fixed in Hollande
Bouin (picric-formol-acetic plus cupric 11 acetate mixture) (Ganter
and Jollcs 1970) for 48 h and then dehydrated and embedded using
standard procedures. Embedded tissue was sectioned at 6 |j.m.
processed through a series of increasing ethanol solutions, and
stained with hematoxylin-eosin (Humason 1962).

RESULTS

Induction of Capsule iMving

Injection of CNS extracts caused the laying of egg capsules.
The pattern of responses to CNS extract and control injections in
one experiment is illustrated in Figure I. Five animals were in-
jected twice with CNS extract at an interval of 2.5 h. At the lime
of the second injection, no capsule had yet been laid; however,
approximately 2 h later (a total of 4.5 h afler the initial injection),
four of the five animals laid an egg capsule. The capsules were soft
and bulb-shaped (Fig. 2A), apparently not having entered the VPG.
Approximately 3 h later (5 h after the second injection), three of
the animals laid another egg capsule, including one hardened cap-
sule allached to the substrate by a typical peduncle (Fig. 28). All
capsules contained eggs, varying from 60-70 eggs in the smallest
capsule to 3000 eggs in the capsule shown in Fig. 28.

Induction of Egg-Laying in Chorus giganteus

907

TIME Chr) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 U 15 16 17 18 19 20 21 22 23 24

CSls

INJECTION

# 1

2

3

4

5

6

7

ANIMAL

#

1

E

E

[SI,

C

c

E

E

CSI5

2

E

E

[S],

[SIjC

c

E

E

E

3

E

E

CS],

tH];C

c

E

E

[Sis

4

E

E

[S],

CSljC

c

E

E

[His

5

E

E

C

c

E

E

CHJs

[ ] - capsule laid; S = soft capsule; H = hard capsule

E = extract injected

C = negative control (filtered sea water) injected

Figure 1. Sequence of injections and capsule laying in response to CNS extracts in Chorus giganteus. Time from the beginning of the experiment
is shown across the top, and the time of extract injections (E) or control injections (C, fdtered sea water) is shown for each animal. [S] or [H]
indicates when a soft capsule or a hard capsule was laid, respectively. Subscripts for each capsule indicate the injection that hypothetically caused
laying of the capsule. Most extract injections resulted in the laying of a capsule, but some did not. One recipient animal (#5) did not lay at all.

Control injections of filtered seawater into tlie same animals
did not cause laying, demonstrating that it was not simply the
effect of injection that caused laying. No capsules were laid within
the same time range that previous extract injections had caused
laying. When extract was injected again several hours later, ani-
mals resumed laying capsules. The fifth animal (i.e.. the one that
did not lay following extract injections) never laid an egg capsule
and died sometime during the next ten days following the above
described experiments.

Experiment 2

Ten days after the above experiment. CNS extracts were in-
jected into the four animals that had laid capsules previously in
response to extract injections. During the intervening 10 days, no
additional capsules were laid; however, all four animals laid a
capsule within 4 h of a single injection of CNS extract. At the same
time, an additional group of animals was injected with the same
extracts. This second group of animals had been maintained at 15
°C in the laboratory but had not previously laid egg capsules. Only
the animals that had laid capsules previously responded by laying
egg capsules after CNS injections. No animal laid in response to
two subsequent sea water control injections; however, three of the
four laying animals laid capsules again within 4 h of a second
extract injection. One of the responsive animals also laid an egg
capsule within the next 10 h during shipment to another location.

Dose-Response Experiment

Three animals that had been maintained by J. Navarro in a
closed aquarium system laid capsules when injected with the usual
dosage (0.25 mL of 2 CNS/mL extract; i.e.. 1/2 CNS per recipient)
of C. giganteus CNS extract. Subsequently, these animals were
injected with 1/32 CNS and successively increasing dosages of
CNS extract at 4-h intervals, and observations were made regard-
ing whether or when capsules were laid. Thus, animals were in-
jected with 0.25 mL containing 1/32 CNS. 1/16 CNS. 1/8 CNS. 1/4
CNS. and 1/2 CNS. One animal did not respond at all (and. in fact.
did not respond to several subsequent maximal dosage injections).
The other two animals laid a capsule only after the highest dose
(1/2 CNS). each laying a single soft capsule approximately four
hours after the injection.

Ambient Temperature and Benzamil Experiment

Three animals that had laid capsules in respon.se to boiled CNS
extracts were injected with an extract that had been incubated at
room temperature for 10 min prior to boiling. None of the recipi-
ents laid a capsule within 4 h of the injection. Subsequent injection
of a benzamil-treated extract elicited laying of a soft capsule from
two of the three recipients approximately 3.5 h after the injection.

In addition to the above experiments, capsules were also laid in
a pilot experiment following CNS injections into two animals but
not following control injections of seawater.

Size of Capsules, Number of Eggs, and Intracapsular Development

Altogether. CNS extracts stimulated 10 animals to lay at least
one egg capsule, with 8 of these animals responding at least two or
more times. These animals laid a total of 36 egg capsules, includ-
ing 28 soft capsules and 8 hard capsules. The hard capsules ranged
in length from 18 to 23 mm (median = 21 mm), whereas soft
capsules were 7 to 20 mm (median = 14 mm) in length. All but
two capsules contained eggs. The number of eggs per capsule in
six hard capsules in which eggs were counted ranged from 550 to
3,050 (median = 1.700), whereas two of the largest soft capsules

Figure 2. Soft and hard capsules laid by Chorus giganteus in response
to extract injections. (.41 capsule IS], of animal #2 (see Fig. 11. (B)
capsule [H], of animal #3 (see Fig. 1). Calibration: 10 mm.

908

Ram et al.

contained 1,100 and 1.550 eggs. The diameters of the eggs in the
hard capsules ranged from 250 to 260 |xm (median = 253 iJim).
whereas the diameters of the eggs in five soft capsules averaged
249. 255, 262, 264, and 323 (jim.

Embryonic development occurred in some induced hard cap-
sules, but in several others development appeared to be arrested or
slower than normal. Eggs in four capsules maintained for 29 to 49
days before fixation had reached only the polar body stage or had
undergone only the first division. However, normal cleavage was
observed in eggs of one capsule that was fixed 1 wk after being
laid. Another hard capsule, maintained at 15 °C for 49 days, con-
tained several veliger larvae (Fig. 3A). along with more than 900
non-developing eggs. By comparison, another hard capsule that
had been laid spontaneously by one animal prior to the beginning
of these experiments contained 8 veliger larvae (Fig. 3B) and
1 .449 uncleaved eggs after being maintained for approximately 50
days.

Intracapsular development also took place in soft extract-
induced capsules. Soft capsules were fragile and more subject to
infection than hard capsules. A few of the soft capsules showed
tears in their wall, even though handled gently. The eggs in these
capsules had a normal appearance. In other soft capsules, embryos
underwent apparently normal initial development. Eggs in one soft
capsule that was fixed 1 wk after laying had embryos that had
achieved a multi-cellular stage, similar to embryos in normal hard
capsules. Nevertheless, soft capsules maintained longer than a
week all eventually became infected with various pathogens (bac-
teria, protozoans, nematodes, etc.) within 5 wk. Of eight soft cap-

sules maintained in the laboratory at 15 °C for 23 or more days, six
contained uncleaved and/or broken eggs, one exhibited .some eggs
with polar bodies, and one that was terminated at day 35 due to
infection had been observed on day 25 to contain trochophore
larvae that had ingested nurse eggs.

Microscopic Structure

Susceptibility to infection and damage could be due to struc-
tural differences in the capsule wall. Micrographs of both hard and
soft capsules are shown in Figure 4. The wall of a hard capsule laid
in response to extracts (Fig. 4A) shows the layered structure of a
normal hard capsule (Fig. 4B). including ( 1 ) the outer lamina, (2)
the middle lamina, and (3) the albumen-retaining lamina. Another
layer, the lax lamina, would be situated between laminae 2 and 3,
but the lax lamina is only 0.5-|jLm thick and therefore not visible in
these micrographs. The middle lamina is the most complex, in-
cluding a compact outer layer, a middle porous vacuolated layer,
and a compact inner layer. In the normal and extract-induced hard
capsules of C. gigcmteu.s. the middle lamina was approximately
20-25-p,m thick. Two soft capsules (Figs. 4C and 4D) had a less-
compact middle lamina, with much thicker vacuolated regions and
total widths of 36 (xm and 45 fxm. In addition, the layered structure
of one of the soft capsules (Fig. 4C) is incomplete in that the two
outermost coverings (both the "outer lamina" of the surface and
the underlying "compact outer layer") are practically absent. In the
other soft capsule (Fig. 4D). the wall layering was relatively com-
plete, although the outer lamina appears relatively less compact
than in a hard capsule.

Fijjurc ^. Intrucapsulur (li'M'lopnit'iil. (A) \ I'liser larviio and non-devciopinj; un^s presi'nl in hard capsulu [Hj^ (set Fig. 11 when lived 49 days
after hein^ laid. (K) Veliger larvae and nnn-develiipinj; vans in a hard capsule spontaneously laid hy animal #.' prior to injection experiments.
Calibrations: 5(10 pm.

Induction of Egg-Laying in Chorus g/ganteus

909

'^O

[

Figure 4. Micrographs of capsule walls from hard and soft capsules:
(Al hard capsule [H]^ (see Fig. 1), induced by extract injection. (B)
hard capsule laid spontaneously by animal #3 prior to extract injec-
tion. (C) soft capsule [SJj laid by animal #3 in response to extract
injection. (D) soft capsule laid by animal #4. in response to extract
injection, in experiment conducted 10 days after the experiment illus-
trated in Fig. 1. Laminae of the capsule walls are labeled as follows: 1,
outer lamina: 2, middle lamina; and 3. albumen retaining lamina.
Calibrations: 30 jim for all.

Histology of Responsive and Non-Responsive Recipients

Histological studies of the gonads of the four responsive fe-
males that laid eggs in response to nervous system extracts showed
clearly that three had a ripe ovary (animals I, 2, and 3 in Fig. 1)
while the other was in active maturation (animal number 4 in Fig.
1 ). Six additional females (previously conditioned for reproduction
at the laboratory in Metri) that were not responsive to injections of
CNS extracts were also studied to address their gonadic stages;
three had ripe gonads and three were in active maturation.

DISCUSSION

This article is the first to demonstrate induction of egg-laying
by nervous system extracts in a muricid snail. Eggs were present

in the first induced capsules of responding animals and in all but

2 of 36 induced capsules altogether, a result that is different from
capsules induced by a similar method in Busycim. another neogas-
tropod (Melongenidae). Because C. giganteus laid normal hard
capsules as well as soft capsules, these experiments enabled us to
study the role that the hardening process has on protecting the
eggs, which has not previously been accomplished. Our micro-
graphic studies illuminate the structural differences between hard
and soft capsules.

Although not every extract injection caused the laying of an
egg capsule, the laying that did occur was clearly in response to the
extracts. In Experiment I (Fig. I ), 17 extract injections resulted in
the laying of 13 capsules; in experiment 2. 8 extract injections
resulted in at least 7 capsules. Animals never laid capsules after
control injections within the same time period as after extract
injections. Furthermore, no capsules were laid by animals during
the 10 days following the end of experiment I, showing that spon-
taneous laying was rare for these animals. In the dose-response
experiment, low concentrations of CNS extract failed to elicit lay-
ing.

We hypothesize that the egg-laying factor in C. giganteus CNS
extracts is a heat stable peptide. Retention of activity after boiling
is similar to egg-laying neuropeptides in Aplysia spp., Lymnaea
spp. (both Heterobranchia. traditionally classified as Opisthobran-
chia and Pulmonata. respectively), and in Biisycon spp. (Caeno-
gastropoda) (Kupfermann 1970. Ram 1977, Ebberink et al. 1985).
The egg-laying hormones of Aplysia and Lymnaea are homologous
36-amino acid peptides known as egg-laying hormone (ELH) and
caudo-dorsal cell hormone, respectively (Chiu et al. 1979,
Ebberink et al. 1985). In Busycon, the inducing agent is protease
sensitive (Ram 1977) and approximately the same estimated size
by gel filtration as ELH (Ram et al. 1982). We hypothesize that the
loss of activity of C. giganteus CNS extracts during a 10-min
incubation at room temperature prior to boiling is due to endog-
enous proteases, consistent with preservation of its activity by
addition of the protease inhibitor benzamil.

Despite the similarities in size, protease sensitivity, and heat
stability of the Biisycon egg-laying factor to Aplysia ELH, they
have distinct differences reflecting the divergence of gastropod
subclasses 350— KJO million years ago (Goodman et al. 1988).
Thus, inter-species injection experiments between Aplysia (Het-
erobranchia) and Busycon (Caenogastropoda) do not cause laying
in the other species (Ram et al. 1982). Determination of the pri-
mary structure of the active factors causing laying in caenogastro-
pods would help identify the structural differences that account for
the lack of inter-class activity. Demonstration of the egg-laying
activity of CNS extracts in C. giganteus suggests that such studies
could also be pursued in this species.

The latency of the egg-laying response to CNS extract injection
was generally longer in C. giganteus than in other gastropods. In
experiment 2, in which each C. giganteus was injected with extract
only once prior to observing laying of an egg capsule, the median
latency was 4 h. The shortest latency observed in any experiment
after single injections was about 3 h. The data on latency in Figure
1 are difficult to interpret, since two extract injections were given
prior to the laying of the first capsule. However, by hypothesizing
that each injection elicited the laying of no more than one capsule
[as previously observed in Busycon (Ram et al. I982)|. the laten-
cies between CNS injection and laying are estimated to vary from

3 to 6 h. In compari-son, latencies to lay after hormone injection
were 30 min in Aplysia (Stuart et al. 1980, Strumwasser 1984,

910

Ram et al.

Ferguson et al. 1989), 100 min in Lymnaea (Dogterom and van
Loenhout 1983, Geraerts et al. 1988), and 2-4 h in Busycon (Ram
1977).

The number and presence of eggs in most induced capsules of
C. gigaiueiis differs from Busycon. In Busycon. the initial 5-15
capsules laid in spontaneous egg-laying episodes are devoid of
eggs (Ram et al. 1982). Subsequently laid capsules average ap-
proximately 35 eggs per capsule (Ram et al. 1982). A single ex-
tract injection into Busycon generally elicits only one egg-less
capsule, and repeated injections over a 24-h period are necessary
to obtain capsules containing eggs (Ram et al. 1982). In contrast,
for C. giganteus almost all capsules contain eggs, and the number
of eggs per capsule is usually >I000 (Castillo and Ulloa 1998,
Gonzalez and Gallardo 1999). Thus, the presence of eggs in nearly
all capsules induced by CNS extract in C. giganteus reflects the
normal pattern of egg deposition in this animal.

These experiments demonstrated that normal development can
take place in induced capsules. Trochophore larvae developed in
one soft capsule before development was disrupted by infection,
and veliger larvae were obtained in an induced hard capsule. De-
velopment of only a small percentage of eggs into larvae is usual
for this species, in which a large proportion of eggs serve as nurse
eggs to developing embryos, and eggs in many capsules may not
develop at all (Castillo and Ulloa 1998, Gonzalez and Gallardo
1999). In the present study, in one spontaneously laid capsule only
eight veliger larvae out of approximately 1,500 eggs developed.
Previous studies of C. giganteus reported 7-12% of encapsulated
eggs developed, the remainder being used as nurse eggs (Castillo
and Ulloa 1998, Gonzalez and Gallardo 1999). Arrested develop-
ment has also been observed in capsules collected from the field,
in which up to 45% of the capsules in a spawn exhibited arrested
development (Castillo and Ulloa 1998, Gonzalez and Gallardo
1999). The number of eggs developing per capsule obviously can
vary a great deal in both natural and induced spawn. A challenge
for aquacultural development of this species will be to attain an
optimal ratio of developing eggs to nurse eggs in every capsule.

Capsules laid by C. giganteus in response to CNS extracts were
successfully transported to the VPG for hardening in only a mi-
nority of cases, providing an opportunity to study the effect of
hardening on capsule wall structure and function. Whereas the wall
structure of induced hardened capsules was similar to spontane-
ously laid capsules, the walls of soft capsules were less compact

and/or were missing layers. This difference in wall structure of the
soft capsules may account for their greater susceptibility to patho-
gens. Previous authors have suggested that protection of embryos
from microorganisms is a major function of gastropod capsule
walls (D'Asaro 1988. Garrido and Gallardo 1993, Rawlings 1999)
and that capsules from Nucella emarginata from which two layers
had been mechanically stripped were much more vulnerable to
protists and predators (Rawlings 1994, Rawlings 1995). Compac-
tion or addition of layers to the capsule wall by the VPG may
impart this biotic protection to the capsule.

Application of this method for inducing the laying of egg cap-
sules to culturing C. giganteus may be hindered by the necessity of
producing hard capsules and by the difficulty of ascertaining
which animals will respond. Our histological studies comparing
responsive and non-responsive animals did not reveal any differ-
ences in gonadal maturity. The lack of responsiveness of seem-
ingly mature animals to effective stimuli has been noted in other
molluscs (Ram 1977, Ram et al. 1993). Determining what addi-
tional factors regulate responsiveness to spawning inducers is one
of the most critical unsolved problems in understanding and reli-
ably controlling reproduction in these animals. The low numbers
of developing embryos in this study may also indicate a limitation
in applying these methods to mass-production of snails. Finally,
the high dosage of nervous system extract needed to induce laying
of egg capsules indicates that this could be an efficient method to
obtain more snails only if the inducing substance (presumed to be
an egg-laying peptide hormone similar to those sequenced in other
gastropods) could be synthesized economically for injection into
responsive animals.

ACKNOWLEDGMENTS

This research was supported by NSF grant INT-9724918 to J.L.
Ram, by a Fondecyt grant (1960488) to C. Gallardo, and a
FONDAP grant to J. M. Navarro (P.I.; C. Gallardo, sub-project
P.I.). We gratefully acknowledge the use of facilities and some C.
giganteus from C. Varela and CEACIMA-METRI, Chile and bio-
chemical equipment from Rudolpho Amthauer of the Universidad
Austral de Chile. We are al.so grateful to Cristian Manque and
Rodrigo Merino for valuable help in the experimental studies and
to German Leiva for providing some adult animals for experi-
ments. Dr. Orlando Gairido gave us technical assistance in pre-
paring the microscopical sections of C. giganteus egg capsules.

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Joiinuil ofSlwllfixh Resi'iinh. Vol. 19. No. 2, 913-917. 2000.

EFFECT OF REPETITIVE DYE EXTRACTION OVER YIELD AND SURVIVAL RATE OF THE
PURPLE SNAIL PLICOPURPURA PANSA(GOVhD, 1853)

JESUS EMILIO MICHEL-MORFIN' - AND
ERNESTO A. CHAVEZ O'

* Centra ile Ecologiu Costera
Universidad de Guadalajara
Gomez Farias 82, San Patricio-Melaque
Jalisco 48980 Mexico.

'Ceiuro liuerdisciplinario de Ciencias Marinas- IPN
Playa El Conchalito s/n
Apdo Postal 592. La Paz BCS. 23000. Mexico

ABSTRACT The purple snail is an important economic species because of the dye obtained from it in western Mexico. This dye has
been used since ancient times to color ceremonial dresses purple. Other snails produce dye. but Plicopiiipiira punsa dye extraction was
done without killing the snail. Repetitive dye extraction is possible. The best time between each milking, dye yield versus milking
frequency, and effect on survival was determined by repetitive milking snail groups several times (7. 14, 21, and 28 days). Mortality
in the most frequently milked groups and dye yield reduction occurred (every 7 and 14 days). When milking frequency occurred every
21 days, the best dye yield and 100% survival rate was observed. These results suggest wild populations can be exploited using
optimum extraction schedules, leaving at least 2 1 days between each dye extraction.

KEY WORDS: mollusks. purple snail, exploitation, natural dyes, PHcopurpiira punsa

INTRODUCTION

Several animal and vegetable products have been used for
milennia by different cultures to supply dyes (Baranyovits 1978).
Among all ancient natural dyes, those using rnarine ga.stropods
were the most prestigious, and the textile industry established was
one of the most important and complex in Europe and Mideast
(Koren 1995). Several species from genera Purpura. PUcopur-
pura, Mitrex, and Thais were used to obtain purple and blue dyes
(Baker 1974, Fox 1966, Ghiretti, 1996). These colors have been
symbolically related with important and powerful people.

At the intertidal zone of the eastern tropical Pacific, purple snail
or dye snail Plicopurpura pansa (Gould, 1853; Synonymous: Pur-
pura pansa). is a valuable species because of the fluid produced in
the hypobranchial gland. After secretion, this dye changes from its
initial white color to purple by a chemical reduction. Unlike other
snail dye producers in which it is necessary to break the shell and
to kill the animal to obtain the dye gland, dye extraction from P.
pansa is made by mechanically exciting the snail foot and oper-
culum, so multiple milkings can be made. Friedlander determined
that 12,000 Murex snails were necessary to obtain only 1.4 g of
Tyrian purple dye, after a complicated extraction process. This
explained the rarity and high cost of such dyes in the past (Fried-
lander 1908 in Baranyovits 1978).

Natural colors are again becoming desirable, and their use for
dying fabric is increasing. In the late 1980s on the rocky shores of
the Mexican Pacific coast, a small-scale fishery of purple shell was
developed to support a Japanese market for dye of expensive ki-
monos. However, this fishery was looking for the highest dye
yield, and the time between milkings was not con.sidered; there-
fore, the local purple shell stock was depleted in a short time
(Turok et al. 1988). Since 1988, the Mexican Government has had
P. pansa under special protection (Anonymous 1988, Anonymous
1994). Currently, the purple snail is used only by the Mixteco
people on the coast of Oaxaca to make ceremonial dresses. This

activity has existed from before the time of Columbus (Turok
1996).

To study reproduction (Acevedo 1995) and feeding aspects
(Memije 1994, Montiel 1993, Renten'a 1996), purple snails were
examined under controlled conditions. Rfos-Jara et al. ( 1994) used
tagged wild purple snails to determine dye yield and recovery time
after being milked. They found a relationship between each of
these variables and the shell length, but the effect of repetitive dye
extraction was not established. Gonzalez (1996). through a com-
plex experimental design, tried to establish the best time between
milking; however, his results are not clear.

The aim of this study was to establish the minimum time be-
tween milking without detriment to snail survival. A 3-month ex-
periment with several snail groups and repetitive milking at dif-
ferent times was carried out. The results can be used for the as-
sessment and planning of potential exploitation of this species.

METHODS

Purple snails {n = 110) were collected by hand in June 1997
from the intertidal zone on three rocky shores of the Mexican
Pacific coast (Fig. 1), on the south side of Isia Socorro, Archi-
pielague Revillagigedo, in Cuastecomates Bay, on the Jalisco State
and at Pescadero Point, State of Baja California Sur (BCS). Many
snails from different areas were collected to consider the condi-
tions of several habitats and the possible intrinsic variability of its
population dynatnics.

Snails were transported wet to the Laboratory of Experimental
Biology at CICIMAR, in La Paz BCS, Mexico. Two fiberglass
200-L aquariums were used for the experiment. Each aquarium
had an opening and semicontinuos sea-water-filtered system. 400-
L/h power-head pumps and different size stones were used to
simulate natural substratum and splash condition. The water tem-
perature was the same of adjacent sea (23-26 °C), the salinity
ranged from 36 to 38 %c and a 1 2-h daylight cycle was established.

913

914

MiCHEL-MORFIN AND CHAVEZ O

^^"^^^^^

.2CC;

Figure. 1. Collect sites of purple snails in Mexican Pacific coast.

In the tidal rocky shores, this species preys upon a variety of
other moliusks. invertebrates, and dead animals. In the laboratory,
they were fed daily with fresh squid chunks. Several other kinds of
food were offered during acclimation period, but squid meat was
the preferred one. Water and laboratory temperatures were re-
corded daily and corrected as required. Full aquarium cleaning was
carried out daily.

Before any manipulation, a 2-month acclimation period was
spent before the experiment. Five groups with 20 snails of different
sizes (shell length range: 19.8-61.6 mm) and both sexes were
conformed at random (feniale:male ratio at field, 1 :0.95). All snails
were kept together, and each snail was tagged with a particular
color group and a plastic number on the conch. Snail size and wet
weight were recorded to the nearest 0.1 g through an electronic
caliper and digital scale, and the data were entered into a database.
Each aquarium had 47 snails, two groups each with 20 snails, and
a control group of seven snails. Most of the mortality recorded
occurred after collection and during acclimation.

After the acclimation period, the groups were submitted to a
series of milking, according to an established schedule. For milk-
ing, each snail was taken from the aquarium, excess water was
removed with a towel, and the expulsion of the dye was stimulated
by blowing and slightly pressing on the foot and the operculum.
The dye obtained from each snail was stored individually in Ep-
pendorf vials.

All groups were submitted to an initial milking on 2S August,
and the last milking took place on 21 November. 1997. Subsequent
lo the initial milking, each group was exploited on different dates
according to the 3-inonth period of the experimental design. The
control group was not milked (Gw). One group was milked every
7 days (07), one every 14 days (G14), one every 21 days (G21),
and one every 2H days (G2S).

At each milking, the dye produced by each snail was collected
and the volume measured. To determine the volume of dye, a
regression equation between volume and the weight of the dye was
made, using the data of initial milkings. Later, the volume pro-
duced by each snail was calculated by determining the weight of
the vials and using the regression equation. A daily control of the
mortality in each group was recorded.

After the .'^-month period experiment, the sex of each snail was
determined, stinuiUiting manually the exit of the body from the
shell and then determining the presence of penis or genital orifice.
Although the sex of each of the snails in the experiment was
identified, we decided not to consider this variable within the
analysis, because secondary sexual tlimorphism is not present, and

therefore it is not easy to distinguish the sex of the snails collected
and milked in a commercially exploited stock.

To determine possible differences in the conformation of dif-
ferent groups, an analysis of variance (ANOVA) and Tukey tests
with untransformed data were carried out to compare the shell
length and the wet total weight of the snails (Zar 1996). Confi-
dence limits were prefixed at 95%. The mean volume of dye of all
snails at initial milking was determined, and the anomalies in dye
production for each size group were calculated with reference to
the initial value.

To compare dye production under different milking regimes,
data on dye production was standardized by dividing the volume
produced per snail by the snail length. Normality tests, analyses of
variance, and Tukey test were completed to compare dye extrac-
tion of each group and among different groups (Zar 1996). The
analysis was done using Statistica for Windows 93.

Regression equations were fitted to the data of the shell length
and volume of dye per animal, the frequency of milking, and the
total dye volume by group. Survival data after milking was de-
scribed by the differential equation of numbers of survivors against
milking times. A polynomial regression was used to describe the
relationship between the frequency of milking as a variable de-
pending upon the product of total dye volume and survival.

RESULTS

The mean temperature during the experiment was 24.5 °C in
the aquaria. The characteristics of shell length, weight, and sexual
proportion of each group of snails is shown in Table 1. The mini-
mum length was 19.8 mm, and the inaximum 61.1 mm. The av-
erage shell length for all the groups was .^5.1 mm. The multiple
comparison test does not show significant differences among the
different groups (P > 0.05), in length or in weight. The sexual
proportion ob.served in the different groups approached a 1:1
male — female ratio.

Figure 2 shows the results of the survival rate observed in each
one of the groups subjected to different milking regimes during the
study. Groups with dye extraction at 21 and 28 days showed no
mortality. In contrast, groups with a milking frequency of every 14
davs (G14). had a 95'/c survival rate, and the beginning of mor-

TABLF. 1.

Size, weight, and sexual proportion of snail groups under different
milking regime.

(i roups

Parameters

G7

G14

G21

G28

Gw

Total

Average si/.e''

.^ .'!..'>

.■^6.0

^b.}

.^4.7

.12.4

}f<.\

Minimum si/e"

25. .1

25.0

24.5

27..^

19.8

19.8

Maximum si/e'

.S.V.'?

51.8

61.1

51..^

52.5

61.1

SD in si/e'

X.5

7.8

11.2

7.4

10.1

8.9

Average weight''

7.x

8..^

y.o

6.9

8.1

8.1

Minimum weight''

2.5

2.(1

2..S

2.9

1,4

1.4

Maximum weight''

21.1

22. .1

.^0.(1

16.9

24.2

.10.6

.SO weight''

5.8

6..1

8.9

4.6

7.8

6.7

n females

II

10

9

II

6

47

// males

9

10

11

9

8

47

■' Length si/c, mm.
'' Weight, g.

Effect of Dye Extraction on the Snail

915

CD
<

o

<
>
>

a:

G28

WEEKS

Figure. 2. Percentage of survival for each purple snail group during
milking period.

tality was shown in the ninth week (at the fourth milking). In the
group (G7) with weekly dye extraction, the lowest values of sur-
vival rate (85%) were observed, and snail mortality began at the
sixth week (at the fifth milking). An accidental death of a snail in
the control group was recorded at the second week of the experi-
ment.

Evidence shows the possible existence of a moderate effect of
milking frequency on survival; however, the values of dye pro-
duction analyzed for each group indicate that repetitive dye ex-
traction is important.

The mean volume of dye produced in relation to shell length is
shown in Figure 3. The volume of dye/snail using the data of the
first milking was determined, and an average value of 0.47 mL/
snail was obtained (SE m 0.03). It was not possible to obtain dye
in 1 2 out of 94 snails ( 1 2.7 %). A power regression establishing the
relationship between the length (L) and the volume of dye (D) of
the snails was established, with a correlation coefficient value of r
= 0.64. The equation follows:

D = 0.0003*L-"'

Figure 4 shows the error (observed value minus the mean) with
respect to the average volume of dye for each group of snails at

o
>

z
<

■

1 8 .

n=82

1

1 6 .

14

1.2 .

6

1 ,

06 .

14

06 .

04

30

31

0.2 .

0 ,

LENGTH (mm)

Figure. 3. Mean dye volume and .size of each group, for all snails at
initial milking.

04

03

0.2

01
0
.01
-02
-02 ■

■oa

04

01 ■

0
.01
-0.2 ■
-03 •
-0 4

G7

}''''■)

^mm

iT^^iiiliP^

014

fsm,

f \

:rii

• 1 (

0.4
0 3
02
01
0
4)1
-02
-03
-04

G21

04
03
0:2 •
01 -

G28

WEEKS
Figure. 4. Errors in average yield for each milking and snail group.

each milking. The dye extraction determines the recovery of dye
production. Thus, in group G7. a decrease in the volume of dye
produced is observed; in G21 and G28 by contrast, an increase in
the average production after the second milking is observed.

There are no significant differences among the different groups
at initial milkings (Tukey test, P > 0.05). but significant differ-
ences at the final milkings (Tukey test, P > 0.05) are shown.

If the accumulated total volume obtained in each milking re-
gime is considered, in G7 the maximum dye volume is obtained
after the 3-month period. Although individual extraction and milk-
ing tend to yield smaller volumes, the higher milking frequency
( 12 in total) implies a greater accrued volume. Group G28 shows
the opposite pattern, high dye yields per milking but a smaller
accrued volume (Table 2). This information is relevant if intense
management of the purple snail is planned. However, when the dye
yields along with the survival rate observed during the study pe-
riod for each group (Figs. 5a and 5b) are analyzed, the product of
dye volume by survival (Fig. 5c), shows the maximum yields
would be obtained by milking every 10 days, which assumes the
possibility of a certain mortality induced by the handling of these
animals by the fishers.

Looking for conservative use wherein the lowest niortulily in-
duced by repetitive dye extraction prevails, we conclude the opti-
muin period between each milking is 21 days.

DISCUSSION

The results obtained depart from the implicit assumption of an
adequate conformation of experimental grt)ups, because differ-

916

MiCHEL-MORFIN AND CHAVEZ O

TABLE 2.

Accumulated dye volume (mL) for each snail group during
milking period.

Date

Week G7 G14 G21 028 Gw

5 September
12 September
19 September
26 September

3 October
10 October
17 October
24 October
31 October

7 November
14 November
21 November

Accumulated dye volume

1

2
3

7.7
9.5
5.8

9.5

8.8

O

CO

4

5.8

12.8

8.0

5

6.3

n-1

6

6.6

7.3

9.3

7

4.0

8

5.8

10.7

9.4

9
10

3.3
7.8

7.3

10.8

<

11

4.6

>
>

12

5.4

10.5

13.5

11.9

6.5

73

58.2

42.4

29.4

6.5

ences in mean length or weight were not observed. Equally, the
sexual proportion is about 1:1.

The values observed in this study of dye yield with respect to
size are lower with respect to those cited in the literature on purple
snails with similar size or weight in the wild ( Michel-Morfi'n et al.
in press, Ri'os-Jara et al. 1994), but are coincident with those of
previous laboratory studies (Gonzalez, 1996). This difference
could have two causes. Dye extraction under controlled conditions
allows complete removal of water that is incorporated into dye
from the mantle cavity. In the intertidal zone, this is difficult to
achieve because of the uncontrolled conditions. This may cause
overestimation of dye volume.

In addition, data obtained under experimental conditions can
differ from values obtained under natural conditions, because the
snails were fed with a monospecific diet, in this case squid chunks.
The nutritional condition of experimental snails could vary with
respect to those from the intertidal zone, which have diverse prey.
This could have an effect on dye production. Even with this pos-
sible effect, the inlluence of milk frequency found should be con-
sidered to obtain the optimum dye yield in the field.

The role of dye in snail physiology should be studied in depth
under specific experimental designs, even though it has been men-
tioned to have a feeding role (Bandel 19H7 in Kool 199.3, Ri'os-Jara
et al. 1994), during the experimental period there was no evidence
recorded for the use of dye for this puipose. We did not detect
traces of dye in the food that was withdrawn from the aquaria, but
experimental purple snails were fed with processed food; they did
not have to catch and crack open their own. Castillo-Rodriguez
(1995) mentions two feeding mechanisms, and the dye is possibly
used for immobilizing prey. More needs to be disco\ercd about
this subject.

A .3-month experinicnial period was taken under the assump-
tion that over this time the effect of repeliti\e milkings would be
seen. In addition, it has been the minimum time in which the
Mixteco tribes from Mexican Pacific coast historically devote to
this activity each year (Turok el al. 1988). However, the possibility
ol carrying out repetitive milks over a longer period could be
evaluated to determine the possibility of dye extraction of wild
stock for more than 3 months.

The results suggest the possihiliiy ofexploilnig purple snails ni

y = -2 09x + 87 43
1^ = 0 99

■ ^__^

-—^

B

08

^r^

06

/

04

y = 0 1B7Ln(x) +0 41
f' = 0 90

02

n

1 1 1—

70

60 •

i 50
>

10 •

0

y = 0 0065x' - 0 41 x^ +6 47x +27 3

Figure,
and C)
quency.

5 10 15 20 25 30 35

MILKING FREQUENCY (bays)

5. Relation between A) dye accumulated volume, B) .survival,
the product of volume-survival with respect to milking fre-

the wild assuming that, if a minimum period of 21 days between
each milking is used, no mortality will be induced by handling the
snails. This should be a decisive element for the definition of a
management strategy, given the explicit requirement to avoid
depletion of any exploited stock. Dye extraction must be done
carefully and snails also must be carefully handled to protect them
from the wave action and isolation, putting them on the same sites
from where they were taken to permit them to attach to the rocks
again. The process from the moment each snail is removed,
milked, replaced, and reattached to the grounds takes between 5
and 10 minutes. As long as these simple rules are followed in a
commercial activity, it is possible to have a lou inipacl on the
purple snail.

The Mixteco people have used this resource for direct dying ot
cotton from ancient times, collecting snails along the (^axaca coast
and leaving 4 weeks between each milking from September to
November. There is some evidence that this activity was more
intensive in the past and was done without any adverse effect on
the populations (Turok et al. 1988. Castillo-Rodriguez and
Ame/cua-l-inares 1992).

Although P. pansa is ciiiTenll\ under special protection by
Mexican law. it is importanl lo do lurther research to determine the

r

Effect of Dye Extraction on the Snail

917

real potential of the snail exploitation. Commercial use of purple
snail could be made on specific zones and under particular condi-
tions using local zones where the stock has high densities and
snails are larger. This activity could be profitable to native people.
In areas such as the coasts of Jalisco and Colima. no indigenous
people make use of this resource. There are some groups re-
evaluating the use of natural dyes and interested in the exploitation
of this dye with a high added value; therefore, this activity could
be reactivated.

ACKNOWLEDGMENTS

The authors thank to D. Kosonoy and V. Landa for help in the
field. Isla Socorro Marine Base and Ship C-74 "Lerdo" from the
Mexican Army. In CICIMAR. thanks to Experimental Laboratory
staff for experimental work help and S. Martfnez for statistical
help. E. Glazier edited the English language text. Eniilio Michel-
Morfi'n is grateful for the Conacyt and PlFl-lPN scholarship .
Ernesto Chavez O was partially supported by COFFA- IPN.

LITERATURE CITED

Acevedo. J. 1995. Aprovechamiento del tinte de Purpura pansa en el
Pacifico Mexicano. Informe. CRIP -Pat/.cuaro. INP. Semarnap.
Mexico. 22 pp.

Anonymous. 19S8. Acuerdo Intersecretarial, entre las Secretan'as de Pesca.
Educacion Piiblica, y Desarrollo Urbano y Ecologi'a, con el que se
regula el desarrollo. conservacion y aprovechamiento del caracol Pur-
pura pansa. beneficiando a los niicleos de poblacion que tradicional-
mente lo ban explotado. Mexico. D.F. 13 de Marzo de 1988.

Anonymous. 1994. Norma Oficial Mexicana NOM-059-ECOL-1994, que
determina las especies y subespecies de flora y fauna silvestres ter-
restres, y acuaticas en peligro de extincion. amenazadas, raras y las
sujetas a proteccion especial, y que eslablece especificaciones para su
proteccion. Diario Oficial. Lunes 16 de Mayo de 1994.

Baker. J. T. 1974. Tyrian purple: an ancient dye. a modern problem. Eu-
deavour 13:1 1-17.

Baranyovits, F. L. C. 1978. Cochineal carmine: an ancient dye with a mod-
ern role. Endeavour 2 (2):85-92.

Castillo-Rodriguez, Z. G. & F. Amezcua-Linares. 1992. Biologia y aprove-
chamiento del caracol morado Plicopurpura pansa (Gould, 1 853) {Gas-
tropoda:Neogastropoda) en la costa de Oaxaca, Mexico. An. Inst.
Cienc. del Mar y Limnol. Univ. Nal. Auton. Me.xico\9(2y.223-234.

Castillo-Rodriguez. Z. G. 1995. A study of the feeding mechanism of the
Plicopurpura pansa (Gould. 1853) (Gastropoda: Muricidae) from the
central Pacific coast of Mexico. Abstract. A. Guerra. E. Rolan and F.
Roch. (eds.l. In: Proceedings of the 12lh International Malacological
Congress, Vigo, Spain.

Fox, D. L. 1966. Pigmeniation ofmullusks. pp 249-274. In: K. M. Wilbur
and C. M.Yonge (eds.). Physiology ofMoUusca, vol 2. Academic Press.
San Diego.

Ghiretti. F. 1996. Bartolomeo Bi/io and the rediscovery of Tyrian Purple.
Experientia 50:802-807.

Gonzalez, G. L. 1996. Tiempo de recuperacion del time del caracol Pli-

copurpura patula pansa (Gould, 1853) bajo condiciones de cautiverio,
Tesis Profesional. Lie. en Biologia. Division de Ciencias Biologicas y
Ambientales. Universidad de Guadalajara. Guadalajara. Mexico.

Kool, S. P. 1993. Phylogenetic analysis of the Rapaninae (Neogastropoda:
Muricidae). Malacologia 35(2): 155-259.

Koren, Z. 1995. High-pert'ormance liquid chromatographic analysis of an
ancient Tyrian purple dyeing vat from Israel. Israel J. Chem. 35:1 17-
124.

Memije, S. 1994. Prueba de alimenlacion y dietas en cautiverio a temper-
aturas controladas del caracol Purpura pansa (Gould, 1853). Tesis
Profesional. Lie. en Ecologfa Marina. Universidad Autonoma de Guer-
rero. Mexico.

Michel-Morfin. J. E., S. Reyes-Aguilera. V. Landa J.. & E. Rios-Jara. In
press. Aspectos relativos al rendimiento y foto-oxidacion del tinte del
caracol purpura Plicopurpura pansa (Gould. 1853). Oceanoiogia.

Monliel, E. 1993. Pruebas de alimentacion y dietas en el caracol del tinte
Purpura pansa (Gould, 1853). Tesis Profesional. Lie. en Ecologi'a Ma-
rina. Universidad Autcjnoma de Guerrero. 73 pp.

Renteria, V. J. 1996. Prueba de seis diferentes dietas en el caracol morado
Purpura pansa (Gould, 1853) en cautiverio. Tesis Profesional. Lie. en
Ecologi'a Marina. Universidad Autonoma de Guerrero, Mexico.

Ri'os-Jara, E., H. Leon, L. Lizarraga-Chavez & E. Michel-Morfi'n. 1994.
Produccion y tiempo de recuperacion del tinte de Plicopurpura patula
pansa (Neogastropoda: Muricidae) en Jalisco, Mexico. Rev. Biol. Trop.
42(3):537-545.

Turok. M. 1996. Xiuhquilitl. nocheztli, y Tixinda. Tintes del M(5xico an-
liguo. Arqueolugia Mexicana 12:26-33.

Turok , M., A. M. Sigler, E. Hernandez, J. Acevedo, R, Lara & V. Turcott.
1988. El caracol Purpura una tradicion milenaria en Oaxaca. Direccion
General de Culturas Populares. SEP. Mexico. 166 pp.

Zar, J. H. 1996. Biostatistical analysis. 3rd ed. Prentice Hall. Upper Saddle
River, New Jersey, 662 pp.

Journal of Shellfish Rcsfcinh. Vol. 19, No. 2, 919-925, 2000.

POPULATION PARAMETERS AND DYE YIELD OF THE PURPLE SNAIL PLICOPURPURA
PANSA (GOULD, 1853) OF WEST CENTRAL MEXICO

JESUS EMILIO MICHEL-MORFIN,' ^ ERNESTO A. CHAVEZ,' AND
VICTOR LANDA-

' Centra Interdisciplinario de Cieiuias Mahnas-IPN.

Playa El Conchalito s/n.

La Paz BCS 23000. Mexico
'Centra de Ecologia Costera.

Universidad de Guadalajara.

Gomez Farias 82.

San Patricio-Melaqiie.

Jal. 48980. Mexico

ABSTRACT The purple snail {Plicoimrpum pama) is a conspicuous rocky shore species of the intertidal zone of tropical western
America. It is considered a potential resource due to the dye it produces. Estimates of population density, sex ratio, growth parameters,
mortality, and recruitment were obtained from bimonthly samplings from 1993 through 1995 on a rocky shore of west central Mexico.
A different growth rate between sexes was observed. Mortality coefficients for the population are estimated for the first time.
Recruitment to rocky shores occurs principally from September through March. The life span (longevity) was estimated as 1 1 y. Dye
production related to si/e and sex was determined and is described by an exponential relation.

KEY WORDS: Gustropoda. popiiUirion parameters. Plicopurpura pansa. purple dye. Mexico

INTRODUCTION

Some species of gastropods mollusks like Purpura. Plicopur-
pura, and Murex are remarkable for their ability to produce dye.
From ancient times, many cultures have used these inks to dye
ceremonial dresses, often associated with religious traditions and
power (Baker 1974. Baranyovits 1978, Turok et al. 1988, Clark et
al. 1993, Ghiretti 1996).

One of this group of species, commonly known as purple snail
or dye snail Plicopurpura pansa (Gould 1853, sinonimous: Pur-
pura pansa), is a common inhabitant of rocky shores in the inter-
tidal zone of tropical western America (Fig. I ). Its distribution is
typical Panamic, ranging from Baja California to southern Colom-
bia and the Galapagos Islands (Keen 1971 ).

The hipobranchial gland of the purple snail secrets a fluid that
turns intense purple on the contact with sunlight and air (Rios-Jara
et al. 1994). In contrast with other dye-producing snails, the dye
produced by P. pansa is easy to extract without sacrificing the
snail, so one can obtain multiple milkings.

In Mexico, the Mixtecos, an indigenous people on the Pacific
Coast, use the dye secreted by P. pansa combining it with other
natural inks such as cochineal carmine, from the pearl cactus insect
Dactylupius coccus, and indigo, from plants of the genus Indigof-
era (Turok et al. 1988, Turok 1996). The extraction of purple dye
is made at the shore by dying a wet cotton mop directly with the
dye purple from the snails.

A few previous studies on the yield production of dye as related
to size and sex have been performed (Turok et al. 1988, Alvarez
1989, Castillo-Rodriguez and Amezcua-Linares 1992, Holgui'n.
1993, Rios-Jara et al. 1994). Among these articles, two have at-
tempted to describe growth rate (Turok et al. 1988, Alvarez 1989).
No estimation of natural mortality has been made.

The purple snail can be considered a potential resource because
of the dye obtained from it. In the late 1980s, a small-scale dye
exploitation to support a Japanese market for expensive kimonos
was developed in west Mexico. However, this activity was made

without technical and biological regulations, and negative effects
on the snail population were evident (Turok et al. 1988).

Some studies on population dynamics and the effects of milk-
ing on snail populations are necessary to evaluate the real potential
of this activity. During the last few years, there has been an in-
creased interest in natural dyes, probably because some of the
artificial ones tend to cause sensitivity and toxicity problems (Lo-
pez 1993).

For this reason, the goal of the present study was to determine
population parameters and dye yield as a first step to obtain basic
information about this species. This information, together with
other work now in progress, can help in determining the viability
of a fishery for purple dye and the best way to manage this re-
source.

METHODS

Bimonthly samples to measure density and length frequency
were made from May 1993 to January 1995 (except November
1994) in Bahia de Navidad, Jalisco, Mexico (19°13'29'N and
I04°43'45'W, Fig. 2). A 50-m long by 2-m wide transect was
established in the intertidal zone along the shoreline. All snails
found were recorded, and each snail was milked by blowing and
slightly pressing on the foot and the operculum to stimulate the
expulsion of dye. The volume of dye obtained from each snail, the
length of each shell, wet weight, and sex were recorded. All snails
were released at the same site after sampling.

Length was recorded from the apex to the most distal point of
the anterior siphon canal (Fig. 1, bottom). Because the purple snail
has no sexual dimorphism, the sex of each snail was determined by
manually stimulating the exit of the body from the shell and then
determining the presence of penis or genital orifice.

The length-weight relation was established by fitting a power
regression to the data. Differences in sex ratios were tested by a
slope-comparing t test (Zar 1984). Growth was described by the
von Bertalanffy growth model (VBGM) with the use of the Fisat

919

920

MlCHEL-MORFIN ET AL.

Figure 1. Purple snail Plicopiirpura pansa dorsal view (top left) ventral
view (top right) points of reference to length of shell (bottom center).

software package (Gayanillo et al. 1995) to fit sampling data and
estimate recruitment and tlie fishing mortality coefficient. Esti-
mates of natural mortality coefficient were obtained by using di-
verse methods described by Sparre and Venema (1992) and
Chavez (1995) using a computerized spreadsheet.

The relationship between length and dye volume was deter-
mined by power regression. Sex differences was tested by a slope-
comparing t test (Zar 1984).

RESULTS

A total of 964 snails was sampled in all the study months, 380
females and 288 males. The .sex of 296 snails could not be deter-
mined. Table 1 shows sexual ratios and abundance by month. The
average female:male ratio was 1:0.75. Although sexual proportion
favored females in most months, sexual proportion was between

1:1 in March to 1:0.53 in May. The mean overall density was 1.7
snails/m" (SD = .34). No relation between density and sexual
proportion was found.

The method for sex assessment in the field is not always ef-
fective (only 70% of snails were sexed from 964 collected) be-
cause it is necessary to sex snails one by one under difficult con-
ditions (rocky terrain with much wave splashing) and it must be
done quickly to prevent snail desiccation and death. The method is
particularly difficult with large snails, and maybe this situation
could affect the sexual proportion values.

Lengths ranged between 7.8 and 79 mm (mean = 32 mm, SD
= 12.6) for females and from 9 to 76.4 mm (mean = 28.9 mm,
SD = 8.6) for males (Fig. 3). The lowest modal value, at 14-18
mm, corresponds to recruits to the rocky shore. Snails with sizes
>50 mm are few and are mainly females.

Estimates of condition factor or a value (.0003 and .0002) and
slope value p (2.85 and 2.9) for females and males suggest iso-
metric growth (Fig. 4). A good fitness to length-weight power
regression is shown for both sexes (/' of 0.94 for females and 0.90
for males). Slopes were significantly different between sexes (P <
.02).

The bimonthly length-frequency distributions for each sex, as-
ymptotic length (L^), and growth coefficient estimate (K) were
calculated (Table 2). There is a different growth rate between
sexes. Females have a higher annual growth rate (K = .27) and a
higher L^ value (110 mm). These values are consistent with the
information obtained from the length-frequency histograms for
each sex.

For the estimation of L^, the Fisat package contains a routine
with the Powell and Whetheral method, and another one with the

19°15"

Navidad Bay

Barra de Navidad

19°10'

104°52'

Coco Beach
I 104°4r

Figure 2, Sliidv arcii. liahia de Navidad, Jalisco.

I

Population Parameters of Purple Snail

921

TABLE 1.

Sex ratio and density of Purple snail Plicopurpura pansa in the
Pacific Coast (May 1993 through January 1995).

Month

May 1993
July 1993
September 1993
November 1993
January 1994
March 1994
May 1994
July 1994
September 1994
January 1995
Mean

Sex Ratio {%)

Females

Males

65

35

58

42

57

43

54

46

58

42

50

50

59

41

57

43

55

45

56

44

57

43

Density
Snails/in'

2.0
1.3
2.0
2.1
2.0
1.5
1.5
1.2
1.3
1.5
1.7

Shepherd method and ELEFAN to estimate K values. The foirner
one provided a better estimation (see Gayanilo et al. 1995 for
details). In Figure 3. the score function a.xis shows the best fit of
the Shepherd function (Pauly and Arregui'n-Sanchez 1993).

Other values were obtained using the Munro and Gulland and
Holt methods in the Fisat package and are consistent with our
estimates done by tagging methods for both sexes together, where
K = .26 and L^ = 1 10 (n = 9). Figure 6 shows the goodness of
fit of the growth estimates of the von Bertalanffy model (VBGM)
for each sex. In addition, through an empirical relation between
longevity and growth rate (K), an estimate of life span as 3/K,
ranging from 11 to 13 y, was determined.

The snail population of the rocky shore at the study site is not
exploited, hence, it is valid to assume that total mortality (Z) is
equal to the Natural Mortality (M). For this reason, several meth-
ods to get estimates of coefficients M and F were considered and
tested. Several mortality estimates are shown in Table 3. ranging
from 0.21 (y"', where K = M. according to Chavez 1993) and
1.47 as given by the catch-curve method, another routine in the
Fisat software package. Most estimated values range between 0.2 1
and 0.47, except those obtained by the Jones and van Zaiinge and
catch-curve methods.

Our field observations show the reproductive season occurs

FEMALES

..BnSm ^ll"^ I

35
O 30

S ^

Z3 20

t 10 -
5-
0 -

,, ,r-rr;:^:

males
- - lu-n-. , n 1=1

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88

Length (mm)

Figure 3. Length-frequency histograms of male and female purple
snails.

Males
n=280

Length (mm)

Figure 4. Length-weight relation for male and female purple snail
Plicopurpura pansa in Bahia de Navidad, Jalisco.

between January and May and is characterized by snail couplings
and clutches of egg capsules in rock crevices. Our own laboratory
observations suggest that the time of larval development inside the
capsule is about two to three months. Later, a planktotrophic larval
stage hatches, whose time length is not known, but it is suspected
that it may take about 6 mo.

In our data series, recruitment occurs from July 1993 to May
1994. A second period of recruitment was seen from September
1993 to January 1993. but this could start eariier (November 1994
was not sampled. Fig. 7). The minimum size observed was 7.8 mm
and the maximum size was 20 mm. The mean length of recruit-
ment to the rocky shores is 15 mm (SD = 3.05). From this data
and our growth parameter estimates, age group I (recruit size) must
be about 1 y after hatching.

Dye yield increased exponentially with length for both males

TABLE 2.

Growth parameters (L=c, K, to, W^c), and length-weight parameters

(a, P) estimated for purple snail Plicopurpura pansa females and

males using the Fisat software package."

Attributes (Units)

Female

Male

Lx (mm)
K(y-')

Wx (g)

110

.27
198
-0.04

0.0003

2.85

102

.21
134
-0.04

0.0002

2.90

' See Gayanilo ei al. (1995) for details.

922

Michel-Morfi'n et al.

FEMALES

i

8 08

f\^

g 06

N \

o

C 0,4

M \

"^ 02

1 1

^ ^^ .

0 -1

-t 1 1 1 1

12

i

CD

8 0.8

A

§0.6

11 / V

_^ .

^_^^

^0.

\ /-^

3

\ /~N /

0,2

\ / ^'^

0

1 ,

0,1 0,2 03 0,4 05 06 07 0,8 0.9 1

Growth-rate value K (year)

Figure 5. Estimates of growtli rate value (K) by Shepherd method for
females and males.

and females (Fig. 8, Table 4). The best comparing slopes did not
show significant differences between sexes (P > A). However, the
large variation in dye produced is evident, especially in large sizes
(>50 mm). This variation may be related to the use of dye by the
snail. The mean dye yield was 1.88 mL/snail (SD = 1.69) for
females, 1.23 mL/snail (SD = .85) for males and 1.81 mL/snail
(SD = 1.6) for all the snails sampled (female, male, and unsexed
specimens).

Females

75

E

E 50

ii 25

1993

1994

1995

Males

75

50

a, 25 ■

0 L

1993 1994 1995

Figure 6. (irowth curve of female and male purple snail PtUnpurpiira
pansa In liahia de Navldad, Mexico for years 199.^ through 1995.

TABLE 3.

Estimates of natural mortality (M) for purple snail Plicopurpura
paiisa population using several methods.

Estimates of Natural Mortality (M)

Method

Females

Males

Alagaraja

.27

.21

Ault and Erhardt

.36

.27

Beverton and Holt

.47

.38

Catch Curve

1.51

L79

Chavez

.27

.21

Hoenig

.39

.36

Jones and Van Zalinge

1.43

L45

Rickther and Efanov

(modified)

..^9

.36

=5

o

UJ

tr

LL

o

o
<

H

z

UJ

o

100 -

I

50 (

too

50
0 ■
100
50
0 •
100 ■

»t

May-93

Jul-93

— Sep-93

Jan-94

Mar-94

May-94

Jul-94

Sep-94

Jan-95

2 4 6 8 10 12 14 16 18 20

LENGHT (mm)
Figure 7. Bimonlly percentage of recruitment for purple snail.

Population Parameters of Purple Snail

923

Females
n=63

Both Sexes
n=124

40 50

Length (mm)

Figure 8. Length-dye yield relation of female, male, and unsexed
(both) snails Plicopurpura paitsa.

TABLE 4.

Estimated power regression values (Y = aS'') between snail size (S,
mm) and dye yield (Y, mL) for purple snail Plicopurpura pansa.

Attribute

Females

Males

Total"

a

.0001

.0005

.0002

b

2.43

2.06

2.38

r"

.52

.25

.52

n

6.^

38

124

" Total includes female, male, and unsexed snails.
DISCUSSION

To compare the purple snail with other mollusks, in Table 5
estimates of growth parameters (Lac and K of the VBGM) for
several species of gastropods were compiled. These parameters
were compared among gastropods following the methods of Pauly
and Binohlan ( 1966). Data of L^ and K for those species and those
of the purple snail were plotted in auximetric grids shown in Fig-
ure 9; in addition, L^ and longevity (as 3/K) is also displayed in
Figure 10.

These comparisons show that the purple snail displays a quite
different strategy from other species because it has a slow growth
rate and is small compared to other species. Although it is small,
it lives longer than the others, which could be a result of its
predatory activity and mobility in the very limiting environment
of the intertidal zone, where animals with a small size may have
a better chance of survival struggling against the impact of
waves.

On comparing our own results with other growth estimates by
graphic methods (Battacharya's). the results are different. K values
obtained seem to be low (.069 and .088 for females, after Turok et
al. 1988, Alvarez 1989). These values suggest great longevity and
are in contrast with estimates obtained by tagging methods by the
same authors (2 mm/mo). However, L^ estimates are similar to
our estimates.

TABLE 5.
Estimates of growth parameters (L, K, and t,,! for the purple snail and several other species of gastropod mollusks.

Common Name

Species

L=o

(mm)

K

Source

Panoclia

Loco

Blue abalone

Asrraea undosa
Concholepcis concholepns
Haliotis fiilgens

Queen Conch

Purple

Stromhiis gigas

P. pansa
Males
Females

103

1.50

189

170

183

175

177

190.2

182.6

187.1

179.6

126

260

201

296

318

102
110

0.12 — Cupul-Magafia and Torres-Moye (1996).

0.32 — Stolz and Perez (1992).

0..34 — Guzman del Proo et al. (1976).

0.36 -0.05 Guzman del Proo et al. ( 1 980).

0.38 — Shepherd et al. (1991).

0.24 -0.43 Turrubiates and Castro-Ortiz (1992).

0.28 -0.24 Turrubiates and Castro-Ortiz (1992).

0.37 — Shepherd and Turrubiates (1997).

0.36 -0.36 Shepherd and Turrubiates (1997).

0.35 — Shepherd and Turrubiates (1997).

0.29 -0.78 Shepherd and Turrubiates (1997).

1.38 -0.07 Shepherd and Turrubiates (1997).

0.51 0 Randall (1964).

0..59 0 Berg (1976).

0.42 -0.05 Berg and Olsen (1989; after Alcolado. 1976).

0.38 -0.08 Hesse (1976).

0.21 -0.04 This paper

0.27 -0.04 This paper

924

MiCHEL-MORFIN ET AL.

1.4

o

1.2

1

0.8

0.6

0

o

0.4

P. pansa

S 6^

o

o

0.2
0

u

dP

o

2.2 2.4

Asymptotic length (log L)

2.5

Figure 9. Auximetric grid showing growth performance expressed as
the parameters K and L^ of the VBGM, of the purple snail (P. pansa)
as compared to the same parameters for the blue abalone [Haliotis
fiilgeiis), the loco iConcholepas coiicholepas), the queen conch {Strom-
bus gigas), and the wavy turban {Aslraea iindosa).

2.2 2.4

Asymptotic length (log L)

Figure 10. Same as Figure 9, where L=c and longevity (determined as
3/K) of the purple snail iP. pansa), are compared to the same param-
eters of the blue abalone {Haliotis fiilgens), the loco iConcholepas con-
cholepas), the queen conch (Sirombus gigas), and the wavy turban
{Aslraea iindosa).

Though several methods are available and useful (Devillers et
al. 1998), we used the VBGM to compare our estimates with
previous works on the purple snail and with other gastropods.
Moreover, in this generally applicable model, parameters have
biological significance because they are based upon metabolism,
not only on mathematical aspects (Bustos et al. 1986).

The mortality of the purple snail is evaluated here for the first
time. After mortality estimates were found by several methods,
ranging from 0.21 to = 0.5, it led us to believe the values obtained
by the catch-curve and the Jones and van Zalinge methods are
probably overestimates because of reasons intrinsic to each
method. These differences could be caused by changes in spatial
distribution of snail population, which are reflected as changes in
length-frequency distributions and considered as mortality by the
catch-curve and by the Jones and van Zalinge methods.

Sexual proportion and density could be affected by changes in
spatial distribution of the purple snail. In the laboratory and in the
field, we observed some changes in relation with reproductive
success — aggregations of males around a female — however, in this
work no gradient by size was observed and the sampling method
was not specific for spatial distribution determination.

The information in this work on the population dynamics and
dye yield of the purple snail can be considered as baseline data and
a first step to answer the question whether a fishery for purple dye
extraction can be established or not. Moreover, this allows us to
look for specific research required in distinguishing critical points
in this activity and about the snail population. Questions as to the
effect of repetitive dye extraction over dye and survival rate, or
physiological role of the dye. should be studied. With this infor-
mation, recent approaches in fishery biology and resource man-
agement, i.e., simulation models, could be used to assess the best
management strategies of this ancient resource from a modem
viewpoint.

ACKNOWLEDGMENTS

The authors wish to thank D. Kosonoy and Luis Gonzalez from
Centre de Ecologi'a Costera, Universidad de Guadalajara, for their
assistance in field work. We also thank two anonymous reviewers
for their helpful comments. Thanks to E. Glazier for editing the
English-language text. E. Chavez was partially supported by CO-
FAA-IPN and J. E. Michel-Morfin by CONACyT and by PIFI-
IPN.

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Jourmil i)f Shellfish Research. Vol. 19, No. 2. 927-931. 2000.

TECHNIQUES FOR ASSESSING REPAIRED SHELL DAMAGE IN DOG COCKLES

GLYCYMERIS GLYCYMERIS L.

KIRSTEN RAMSAY* and CHRISTOPHER A. RICHARDSON

School of Ocean Sciences,
Universit)' of Wales Bangor.
Menai Bridge. Anglesey
LL59 5EY. United Kingdom

ABSTRACT Three techniques for assessing repaired shell damage in dog cockles Glycymeris glycymeris (visual assessment, x-rays,
and shell sectioning) were examined for objectivity and repeatability. Visual assessment of the number of scars was found to be
inaccurate and highly subjective. Image analysis of x-rays suffered from inconsistencies in film development and image quality.
However, this technique provided an estimate of the area of the shell affected by scarring, rather than simply a record of the number
and/or severity of scars. Examination of shell cross-sections provided the most objective and repeatable technique, although the
methodology had a number of disadvantages as it was time consuming and scar severity could be underestimated depending on the
position of the line of section through the shell. This technique also offers the opportunity to date the formation of the scars using
internal annual growth lines.

KEY WORDS: Shell damage, Glycymeris glycymeris. x-rays, shell sections, visual assessment

INTRODUCTION

Repaired shell damage, or scarring, in tnolluscs has been used
to provide information about a range of biotic and abiotic distur-
bances. In this paper scars are defined as indentations in the shell
surface where the shell margin has apparently been chipped and
subsequently repaired. In gastropods, shell damage has often been
used to infer predator activity both in modem populations and
those from the palaeontological record (Vemieij et al. 1981. West
et al. 1991. Cadee et al. 1997). Several studies have investigated
the role of fishing disturbance by towed demersal gears (trawls or
dredges) in causing non-lethal shell damage in both gastropods and
bivalves (Caspar et al. 1994. Witbaard and Klein 1994. Mensink et
al. 2000. Ramsay et al. 2000). These studies have suggested that it
may be possible to use repaired shell damage as an indicator of
fishing intensity.

If repaired shell damage is to be used to infer levels of histori-
cal disturbance (either natural or anthropogenic), a reliable method
for quantifying this damage is required. Previous investigators
have largely relied on a visual assessment of shell damage and this
technique has been successfully used for gastropods (Preston et al.
1993, Cadee et al. 1997, Mensink et al, 2000). For the bivalve
Glycynteris glycymeri.'i (L.) however, visual assessment presented
difficulties, as minor scars could not be readily distinguished from
marks of annual origin in an objective manner (Ramsay et al,
2000),

G. glycymeris is a largely infaunal bivalve and is commonly
found in gravelly sediments, although animals are also found in
muddy and sandy sediments (Tebble 1966), The depth to which
these animals bury appears to vary according to the substratum
type, with the deepest depths of several centimeters being reached
in gravel (Ansell and Trueman 1967). Shells of G. glycymeris
often exhibit signs of repaired damage and Ramsay et al. (2000)
found a correlation between the occurrence of scarring and tlshing
effort around the Isle of Man. Irish Sea. whilst Steingrfmsson
(1989) suggested that scars may be caused by unsuccessful preda-

*Current address: Countryside Council for Wales. Plas Penrhos. Ffordd
Penrhos. Bangor, Gwynedd LL57 2LQ, UK; e-mail k.ramsay@ccw,gov.uk.

tor attacks or storm damage. In this paper we have compared the
accuracy and repeatability of three methods for assessing shell
scars in G. glycymeris: ( 1 ) visual assessment, (2) image analysis of
x-rays, and (3) examination of shell cross-sections,

MATERIALS AND METHODS

A sample of 40 live G, glycymeris was collected from a site off
the East Coast of the Isle of Man (water depth of 50 m. sediment
of mainly gravel and coarse sand) in October 1997. Repaired shell
damage (scarring) was assessed in dry. clean G. glycymeris shells
(shell height [maximum measurement from the dorsal to the ven-
tral edge] 4-5 cm) from which the periostracuni had been removed
by gentle brushing.

Visual Assessmenl

A six-point damage scale (Table 1 ) was used by two assessors
(with prior experience of working with G. glycymeris) and three
small groups of up to five students to categorize damage in the
shells. Agreement between recorders was analysed using a gener-
alization of Cohen's kappa statistic (Fleiss 1971, Banerjee et al
1999),

Image Analysis ofX-Rays

Shells were x-rayed (height of the x-ray source above the shells
75 cm, power and exposure of 60 kV for 0,04 sec), 20 shells each
time on a photographic filin measuring 30 x 24 cm. One shell was

TABLE 1.

The scale used for the visual assessment of shell scars.

Score Description

0 No scars

1 Very mild damage, e,g. 1 small scar

2 Mild damage, 2 or more .small scars

? Moderate damage, 1 larger scar, possibly also small scars

4 Moderate/severe damage, several large scars

."i Severe damage, lots of large scars or large chunks missing

927

928

Ramsay and Richardson

Figure 1. Shell x-rays. (A) Area of repaired damage indicated by arrow. (B) Poor resolution created dilTiculties in distinguishing areas of
repaired damage. Scale bar = I cm.

included on both x-ray sheets to allow calibration in the case of
inconsistencies in film development. The damaged areas of the
shell (scars) (as seen in visual inspections) appeared as darker
areas on the x-ray (Fig la). These x-ray images were captured as
digital images and each image was examined using image analysis
software {Sii-iiia Scan. Jandel Scientific). Possible scars were iden-
tified by eye and greyscale measurements (a measurement of dark-
ness/lightness with a scale of 0 (nearly black) to 255 (nearly white)
IJandel Scientific Software. I995|) were taken along a line of eight
points within the scar and eight points either side of the scar (Fig
2). Wherever the difference between the average greyscale values
(within the scar and outside the scar) exceeded 15. the area that
was as dark or darker than the within-scar average was measuretl
The surface area of the shell was also determined using image
analysis and the proportion of the shell that was damaged calcu-
lated.

Figure 2. Procedure for deterniinini; shell scars from x-rays. The area
was considered to be a scar if the difference between the averajjc
greyscale values within the scar (central line of dots! and outside the
scar (outer two lines of dots! exceeded 1.^. .Scale bar = I cm.

Examination of Cross-Sections

Shells were embedded in resin and sectioned using a diamond
saw along a line from the umbo to the centre of the ventral edge.
The section containing the posterior portion of the shell was

Figure i. Acetate peels of shell scars viewed in cross-section. Line(sl is the
measurement of the scar depths. .\ was estimated to be 19-years-old »ilh
a scar al the age of y-years-old; and B is 19-ycars-()ld, scars at !•>. IS, and
17 ^ears ( 17- and 18-\ scars indicated by arrows). Scale bar = 0.5 mm.

Assessing Shell Damage

929

ground smooth, polished, and etched tor 3 min using 0. 1 M hy-
drochloric acid. Acetate peel replicas were prepared of the cross-
sectioned surface (Kennish 1980) to allow microscopic examina-
tion of shell damage and internal growth lines (Fig. 3). Repaired
shell damage was visible in these acetate peels as an indentation in
the normal growth line of the outer shell surface (Fig 3).

Since the shell was only sectioned along the line of maximum
growth, estimates of shell damage through this single cross-section
might lead to scars being missed if they occurred elsewhere around
the shell margin. However, it seems likely that severe damage to
one part of the shell edge would result in a growth disturbance
across the entire shell margin. To test this hypothesis, a sample of

bJ^^H

H^l

t-i

Figure 4. Photographs of shells with repaired damage showing the two lines of sectioning through the most severely damaged and least damaged
areas. Scale bar = 1 cm. The corresponding cross-sections are shown to the right of the shell photographs. Scale bar = 0.5 mm. Each figure
corresponds to the following shell number on Table 2: A. Shell 1; B, Shell 3: and C, Shell 5.

.

930

Ramsay and Richardson

10 live animals (23-36 mm shell height) that had visible chips
around the shell edge were selected from a sample collected by
dredging. These shells were placed in a laboratory upwelling sys-
tem (Spencer 1988) for 9 mo to allow the animals to grow and
repair the shell damage. Shells were cleaned and dried, photo-
graphed (to record the appearance of the shell surface), embedded
in resin, and sectioned along two directions from the umbo to the
shell margin. The first section was placed through the centre of the
most severely damaged area, whilst the second section was placed
through an apparently undamaged region (Fig 4). Acetate peel
replicas of the two polished shell sections were prepared and the
dimensions of the damage were compared.

RESULTS

Visual Assessment

Researchers found it extremely difficult to quantify scars
through a visual assessment, as it was difficult to distinguish be-
tween smaller scars and annual growth lines on the shell surface
and this led to the development of the damage scale. However,
visual assessment using the damage .scale in the 40 shells was still
highly variable with only one shell receiving the same score in all
five assessments and two shells being given the same score by four
out of the five assessments. The kappa statistic returned a value of
0.07 (perfect agreement = 1, a random set of numbers = 0, and
agreement between 3 assessments for every shell = 0.16). When
analysis of shell damage was restricted to the assessments of the
two researchers who had substantial prior experience of working
with G. glycymeris shells, the kappa statistic was still low (0.25;
52% of shells received the same score from both researchers).

Image Analysis nfX-Rays

The x-ray images generally had poor resolution or "blurring" of
the shell margins (Fig. 1, a and b). For this reason, the number of
scars identified by the x-ray method was generally low (mean 1 .2
± 0.2 scars per shell). The black-and-white contrast appeared to
vary between the two sheets, probably as a result of inconsistencies
in x-ray development. However, the inclusion of a "standard" shell
on each sheet helped to minimise, but not eliminate, these prob-
lems.

Examination of Cross-Sections

Shell damage was evident in many (95%) of the 40 acetate
peels of shell cross-sections as a break in the continuity of the shell
edge (Fig. 3). The depth of these breaks, or scars, could easily be
measured and these nieasuremcnls proved to be repealable be-
tween recorders with a small degree of error (the average error
between two recorders who measured 30 scars was 16 \xm (scar
sizes ranged from 25 -1.150 p.m|). The average number of scars
per shell was 2.4 ± 0.6 for scar sizes greater than 250 \xm and 3.2
± 0.8 for scars between 1 25 and 250 jjim. Thus more scars were
identified using this methodology than using the x-ray technique.

The experimental shells from the upwelling system had re-
paired the shell margin chips by the end ol' the 9-mo experimental
period. The repaired shell damage was apparent in cross-sections
taken along two directions, including the one through the region
that appeared undamaged from a visual inspection. However, the
scars from the visually undamaged area were smaller than those of
the damaged section (Table 2 Fig 4).

TABLE 2.

Depth of scars from shells damaged by a dredge. Sections were cut

through the area of worst damage and an apparently undamaged

area (from visual inspection). See Figure 4 for photographs and

acetate peels of shells L 3, and 5.

Scar Depths ((im)

Shell no.

Worst damage

Least damage

I

2
3
4
5
6
7
8
9

in

175
250
2.350
200
725
175
550
150
100
650

150
225
675
100
300
150
250
150
50
75

DISCUSSION

The results demonstrated that analysis of acetate peels of shell
cross-sections was the most reliable and repeatable method for
quantifying repaired damage in the shells of G. glycymeris. Shell
damage gives rise to a recordable growth anomaly around the
entire shell margin, although the anomaly was most pronounced in
the area where damage appeared most severe from an external
visual inspection. The most accurate method for assessing the
frequency of shell damage might be to produce multiple cross-
sections through each shell (although this would be highly time
consuming [about a 35-min preparation per section]). This tech-
nique also offers the opportunity to date the formation of the scars
using the internal annual growth lines (Witbaard and Klein 1994,
Ramsay et al 2()()0).

X-rays also proved to be a potentially useful method of assess-
ing the frequency of shell scars, although this method tended to
pick up fewer scars in comparison with the analysis of cross-
sections. However, the technique has the advantage of analysing
the entire shell rather than a single cross-section and estimates
shell damage as a percentage of the total shell surface area, unlike
the acetate peel technique, which can only quantify the number of
scars. The method is also non-destructive, which may be useful
when analysing valuable palaeontological or archaeological
samples. X-rays of bivalve shells have also been used successfully
to assess the extent of infestation by shell-boring parasites (Am-
bariyanto and Seed 1991).

Visual assessment of shell .scars in G. glycvmeris tended to be
unreliable with poor repeatability compared with the other two
techniques. However, it is possible that visual inspection may be
useful if an attempt were made to differentiate between causes of
scarring (e.g. predator attacks, fishing disturbance, and/or storm
damage) from the appearance of scars, although this has proved
difficult to dale (unpublished data).

It appears thai microscopic cxaminalion of shell cross-sections
is the most reliable method for quantifying repaired shell damage
in O'. glycymeris and this could possibly be used in conjunction
with a visual assessment to provide additional information about
the nature of the scars. The combination of x-rays and image
analysis may al.so be a technique worthy of further development
and could afso be used in conjunction with the analysis of shell
cross-sections to allow an initial count of the number of .scars fol-
lowed by a measurement of the area covered by the more severe .scars.

Assessing Shell Damage

931

ACKNOWLEDGMENTS

This study was funded by the Ministry of Agriculture Fisheries
and Food. Fisheries Division III. project code MF07I4. We would

like to thank Dr. Rohan Holt for suggesting the use of x-rays. Dr. hin
Lucas for his help with the image analysis, and Samantha Vize and
Jessica Taylor for their help with shell sectioning. We also thank the
Four Crosses Veterinary Practice. Menai Bridge for the shell x-rays.

LITER.4TURE CITED

Ambariyanto. Seed. R. 1991. The infestation of Mytiliis ecliitis Linnaeus by

Polydora ciliahi (Johnston) in the Conwy estuary. North Wales. J.

Molliis. Stud. 57:413-t24.
Ansell. A. D. & E. R. Trueman. 1967. Observations on burrowing in G/v-

cymeris glycymeris (L.) (Bivalvia. Arcacea) J. Exp. Mar. Biol. Ecol.

1:65-75.
Banerjee. M.. M. Capozzoli. L. Mcsweeney & D. Sinha. 1999. Beyond

kappa: a review of interrater agreement measures. Can. J. Staii.si. 27:

3-23.
Cadee. G. C. S. E. Walker & K. W. Flessa. 1 997. Gastropod shell repair in

the intertidal of Bahia la Choya (N. Gulf of California). Pultwogeogi:

Paliiemiimawl. Pahieoecol. 136:67-78.
Fleiss. J. L. 1971. Measuring nominal scale agreement among many raters.

Psychol. Bull. 76:378-382.
Caspar. M. B.. C. A. Richardson & C. C. Monteiro. 1994. The effects of

dredging on shell formation in the razor clam Bp.s/.v silicjiia from Bar-

rinha. southern Portugal. J. Mar. Biol. A.ssoc. UK 74:927-938.
Jandel Scientific Software. 1995. SigmaScan and SigmaScan Pro User's

Manual. Jandel Corporation. USA.
Kennish. M. J. 1980. Shell microgrowth analysis: Mercenaria mercenaria

as a type example for research in population dynamics, pp. 255-295.

In: D C. Rhoads & R. Lutz (eds.). Skeletal Growth of Aquatic Organ-
isms. Plenum Press, New York.
Mensink, B., C. V. Fischer. G. C. Cadee. M. Fonds & C. C.Ten Hallers-

Tjabbes. 2000. Shell damage and mortality in the common whelk Buc-
cinum undatum. caused by the beam trawl fishery. J. Sea Res. 43:53-64

Preston, S. J., D. Roberts & W. 1. Montgomery. 1993. Shell scarring in
Calliostoma ziziphinwn (Prosobranchia. Trichidae) from Strangford
Lough, Northern Ireland. J. Mollus. Stud. 59:21 1-222.

Ramsay, K., M. J. Kaiser, C. A. Richardson. L. O Veale & A. R. Brand.
2000. Can shell scars on dog cockles [Glycymeris glycymeris L.) be
used as an indicator of fishing disturbance? J. Sea Res. 43:167-176

Spencer. B. E. 1988. Growth and filtration of juvenile oysters in experi-
nienlal outdoor pumped upwelling systems. Aqiiacidnire 75:139-58

Steingrfmsson, S. A. 1989. A comparative ecological study of two Gly-
cymeris glycymeris (L.) populations off the Isle of Man. PhD Thesis,
University of Liverpool. United Kingdom.

Tebble, N. 1966. British Bivalve Shells. British Museum (Natural History),
London. 212 pp.

Vermeij. G. J.. D. E. Schindel & E. Zipser. 1981. Predation through geo-
logical time: evidence from gastropod shell repair. Science 214:1024-
1026.

West, K., A. Cohen & M. Baron. 1991 . Morphology and behaviour of crabs
and gastropods from Lake Tanganyika. Africa: implications for lacus-
trine predator-prey coevolution. Evolution 45:589-607.

Witbaard, R. & R. Klein. 1994. Long-term trends on the effects of the
southern North Sea beamtrawl fishery on the bivalve mollusc Arctica
isUmdica L. (Mollusca. bivalvia). ICES J. Mar. Sci. 51:99-105.

I

i

I

i

Joiinuil i>f Shclltlsli Rrscinli. Vol. 14. No. 2, 933-942. 2000.

POPULATION BIOLOGY OF GAPER (HORSE) CLAMS, TRESUS CAPAX AND T. NUTTALLII,

IN SOUTHERN BRITISH COLUMBIA, CANADA

A. CAMPBELL AND N. BOURNE

Science Branch,
Fisheries and Oceans Canada.
Pacific Biological Station.
Nanaimo. British Columbia,
Canada V9R 5K6

ABSTRACT Growth and mortality rates, and densities of gaper (horse) clams, Tresiis capax and T. nuuallii. were estimated from
several areas in southern British Columbia (B.C.). Growth rates of T. capax from low intertidal and subtidal zones were greater than
those from mid intertidal zones reported in other studies in B.C. Growth rates for T. niimillii varied between areas. Mean natural
mortality rates for adult T. capax were .15 to .20 from Seal Island, and for adult T. nultallii were .44 from Ritchie Bay and .20 from
Klaskino Inlet. Densities and biomass of T. capax were higher in the low intertidal zone than the subtidal zone at the Seal Island study
area and for T. initiallii in the subtidal at Ritchie Bay. The relati\e abundance of T. capax and T. luiiiallii in the subtidal varied
considerably between locations.

KEY WORDS: horse clam, fat gaper, Tresiis capcLX. Pacific gaper. T. nultallii. growth, mortality, density

INTRODUCTION

The two horse clam species, the fat gaper, Trcsii.s capax (A, A.
Gould 1850) and Pacific gaper. T. nultallii (Conrad 1837) (Bi-
valvia: Mactridae), are found from Alaska to Californiu and are
common in mud, sand, and gravel substrates along British Colum-
bia (B.C.) coastal waters (Quayle 1960. Bernard 1983. Coan et al.
2000). Tresiis capax is found from mid-intertidal beach levels to
subtidal depths of at least 20 m (Bourne and Smith 1972b. Haderlie
and Abbott 1980). whereas T. mittallii is found from the low
intertidal to subtidal depths of 50 m (Haderlie and Abbott 1980.
Campbell et al. 1990. Coan et al. 2000). Both species are com-
mercially harvested subtidally (at depths >3 m) in B.C; this mod-
est fishery (started in 1979 with landings of 37t and ranged from
355t in 1987 to 3t in 1995) has been liinited due to lack of markets
for the processed product and lack of stock assessment (Harbo and
Hobbs 1997. Lauzier et al, 1998). Although industry has requested
expansion of this fishery, management has resisted increase in
exploitation until further information on abundance and biology of
these two species was obtained (Lauzier et al. 1998). Some infor-
mation is available on the biology and abundance of T. capax and
T. mittallii in B.C. (Quayle 1960. Quayle and Bourne 1972.
Bourne and Smith 1972a. Bourne and Smith 1972b, Bourne and
Harbo 1987, Campbell et al. 1990, Bourne and Cadwell 1992, Rice
et al. 1993, Bourne et al. 1994). Data on horse clam biology from
other areas come mainly from subtidal populations of T. niittallii
(Harrington and Griffin 1897. MacGinitie 1933. Swan and Finu-
cane 1952, Fitch 1953. Addicott 1963. Armstrong 1965. Pohlo
1964, Pearce 1965, Smith and Davis 1965. Stout 1967, Stout 1970,
DesVoigne et al. 1970. Laurent 1971, Clark 1973. Clark et al,
1975. Kvitek et al. 1988) and mostly from intertidal and a few
subtidal populations of T. capax (Pearce 1965. Pearce 1966. Reid
1969, Machell and DeMartini 1971, Stout 1967. Stout 1970. Arm-
strong and Armstrong 1974, Wendell et al. 1976. Gaumer 1977.
Goodwin and Shaul 1978, Breed-Willeke and Hancock 1980, Rob-
inson and Breese 1982. Kvitek and Oli\er 1992). A third species,
the strange gaper. T. alloiiixax Coan & Scott. v\hich Coan el al.

(2000) indicated was incorrectly named T. pajaroanus (Conrad.
1875) by Dinnel and DeMartini (1974). has a limited distribution
from Oregon to California.

The purpose of this paper is to present estimates on density,
growth, and mortality of inter- and sub-tidal T. capax and subtidal
T. niittallii populations, which will be useful in fishery manage-
ment of these species in B.C.

MATERIALS AND METHODS

Horse clam densities were estimated from a study plot in
Ritchie Bay, northwestern Meares Island, near Tofino (Lat.
49°13.43'N Long. 125°54.99'W) during June 3-9, 1993, and in
the northwestern side of Seal Island (Islets), near Comox (Lat.
49°37.835'N Long. 124°51.892'W) (Fig. 1) during June 3-7,
1993, between the low intertidal depths of -1 m to subtidal depths
of about 10 m subtidal for Ritchie Bay and 1 1 in for Seal Island.
Study plots were characterized by sand and broken shell substrates
at all depths. Eelgrass (Zostera marina L.) abundance, in shallow
water, was dense at Ritchie Bay and low at Seal Island. Both study
plots, 0,5 ha in area (50 m x 100 m). were delineated with lead
lines and subdivided by 10 (50 m) transect lines 10 m apart run-
ning from shallow to deep. The survey of horse clam density
involved counting horse clam siphons showing at or above the
substrate in 5 m" (5 x 1 m) quadrats along both sides of the inner
subdivision transect lines and on the inner sides of the study plot
boundaries. Each transect was assumed to be a sample with quad-
rats as secondary sample units. Depths recorded by divers at each
quadrat were corrected for a standard tidal height at datum (mean
low lower water) at the time (±5 min) each quadrat was sampled.
Density data for the west boundary of the Seal Island study plot
were missing for some unknown reason.

Samples for size and age frequency distributions and growth
estimates were obtained by randomly collecting horse clams
within two depth zones (shallow 2 to 3 m and deep 4 to 10 m
subtidal) in the Ritchie Bay study plot and (low intertidal -1 to 1

933

934

Campbell and Bourne

126

124

122

o
in

British Columbia

L/1

International
Boundary

132

130

128

Figure I. Map of British Columbia indicatin!> j^enera! sample locations. D = Doyle Island, I = Kitkatia Inlet, K = Klaskinii Inlet. I.
Inlet. N = Newcastle Island. R = Ritchie Uav. S = Seal Island or Seal Island.

Lemmens

111 and Miblidal 2 to II m) in the Seal Island study plot. Horse
clams were hand coIIclIciI with a diver operated "stinger" (l-m
long stainless steel pipe, atlaehed lo a high pressure water hose,
that provides a water jet lo allow horse clam removal from the
substrate) (Goodwin 1973). A commercial sample of horse clams
was obtained from Klaskino Inlet (I. I.at. .'^0"l7.y'N Long.
127"48.8'W) on June 23. 1993. An additional sample from as wide
a size range as possible of T. cuptix. to determine growth, was
obtained from l.emmens Inlet (I.at. 49"I2.2'N Long. l2.'i"52.3'W)
May 2.S and August 10. I9S9.

Sampling methods, for counting density or collecting indi-
vidual animals, in this study relied on visual detection of horse
clam siphons in the substrate uiulcr water by experienced com-
mercial dive fishers, which generally only included horse clams of
commercial si/.e OlOO mm shell length. SL). Consequently den-
sity estimates should not be considered as absolute but rather mini-
mum estimates of abundance. Small horse clams (especially <50
mm SL) were usually difficult to see and collect by divers and a
few horse clams may not have shown their siphons clearly above
the substrate surface. Additional samples of 7. cupcLx ( 1 1-50 mm

Gaper Clams in British Columbia

935

SL) were obtained, for dissection, by using a venturi dredge in the
Seal Island study plot.

For each collected horse clam. SL was measured as the straight
line distance between the anterior and posterior margin of the shell
to the nearest 1 mm with vernier calipers, total wet weight and
shell weight were recorded to the nearest 0.1 g. In addition, when-
ever possible subsamples. from as wide a SL range as possible,
were obtained from horse clams collected in each area and wet
weights of the drained total body and shell, shell only, whole soft
body and siphon (neck) only (cut at base of siphon) were recorded
to the nearest 0.1 g within 24 h of collection. For Klaskino Inlet,
only the total wet weight (Wt). shell length, and weight were
recorded for each individual animal; total drained weight (Wd)
was estimated as Wd = Wt C. where C is the mean free-water
weight loss conversion ratio (Wd/Wt = 0.74) calculated from
horse clams from the other areas in this paper.

Age of horse clams was determined by counting the number of
annuli on the shell, and growth was determined by measuring shell
length at each annulus after Weymouth et al. (1925) and discussed
by Bourne and Smith ( 1972b). Horse clams had pronounced annuli
up to about 20 years of age; a few clams older than 20 years had
annuli spaced close together and accuracy in age determination of
the clams was estimated at about ±2 y. Horse clams with broken
shells were discarded.

Mean density, d (number / ni"). was calculated as

Sa,

Standard error of the mean density. se(d). was calculated as

se(d)^

where for each i"' transect. C; is the number of horse clams ob-
served in a transect, a; is the area of transect surveyed in square
metres, a is the mean transect area for all transects and n is the
number of transects sampled. This method was also used to cal-
culate mean and standard error of density from a depth interval by
subsampling each transect in the particular depth range (i.e., ( 1 ) s
0.0 m. (2) >0.0-2.0 m, (3) >2.0-3.0 m, (4) >2.0^.0 m. (5) >4.0-
6.00 m. (6) >6.0-8. 0 m, (7) >8.0-10.0 m, and (8) >10.0-12.0 m).
Total mortality rate (Z) was estimated in the usual way (Ricker
1975) by calculating the slope of the regression relationship be-
tween the natural log of the frequency and age of horse clams >l()
y, which would include mature (Bourne and Smith 1972b, Camp-
bell et al. 1990) horse clams fully recruited to the fishery and
mainly the descending right limb of the age frequency curve. We
assumed that the mortality rate reflected the natural mortality rate
because most of the age frequencies of horse clams sampled from
areas that had little or no commercial fishing history were used.
Ritchie Bay was designated as a research study area since the early
1980s and has had no commercial dive fishing activity for horse
clams. Although Seal Island is in an area (bed 4801 ) where horse
clam landings have been recorded from the subtidal (dive fishing
for horse clams in waters shallower than 3-m depths is prohibited
to protect eelgrass habitat) (Harbo and Hobbs 1997). fishing for
horse clams in the low intertidal areas of Seal Island probably has

not occurred. An insignificant amount of harvested horse clam
landings have been reported from Klaskino Inlet (statistical man-
agement area 27) (Harbo and Hobbs 1997). Lemmens Inlet (sta-
tistical management area 24) has had considerable amounts of
horse clams harvested (Harbo and Hobbs 1997) and were not used
to estimate mortality rates because of low sample sizes.

Average von Bertalanffy growth curves were fitted to data
points of size at age using the equation:

where t is age in years, L, is the shell length at t. L,^ is the
theoretical maximum size, k is a constant, determining rate of
change in length increments, and t,, is the hypothetical age at which

LU

I-

LU

LU

a:
<

Z)
O
C/3

a:

LLl
Q.

a:

LU

1.0

0.8-

0.6

0.4-

0.2

0.0

1

1

1

1 1

1 1 1 1 1

1

-

'

—

"

^

V'

_

11 (

^

\

_

-

\l1

-

-

V 7 -

—

8/

^

1

5

^

1

1

-2-101 23456789 10
MEAN DEPTH (M)

LU

a:

I-

UJ

UJ

a:
<

O
CO

a:

LU
Q.

en

LU
CD

14

1 1

1 1

1 1

1 1

1 1
B

1

12

^

-

10

-

/

I 10

-

8

-

/

\

-

6

" 10.

/

\

-

4

-

[ \

\,0

~

2

n

1 1 1 1

1 1

10

9

1 1
oo ■

-2 -10123456789 10
MEAN DEPTH (M)

Figure 2. Mean density of horse clams by depth at the study areas in
(A) Ritchie Bay (99.2 % were T. nullallii at all depths) and (B) Seal
Island (7". capax were represented 11)0% in <2 m depths and 99.5% in
>2 m depths). Vertical lines are ± 2 SE: numbers beside dots are
number of transects represented at each depth range.

936

Campbell and Bourne

the organism would be at zero length. The parameters L.^, k, and
t|| were estimated using a nonlinear least squares method
(SYSTAT 1996).

Allometric relationships between total, body, neck and shell
weights (Y), and shell length (X) were determined using the power
equation of the linear form log^ Y = log^.a + b log^.X. where a and
b are constants calculated using the least squares method. Com-
parison between sampled areas for each relationship was accom-
plished testing for homogeneity between slopes and subsequently
comparing intercepts of lines by adjusting the Y variables and
testing for differences by analysis of covariance (ANCOVA) using
shell lengths or age as covariates (Snedecor and Cochran 1967)
with SYSTAT.

Mean weight of the size frequency sample was calculated by
2(N^ W, )/SN, where N, is the number of animals per SL interval,
SN is the total number of animals in the size frequency sample,
and Wl is the predicted mean weight for a particular SL estimated
from power equations. For Klaskino Inlet, the total wet weight of
each individual horse clam was measured and the drained total
wet weight was calculated by multiplying the total wet weight
by .74 (the mean free-water weight loss conversion ratio). The
estimated mean biomass per m~ was calculated as the product
of the mean weight and the mean density of horse clams in the
study area.

RESULTS

Density

At Ritchie Bay, density was higher in the 3-6 m range than at
other depths sampled; few horse clams were found in the intertidal
<1 m depth, especially in the dense eelgrass (Fig. 2A); most horse
clams sampled (99.227c, n = 511) were T nuttallii. At Seal Island,
densities were highest at the low intertidal and <l m depth and
were low at >2 m depths (Fig. 2B) (no horse clams were found at
10.5 m, n = 4); T. copav represented 100*^ in samples (n = 525)
from the shallow waters and 99.50% (n = 602) from >2 m depths.
Overall mean density of horse clams was lower at Ritchie Bay,
0.32 per rir (±0.03 SE, n = ID, than at Seal Island, 4.54 per m-
(± 0.85 SE, n = 10). Distribution of density changed with depth
at both study areas (Fig. 2).

Size and Age Distributions

Most horse clams sampled were between 100 and 200 mm SL
(Fig. 3). Average size and age were higher for T. nultallii than for
T. capax (Figs. 3 and 4). The largest (230 mm SL) and oldest (24
y) T. nuttallii were from Klaskino Inlet (Fig. 3B and Fig. 48). The
largest (187 mm SL) and oldest (21 y) T. capax were from Seal

50
40

_ 30

c

o
"20

10

1

A
NUTTALLII
RITCHIE BAY
MEAN = 159

N = 507

50 100 150 200
SHELL LENGTH (MM)

250

25

009

0.08

20

0.07 ?

0.06 1
0.05 §

c
o

15

0 04 1

o

10

0.03 g"

^

0.02

5

0.01

0.0

0

1 r

B

NUTTALLII
KLASKINO
MEAN = 176
N = 332

50 100 150 200

SHELL LENGTH (MM)

0.07

0.06

005

H004

0.03

-0 02

-0 01

0.0

250

40

30

20

10

c

CAPAX
SEAL ISL
INTERTIDAL
MEAN = 122
N = 525

50

100

150

200

SHELL LENGTH (MM)

0.07

1 1

D

CAPAX

0.06

60

. SEAL ISL

0.05 -i
o

SUBTIDAL

MEAN = 113

0.04 o

3

c

3
O

40

N = 599

•a

CJ

1

003 <B

DD

1

0.02 ^

20

1

001

J

0.0

0

.m

250

_L.

50

100

150 200

SHELL LENGTH (MM)

-0,12

-0 10 T)
o

0 08 o^

o'

- 0 06 -^

0.04 S

0 02
00

250

Figure 3. Size freqiiem-y dislrihutions lor (,\) T. nuttallii al Kilchie Bay, (B) T. nuttallii at ^ cilow Bank, (t) /'. capax Seal Island from low
intertidal /.one to Im depth, (1)1 /'. capax Seal Island, (K) /'. nultallii al Klaskino Inlet location I. and (Kl T. nultallii al Klaskino Inlet, locations
2 and 3 combined. All .samples are from subtldal l>2 m depth) localicms except the intertidal sample at Seal Island (t'l.

Gaper Clams in British Columbia

937

90
80

1 1 1

A

NUTTALLII

1

-

0.16

70

- RITCHIE BAY

-

0.14

60

MEAN = 12.1 ■
~ N = 507 J

-

0.12 o

1 50
S 40

: mh

-

rtion pe

O CO

d d

30

-

0.06 en

01

20

-

-

0.04 "

10
0

(

L.-J

-

0.02

) 5 10 15

20

25

AGE (YEARS)

10 15

AGE (YEARS)

150

100

3
o
o

CAPAX
SEAL ISL
INTERTIDAL
MEAN = 8.7
N = 525

10 15

AGE (YEARS)

0.2

■a

c

o

o

1
D

1 1 1

CAPAX
SEAL ISL

-

180

SUBTIDAL
MEAN = 6 5

-

1

N = 599

90
n

ii

L

-

10 15

AGE (YEARS)

20

0.4

03

o

T3
O

o'

-0.2

CO
0)

-0.1

25

00

Figure 4. Age frequency distributions for (A) T. nuttallii at Ritchie Bay, (Bl T. nuttallii at Yellow Bank, (C) T. capax Seal Island from low
intertidal zone to Im depth, (D) T. capax Seal Island, (E) T. niillallii at Klaskino Inlet location I, and (F) T. nullallii at Klaskino Inlet, locations
2 and 3 combined. All samples are from subtidal (>2 m depth) locations except the intertidal sample at Seal Island (C).

Island (Fig. 3C and D. Fig. 4C and D). Mean size and age of the
four 7". capax from Ritchie Bay were 148 mm SL and 10.8 y, and
the three T. numdiii from the Seal Island subtidal were 186 mm SL
and 17.3 y. respectively.

Mortality

Mean mortality rates (with 95% confidence limits) for T. nut-
tallii > 10 y were estimated to be 0.44 (0.26 to 0.63) at Ritchie
Bay. 0.20 (0.06 to 0.33) at Klaskino Inlet, and for T. capax 0.20
(0.04 to 0.35) at the Seal Island intertidal zone. Since a significant
(P < .05) relationship between the log^. of the frequency and age
could not be obtained for T. capax >10 y from the Seal Island
subtidal zone, we estimated mean mortality rates (with 95% con-
fidence limits) for T. capax at >3 y [which provided for additional
samples on the descending right limb of the age frequency curves
(Fig. 4C and D)] to be 0.16 (0.05 to 0.26) for the subtidal and 0.15
(0.09 to 0.2 1 ) for the intertidal zones of the Seal Island study area.

Growth

Age-Shell Length

Tresits nuttallii from Klaskino Inlet had similar growth rates to
those from Lemmens Inlet, but were higher than those from

10 15

AGE (YEARS)

25

Figure 5. Relationship between mean shell length and age for T. nut-
tallii sampled from: 1. Klaskino Inlet during 1993 ( + ) (this study); 2.
Lemmens Inlet during 1989 (•) (after Campbell et al. 1990); 3. Ritchie
Bay during 1993 (Al (this study); 4. Newcastle Island during 1989 (x)
(after Campbell et al. 1990). Equations are presented in Table I.

938

Campbell and Bourne

TABLE 1.

von Bertalanffy growth parameters for horse clams from British Columbia. Depth zone for subtidal was greater than 2 m, low intertidal

was -1 to 1 m, and high intertidal was less than -1 m.

Area

Year

L^

k

to

Source

T. mmallii (subtidal)

Ritchie Bay

1993

200 (±13)

0.139 (±0.024)

-0.15 (±0.33)

This study

Klaskino Inlet

1993

231 (±6)

O.I 16 (±0.010)

-0.15 (±0.23)

This study

Newcastle Island

1989

183 (±5)

0.168 (±0.012)

0.51 (±0.10)

Campbell et al. (1990)

Lemmens Inlet

1989

202 (±3)

0.167 (±0.006)

0.50 (±0.05)

Campbell et al. (1990)

T. capax (subtidal)

Seal Island

1993

192 (±S)

0.148 (±0.013)

-0.13 (±0.22)

This study

Lemmens Inlet

1989

195 (±7)

0.154 (±0.016)

-0.01 (±0.18)

This study

T. capax (low intertidal)

Seal Island

1993

196 (±13)

0.139 (±0.027)

-0.26 (±0.41)

This study

T. capax (high intertidal)

Kitkatia Inlet

1990

149 (±15)

0.180 (±0.043)

0.17 (±0.29)

Bourne & Cawdell (1992)

Seal Island

1969

155 (±5)

0.189 (±0.021)

-0.11 (±0.23)

Bourne & Smith (1972)

Doyle Island

1971

169 (±4)

0.132 (±0.008)

-0.10 (±0.13)

Bourne & Smith (1972)

Values in brackets are approximate 95% confidence intervals.

Ritchie Bay and Newcastle Island (Campbell et al. 1990) (Fig. 5.
Table 1 ). There were no differences in growth rates between 7'.
capax collected between the low intertidal and subtidal zones from
Seal Island (Table 1 ) so data were combined for graphical pur-
poses (Fig. 6). Growth rates were similar for T. capax from Seal
Island and Lemmens Inlet (Fig. 6, Table 1 ). Growth was more
rapid for T. capax in the low intertidal and subtidal areas studies
than for those sampled in the high intertidal areas in other studies
in B.C. (Fig. 6. Table I).

250

10 15

AGE (YEARS)

I'igure 6. Relationship between shell lenKlh and age for /'. capax
sampled from: I. .Seal Island during 1993. low intertidal and subtidal
combined (T) (this study); 2. Lemmens Inlet subtidal /.one during 1989
(+) (this study); 3. .Seal Island during 1969 (•• (after Bourne and
Smith 1972b); 4. Doyle Island during 1971 (x) (after Itourne and Smith
1972b); 5. Kitkatia Inlet during 199(1 (A) (after Bourne and Cawdell
1992). Areas 3, 4, and 5 are from the high intertidal beach /.one.
Equations are presented in lable I.

Length-Weight

All length-weight relationships were positively and highly cor-
related, indicating that weights increased with SL increases (Table
2, Figs. 6 and 7). There were no significant differences
(ANCOVA, P > .05) in slopes or elevations for all length-weight
relationships between T. capax from the low intertidal and those
frotn the subtidal in the Seal Island study area, so the data for each
depth zone were combined. For total wet weight and whole drained
wet weight and SL relations, there were no differences in slopes
between all areas and although there were no differences in eleva-
tions between Lemmens Inlet and Seal Island for T. capax. weights
were significantly higher for T. mirtallii from Klaskino Inlet and
significantly lower for T. nuttallii from Ritchie Bay than for T.
capax from the other two locadons (ANCOVA, P < .01 ) (Table 2,
Figs. 6 and 7). The shell weight-SL relations were similar for horse
clams frotn Klaskino and Letiimens Inlets and Seal Island, how-
ever they were heavier at all three sites compared to those from
Ritchie Bay. Although body weights were heavier than shell
weights at all SL for T. capax at both Lemmens Inlet and Seal
Island (Fig. 8C and D), shell weights becatne heavier than body
weights for T. nuttallii at about SL >17() mm for Ritchie Bay and
SL > 150 mm for Klaskino Inlet (Fig. 8A and B). Neck weights
grew less than the other body parts studied and were similar for the
three areas sampled (Tabic 2, Fig. 8).

Age-Weight

All the age-weight relationships were positively and signifi-
cantly correlated, indicating that weights increased with increasing
age (Table 2). Although the age-weight relationships showed simi-
lar trends to the weight-RL relationships there were considerably
more variation in the R' and intcr-area comparisons in the former
than the latter (Table 2).

Mean Weights and Hiomass

Meat! total wet weight and drained total weight (g) (±1 SE in
brackets) of horse clams was 67 1 .4 (9.3) and 493.7 (7.0) (n = 507)

Gai'kr Clams in British Columbia

939

TABLE 2.

Regression coeftlcients for different morphological relationships of T. nuttalUi from (1) Ritchie Bay and (2) Klaskino Inlet and T. capax from
(3) Lemmens Inlet and (4) Seal Island for equation log,. Y = log,. A + B log, X, where X is the shell length (SI in mm) or age (y) and Y

variables arc weights (g).

Variables

Area

Regression Coefficients

B

R-

Tolal

Drained

Body

Neck

Shell

Drained

Body

Neck

Shell

SL

SL

SL

SL

SL

Age

Age

Age

Age

-8.980
-9.442
-9.865"
-8.858"
-10.115
-9.743
-10.167"
-9.125"
-8.330
-8.067
-9.601"
-9.337"
-8.381"
-10.407"
-8.963""
-13.759
-12.563""
-12.772-'
-11.840"
2.587"
2.685
2.163
2.922"
2.429
2.698"
1.889
2.568"
1.966
0.941
1.820
1.224
1.396
0.859
1.773

3.053"

3. 1 85"

3.290"

3.092"

3.213"

3.185"

3.289"

3.084"

2.729"

2.709"

3.063"

3.020"

2.594"

3.006"

2.731""

3.786"

3.608"

3.638"

3.459"

1 .424""

1.486"

1.851

1.306"

1.225"

1.193"

1.218"

1.225""

1.112"

1.643

1.101"

1.671"

1.728"

2.051

1.418

913

90

893

295

955

73

989

124

964

90

893

295

912

73

979

124

934

90

720

295

906

73

976

111

884

90

881

73

955

111

963

90

886

295

907

73

974

111

858

66

771

332

908

70

930

99

838

66

567

291

894

70

912

99

799

66

823

70

880

99

852

66

792

291

909

70

939

99

Total is whole wet weight. Drained is whole drained total wet weight. Body includes all soft body parts. Minimum and maximum sizes and ages used
for equataions, respectively, were 75-196 mm SL and 3-20 y, for Ritchie Bay, 1 10-229 mm SL and 6-24 y for Klaskino Inlet, 51-169 mm SL and 2-13
y for Lemmens Inlet, and 1 1-183 mm SL and 1-21 y for the Seal Island. All R" values are significant at /" < .01. Neck weights for Klaskino Inlet were
not measured. Coefficients, within the same X and Y combination and in the same column, thai are followed by the same letter are not significantly
different (ANCOVA. P > .05), those not followed by the same letter are significantly different (ANCOVA. P < .05).

for Ritchie Bay. 1,190.2 (24.8) and 880.7 (18.4) (n = 341) for
Klaskino Inlet. 459.5 ( 13.1 ) and 338.3 (9.6) (n = 525) for the low
intertidai. and 355.0 (10.3) and 261.6 (7.6) (n = 599) for the Seal
Island subtidal area, respectively.

Mean biomass (g/m") for T. nuuallii was 157.5 for ail depths at
the Ritchie Bay study area, and for T. capax was 2.756.6 for the
low intertidai zone and 130.7 for the subtidal zone in the Seal
Island study area.

DISCUSSION

Mean densities and biomass were greater for T. capax in the
low intertidai zone than in the subtidal area at Seal Island and for

T. nuttaUii in the subtidal at Ritchie Bay. Results confirm that T.
capax was generally found to be more abundant in shallow waters
than deeper waters in some locations, and although T. nuttaUii may
be found intertidally (Haderlie and Abbott 1980). T. nuttaUii was
most abundant subtidally. Bourne and Cawdell (1992) and Bourne
et al. (1994) found horse clams sampled from intertidai sites in
northern B.C. were all T. capax. The relative abundance in subtidal
zones between T. capax and T. nuttaUii can vary considerably
between locations: in some areas one species may be overwhelm-
ingly more abundant than the other (e.g. >99% of T. nuttaUii at
Ritchie Bay and Klaskino Inlet compared to >99% of T. capax at
Sandy Islets) or there may be a considerable species mix (e.g.,
78.3% 7". nuttaUii and 2 1 .7% T. capax from a commercial sample
(n = 783) at 5-9 m depths al Lemmens Inlet during 1989, A.

940

Campbell and Bourne

2000

O

I-
X

g

1500-

uj 1000 -

<
a:

Q

<

I-
o

1 1
A

1

1

_ NUTTALLII

_

RITCHIE

/

s

^

._^^^

1

h
1

-

2000

50 100 150 200

SHELL LENGTH (MM)

250

CD

1500

C2

LU

2 1000

<

tr
a

o

500

50 100 150 200

SHELL LENGTH (MM)

250

1 1

1 1

o

C

H

5 1500

_ CAPAX

-

lD

LEMMENS

§

Q

m 1000

-

-

z

/

<

cJ

ce

(^ y/O

Q

o /.

_i 500
<

-

■^dlo

H

jy^

o

jg^

n

.^..^

1 1

2000

50 100 150 200
SHELL LENGTH (MM)

250

50 100 150 200
SHELL LENGTH (MM)

250

Figure 7. Total drained and shell length relationship for T. iiutlallii collected from (A) Ritchie Bay, and (B) Klaskino Inlet, and T. capax from
(C) Lemmens Inlet, and (D) Seal Island (low intertidal and subtidal samples combined). Equations are presented in Table 2.

Campbell, unpublished data) (Stout 1967, Wendell et al. 1976).
Distribution and density of both species have been found to vary
considerably and their distribution was often aggregated (e.g..
Stout 1967, Wendell et al. 1976, Goodwin and Shaul 1978).

The large mean sizes and ages of T. numdlii sampled from
Ritchie Bay and Klaskino Inlet were probably a result of an ac-
cumulation of older individuals in an unharvested population, with
probably little or no recruitment having occurred within 5 y prior
to sampling. In contrast, the age frequencies of T. capax from Seal
Island were dominated by large numbers of 4-6 y individuals.
There may have been many more small individuals <3 y, which
could not be monitored by the sampling method used, conse-
quently this study could not examine settlement, abundance and
mortality of horse clams in their first few years o{ life. Wendell et
al. (1976) found that recruitment of recently settled horse clams
varied spatially and annually between beds.

Mean natural mortality rate estimates for adult horse clams
were between 0.15 and 0.44 depending on location and species
examined. These mortality values are within the values predicted
with lloenig's (198.^) generalized mortality equation (()..^(i lor a
max age of 15 y, 0.26 for max age of 22 y, and 0.24 for a max age
of 24 y). Wendell et al. (1976) suggested a theoretical maximum
age of over 20 y and an average longevity of 15 y for T. capux in

Humboldt Bay, California. Wendell et al. (1976) found that mor-
tality of young of year recruits was sufficient to inhibit successful
recruitment for 2 y in some Humboldt Bay T. capax populations.
Natural mortality in 7re.TO.9 juveniles and adults may be caused by
haplosporidian parasites (Armstrong and Armstrong 1974), inver-
tebrate predators (e.g., sea stars, Pisaster brevispinus Stimpson
1857. moon snails, Polinices lewisii Gould 1847. crabs such as
Cancer magister Dana 1 852 (Wendell et al. 1 976, Sloan and Rob-
inson 198."^). fish (Stout 1967. Laurent 1971) and sea otters. En-
hydra hilris Merriam 192.^ (Kvitek et al. 1988. Kvilek and Oliver
1992. Watson and Smith 1996).

Growth rales for T. capax in the low intertidal and subtidal
areas reported in this paper were faster than those reported for
other mid intertidal areas in B.C. (Bourne and Smith 1972b.
Bourne and Cawdell 1992). Breed-Willeke and Hancock (1980)
showed that T. capax from subtidal regions grew more rapidly than
those from intertidal areas in Yaquina Bay. Oregon. Wendell ct al
(1976) showed intertidal and subtidal /'. capax growth rates to
dilTer significantly between beds and between year classes within
a bed in Humboldt Bay. There was considerable variation in
growth between populations of T. nuttaUii (this study. MacGinitie
19.35. Man-iage 19.54. Laurent 1971. Clark 197.3, Campbell et al.
1990). In this study, with increasing size, shells became heavier

Gaphr Clams in British Columbia

941

800

600-

O

5 4001-

200

1
A

'

1

NUTTALLII

RITCHIE

SHELL

-

-

BODY^

/ .-'

_^

' 1

''NECK

1

800

50 100 150

SHELL LENGTH (MM)

200

600

O

g400^

200

1 1
B

1

NUTTALLII
KLASKINO

/

SHELL / /

-

/ / -

//

J^ODY

^^.^

1

50 100 150

SHELL LENGTH (MM)

200

800

600

O

^400

m

200

1

c

'

'

CAPAX
LEMMENS

/ '

body//^
//

//SHELL
/ /

/:r^f,--'NECK

50 100 150

SHELL LENGTH (MM)

200

1

D

I

600

CAPAX
SEAL

//

X400

-

BODY//
/ /

UJ

/ /

^

/ /^W'EVL

200

-

n

_^

^_, ■' NECK

1

50 100 150

SHELL LENGTH (MM)

200

Figure 8. Body, shell, and neck weight and shell length relationships T. nuttallii collected from (A) Ritchie Bay. and (B) Klaskino Inlet, and T.
capax from (Cl Lemmens Inlet, and (D) Seal Island (low intertidal and subtidal samples combined). Equations are presented in Table 2.

than the soft body parts for T. niitlallii compared to T. capax. The
reasons for the differences in growth rates in T. iiunallii and T.
capax are unknown, but may be attributed to differences in a
variety of environmental factors associated with habitat, such as
food availabiHty. temperature, current patterns, and substrate
types.

ACKNOWLEDGMENTS

The authors thanl< R. Antifave. D. Brouwer. W. Carolsfeld, B.
Clapp, G. Dovey, L. Flostrand, D. Larsen, J-M Leguerrier. G.
MacDonald, D. Miller. S. Renshaw. J. Rogers, L. Sorensen. and J.
Wasilewski for diving and technical assistance.

LITERATURE CITED

Addicott. W. O. 1963. An unusual occurrence of Tresiis nuiralli (Conrad.

1837) (Mollusca: Pelecypoda). Veliger 5: 143-145.
Armstrong. D. A. & J. L. Armstrong. 1974. A haplosporidian infection in

gaper clams. Tresiis capax (Gould), from Yaquina Bay. Oregon. Pmc.

Nal. Shellfish Assoc. 64: 68-72.
.Armstrong. L. R. 1965. Burrowing limitations in Pelecypoda. Veliger. 1:

195-200. Bernard. F. R. 1983. Catalogue of the living Blvalvia of the

eastern Pacific Ocean: Bering Strait to Cape Horn. Can. Spec. Puhl.

Fish. Aqua!. Sci. 61: vii + 102 pp.
Bourne. N. & G. Cawdell. 1992. Intertidal clam survey of the north coast

area of British Columbia — 1990. Can. Tech. Rep. Fi.sh. .iipuil. Sci.

1 864: xi + 151 pp.
Bourne. N. F.. G. D. Heritage & G. Cawdell. 1994. Intertidal clam surveys

ot British Columbia— 1 99 1. Can. Tech. Rep. Fish. Aqual. Sci. 1972: x

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JoKincil ot Shcllfi'^li Ri-.sfiiirh. Vol. 19. No. 2. 943-947. :(K)(1.

SIZE AND AGE AT SEXUAL MATURITY AND SEX RATIO IN OCEAN QUAHOG, ARCTICA
ISLANDICA (LINNAEUS, 1767), OFF NORTHWEST ICELAND

GUDRUN G. THORARINSDOTTIR AND
SIGMAR A. STEINGRIMSSON

Marine Research Institute
P. O. Box 1390
Skiilagata 4
121 Reykjavi'k. Iceland

ABSTRACT Ocean quahogs, Arcrica islandica. were collected in February 1994 from near-shore populations off north-west Iceland
for determination of developmental stages relative to size, age. and sex. A microscopic examination of histologically prepared tissue
of 206 ocean quahogs showed that s6 specimens (3 1-70 mm shell length) were immature and could not be sexed. Sexual differentiation
was evident in 200 individuals. 17 were in the intermediate stage and 183 were mature. On the basis of annual internal growth bandings
in the shells of the specimens, the age ranged between 7 and 32 y in the intermediate stage and the individuals were from 24 to 60 mm
in shell length. The smallest and youngest individuals that could be .sexed were males. The smallest mature male was 36 mm in length,
but the youngest aged individual was 10-year-old (49 nun length). The youngest mature female was -14 mm and 13-year-old. Age and
size of maturity showed a wide range and may be dependent on growth rate and environmental conditions. Sex ratio between males
and females was examined relative to size in 200 quahogs. The male to female (M:F) ratio varied between size classes, with males
dominating in the smallest size classes, which may be related to their earlier development of germinal cells. After 40 mm length was
reached, the females dominated in all size classes except 65 to 69 mm (1.3:1) and 70 to 74 mm (1:1).

KEY WORDS: Arcrica islandica. ocean quahog, sexual maturity, sex ratio. Iceland.

INTRODUCTION

The ocean quahog, Arctica islandica, is vi/idely distributed over
the continental shelves of both Europe and North America. In
Icelandic waters there have been reported great densities of com-
mercial potential (EiriTcsson 1988, Thorarinsdottir & Einarsson
1996).

The ocean quahog has been the focus of an important commer-
cial fisheries in the United States since 1976 (Murawski & Serchuk
1989) and with growing demand for this species an interest has
arisen in Iceland to investigate the possibility for developing an
Icelandic fishery for human consumption. As a result of this in-
terest a study was undertaken in 1994 to assess the distribution,
abundance, and biology of this species in Icelandic waters (Tho-
rarinsdottir & Einarsson 1996, Thorarinsdottir & Johannesson
1996). The examination of size and age of sexual maturity and sex
ratio is reported on in this paper.

Studies on size and age at sexual maturity in ocean quahog
have been undertaken in the Mid-Atlantic Bight area of the United
States (Thompson et al. 1980b, Ropes et al. 1984) and in Nova
Scotia, Canada (Rowell et al. 1990), but such studies have not been
made in ocean quahog from Icelandic waters before.

MATERIALS AND METHODS

Specimens of/i. islandica were dredged off the northwest coast
of Iceland during a shellfish assessment survey in February 1994.
In each of 7 locations visited during the survey, about 30 speci-
mens were sampled for the study of shell length, age, and sexual
maturity (Fig. 1). The size-frequency distributions of A. islandica
captured by sampling dredges are normally dotiiinated by large
individuals with small shells being rare (Fogarty 1981, Murawski
et al. 1982, Thorarinsdottir & Einarsson 1996). In this study few
specimens smaller than 40 mm in shell length were collected by
the hydraulic dredge. Out of the 206 individuals investigated in the
present study, only 10 had a shell length less than 40 mm.

The samples from the various sampling sites were pooled to-
gether and returned to the laboratory where a piece of the gonads
4- to 5-mm wide was cut from the hinge region to the ventral

region of the mantle edge. In small animals sections included the
whole gonads. The gonads were preserved in 10% formaldehyde in
seawater. Histological preparation of the gonads included embed-
ding in paraffin, sectioning at 8 |jim, and staining with haematoxy-
lin and eosin. The stained preparations were examined microscopi-
cally for the presence of differentiated gametes.

Those specimens having little follicular development, no cel-
lular structures definable as male or female, and much of the gonad
filled with connective tissue were designated as undifferentiated.
Those with sufficient development to be differentiable as males or
females were further classified as intermediate or mature in their
gonadal development according to the criteria used by Rowell et
al. (1990). Intermediate specimens were typified by reduced-to-
sparse follicular development with follicles widely spaced and
separated by vesicular connective tissue. The follicles themselves
displayed varying degrees of development, from those with small
diameter and lacking germinal cells in portions of the epithelium to
those typifying the mature condition with large diameter, little
connective tissue, and a completely filled the gonadal area.

The height of the shells was measured with vernier calipers to
the nearest millimeter. For the purpose of comparison with other
investigations dealing with size and age at sexual maturity, the
shell height estimates in the present study were converted to shell
length using a ratio height:length of 0.91 (Witbaard 1997).

Sex ratio of the clams was examined relative to size in 5-mm
size classes for a total of 200 animals having shell-lengths ranging
from 24 to 119 mm.

The age data was reached by examining acetate peels of a
cross-section of the left valve of each individual and by counting
the growth lines in the hinge tooth (Ropes 1987). The growth lines
in the hinge tooth have been shown to correspond in number to
those in the valve itself (Ropes et al. 1984, Thompson el al. 1980a,
Thompson et al. 1980b).

RESULTS

Of the 206 quahogs studied, 6 ranging in length from 31 to 70
min were found to be sexually undifferentiated. Only 2 of these

943

944

Thorarinsdottir and Steingri'msson

, Araarfjorflur
• TiUuia^orOur

Figure 1. Sampling sites of A. islandica off northwest Iceland used for
determination of size and age for sexual maturity.

individuals, 31 and 70 mm in length were aged and had the age of
6 and 43 y, respectively (Table I). The gonia were embedded in the
germinal epithelium and lacked definable cellular features. Go-
nadal follicles were sparse, of small diameter, and were sur-
rounded by an abundant loose vesicular connective tissue. The
lumen of the follicles were empty (Fig 2a).

Sex could be determined in 200 specimens: 92 males and 108
females. Seventeen quahogs, 7 males and 10 females were deter-
mined to be intermediate in their gonadal development. The males
ranged in length from 24 to 60 mm. with a mean of 44 mm. Of
these, 2 animals 34 and 45 mm in length were aged 7- and 9-year-
old, respectively. Intermediate females ranged from 35 to 58 mm
in length. Of these, 1 52-mm female was aged 32-year-old (Table
I). For both sexes the follicles were of small diameter and sparsely
scattered among a loose vesicular connective tissue. The lumina of
the follicles were often free of cells. Females were distinguishable
from males by the presence of enlarging oocytes and some sper-
matogenesis could bee seen in the males (Fig. 2, b and c). There
were 183 mature quahogs found; 84 males and 99 females. Males
ranged in the length from 36 to 103 mm with a mean of 75 mm,
while females ranged from 44 to 1 19 mm in length with a mean of
74 mm. Of those that could be aged there were 1 14 mature speci-
mens; 53 males and 61 females. Mature males with lengths of 49
to 99 mm ranged from 10 to 108 y with a mean of 50 y. Mature
females with lengths of 44 to 104 mm ranged from 13 to 120 y
with a mean age of 53 y (Table 1 ). In these quahogs the follicles
were greatly expanded and often filled the gonadal area, with little
connective tissue occurring between adjacent follicles. In females

the oocytes crowded into the lumen of follicles. The diameter of
fully ripe oocytes that were measured ranged from 54.2 to 74. 1 (xm
and the average was 63.9 (xm (Fig. 2d). In males, spermatocytes
and spermatids proliferated from the germinal epithelium and
sperm predominated the lumen of the follicles (Fig. 2e). However,
the intermediate and mature specimens displayed all phases of the
gametogenic cycle between early active and partially spawned, and
the same individual could show more than one phase. The 91
males and 109 females studied yielded a male:female ratio of 0.8
(Table 2). No evidence of hermaphroditism was observed. When
males and females were pooled by 5-mm-length categories, the
males dominated in the smallest size categories, as no female was
identified smaller than 35 mm in length. With increased shell
length, females dominated and no males were observed having
shell length greater than 103 mm. The largest female was 1 19 mm
long.

DISCUSSION

Investigations made on A. islandica have shown a continuous
annual gonadal cycle (Loosanoff 1953, Jones 1981, Mann 1982,
von Oberlzen 1 972, Rowell et al. 1990) with well-defined sex cells
present in the follicles throughout the year, even immediately fol-
lowing spawning. This makes it possible to estimate age and size
at sexual maturity in samples collected at any time of the year.

In the present study male ocean quahogs seem to begin pro-
ducing germinal cells at a smaller size and probably younger age
than females, as reported for the eastern coast of the United States
and Canada (Ropes et al. 1984, Rowell et al. 1990).

The minimum age and size of maturation from undifferentiated
to intermediate stage and from intermediate to mature stages was
very variable. The variability observed might be attributed to dif-
ferences in growth rate due to different environmental conditions,
as the longest distance between sampling sites was about 100 km
(Fig. 1). However, Rowell et al. (1990) had similar results for
ocean quahogs from a single locality, suggesting that local condi-
tions are less important than suggested for this species before
(Thompson et al. 1980). The explanation of the variability ob-
served might be variability inherent to the individuals making up
the population. Dahlgren et al. (2000) assessed genetic subdivision
in A. isiaiuUca from the North American coastline, Icelandic wa-
ters, Faroe Islands, Norway, and Sweden, The greatest diversity
(haplotypes per individual sampled) was found in the population
from Iceland.

All undifferentiated or immature individuals observed in the

TABLE 1.

Stages of gonadal development relative to shell length, age, and sex.

Undifferentiated

Intermediate

Mat

lire

Males

Females

Males

Females

Total

Aged

Total

Aged

Total

Aged

Total

Aged

Total

Aged

Number

6

->

7

■>

10

1

S4

51

49

(il

I.englh (mm)

31-70

3 1 . 70

24-ft()

34. 4.'S

.W.'iX

52

.16-103

4y-'w

44-11')

44-104

X Lcnylh

■iy

—

44

—

46

—

7.5

73

74

74

SD Length

14

—

14

—

7

—

10

10

14

13

Age (y)

—

6. 43

^

7.9

—

32

—

10-1 OS

—

1.1-121)

X Age

—

—

—

—

—

—

—

50

—

43

SD Age

—

—

—

—

—

—

—

20

—

26

Size and Age at Sexual Maturity and Sex Ratio of Ahctica isiandica

945

V*''

Figure 2. Photomicrographs (x30) of transverse sections through gonads of A. isiandica at various stages in gamete development, (a) Undiffer-
entiated gonadal tissue from a 6-year-old individual 31 mm in shell length, (b) Intermediate gonadal tissue from a 32-year-old female, 52 mm
in shell length. Ic) Intermediate gonadal tissue from a 9-year-old male, 45 mm in shell length, (d) Fully mature gonadal tissue from a 45-year-old
female. 64 mm in shell length, (e) Fully mature gonadal tissue from a 44-year-old male, 62 mm in shell length.

present study had shell length less than 61 mm except 1 immature
individual of shell length 70 mm and 43 years of age. It is difficult
to explain the immaturity of this large and old individual, but it
might indicate that some individuals do not reach maturity at all.
TTie observed size and age at sexual maturity is somewhat higher
than reported from studies on quahogs off the east coast of North
America. However, it is difficult to compare the age of the imma-
ture individuals in the present study to the age observed off the east
coa.st of America, as only 2 specimens were aged and I of them
seems to be exceptional both in age and size. Immature ocean
quahogs were observed to a maximum length of 47 mm and 14
years of age off Rhode Island (Thompson et al. 1980b) and to 46
mm length and 8 years of age off Long Island (Ropes et al. 1984).
From coastal waters in Nova Scotia, the maximum shell length and

age for immature individuals was 45 mm and 12 y. respectively
(Rowell et al. 1990).

The size range of ocean quahogs entering the intermediate
stage of maturity in the present study was 24 to 60 mm for males
and 35 to 58 mm for females with the mean size of 44 and 46 mm,
respectively. Only 3 of 17 individuals were aged and the age
ranged between 7 and 32 y. For ocean quahogs from Canada this
size has been found to be somewhat lower, or 21 to 48 mm for
males and 25 to 53 mm for females with the mean length being 30
and 34 mm, respectively. The age in these individuals ranged from
3-24 y (Rowell et al. 1990). Off the east coast of the United States
the size range for males from Long Island was 21^8 mm and
36-45 mm ftir females and the age ranged from 3 to 1 0 y for the
males and 5 to 8 y for the females (Ropes et al. 1984). Thompson

946

Thorarinsdottir and Steingri'msson

TABLE 2.
Male:female sex ratio relative to lengtli.

Numbers

M:F

Length (mm)

Males

Females

Ratio

20-24

1

0

—

30-34

2

0

—

35-39

1

1

1:1

40-44

2

4

0.5:1

45-49

2

4

0.5: 1

50-54

3

3

1:1

55-59

3

12

0.3:1

60-64

6

10

0.6: 1

65-69

22

17

1.3:1

70-74

14

14

1:1

75-79

15

17

0.9:1

80-84

7

8

0.9:1

85-89

7

9

0.8:1

90-94

2

3

0.7:1

95-99

3

4

0.7:1

100-104

1

2

0.5:1

105-109

0

1

—

115-119

0

1

—

Total

91

109

0.8:1

et al. (1980b) found intermediate-stage individuals from 25 to 50
mm length and 6- to 15-year-old off Long Island.

In the present study a male as small as 36 mm was considered
sexually mature. The smallest mature male was aged 10-year-old
and 49 mm in shell length and females aged 13-year-old and 44
mm were considered mature. This size and age at maturity is
similar to records from the eastern coast of the United States.
Furthermore, Thomp.son et al. (1980b) reported the smallest ma-
ture male ocean quahog to be 42 mm and 1 1 -year-old and the
smallest female 50 mm and i 1-year-old. Ropes et ul. ( 1984), how-
ever, reported 36 mm and 5 y for males and 41 mm and 6 y for
females. Rowell et al. (1990) found the smallest mature quahog in
Nova Scotia to be of 27 and 30 mm length for males and females,
respectively. The smallest aged mature individuals were of 7 y and
40 mm for both sexes.

For the 200 individuals in the intermediate and mature stage of
gonadal development, all phases of the gametogenic cycle were
displayed and some were approaching ripeness or were ripe. The
main spawning time for A. islandica in Icelandic waters is from
June to August, but the spawning is protracted (Gudrun Thorarins-
dottir 2000), as previous studies off the United States and Canada
have also indicated (Mann 1982, Rowell et al. 1990).

The life span of A. islandica is very long. Individuals over 100
years of age are common and the oldest individual from Icelandic
waters was 202-year-old (Steingri'msson & Thorarinsdottir 1995).
The development of the continuous reproductive potential in A.
islandica after the age of 7 to 32 years and the length of 24 to 60
mm seems consistent with the estimates of the species long life
span. Continuous reproduction during a long life span can be ben-
eficial for A. islandica and may be an evolutionary strategy in
response to uncertain larval and juvenile survival. Thompson et al.
( 1980b) conclude that there is no obvious indication of senility for
A. islandica of 100 or more y in regards to spawning. However, in
the present study the gonads of the oldest individuals contained
smaller follicles than the younger mature individuals, possibly
indicating senility.

The .sex ratio showed predominance of males in the smallest
size classes, which may be explained by the smaller size at which
males reach the intermediate stage or it may be due to small
numbers of individuals in these classes. The females were domi-
nating in all the size classes bigger than 40 mm in shell length
except in the 65 to 69 mm size class. Previous .studies of sex ratios
in ocean quahogs have generally indicated ratios in favor of males
(Jones 1981, Ropes et al. 1984, Rowell et al. 1990), although
results have been quite variable. The hypothesis that female ocean
quahogs may live longer than males based on predicted ages of
ocean quahogs at a marking site (Murawski et al. 1982) and ob-
served shift in sex ratio from male to female in the highest age
classes (Fritz 1991, Ropes et al. 1984, Rowell et al. 1990) has
some support in the present study, but due to the small sample
number in the bigger size classes it can not be ascertained.

ACKNOWLEDGMENT

The manuscript was critically reviewed by Karl Gunnarsson
to whom we owe our sincere thanks.

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NMFS 50:5 pp.
Ropes. J.W.. A. Murawski & F. M. Serchuk. 1984. Size. age. sexual

maturity and sex ratio in ocean quahogs. Arctica islandica Linne, off

Long Island. New York. Fish. Ball. 82:2.
Rowell, T.W., D. R. Chaisson & J. T. McLane. 1990. Size and age of the

sexual maturity and annual gametogencsis cycle in the ocean quahog.

Arctica islandica. (Linnaeus. 1767) from coastal waters in Nova Scotia,

Canada. J. .Shellfish Rp.v. 9:195-203.
Steingri'msson. S.A. & G. G, Thorarinsdcittir. 1945. .Age structure, growth

Size and Age at Sexual Maturity and Sex Ratio of Arctica islandica

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and size al sexual maturity in ocean quahog. Arcticii isUiiuliai (Mol-

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Thompson. I.. D. S. Jones & J. W. Ropes. 1980b. Advanced age for sexual

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population structure and meat yield of the ocean quahog, Arctica is-

landica (Linneaus. 1767) in Icelandic waters. Mar. Biol. As.soc. U.K.
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Joiirmil of Shellfish Rcmiich. Vol. 19. No. 2. 949-956. 2000.

GROWTH AND REPRODUCTIVE PATTERNS IN VENERUPIS PVLLASTRA SEED REARED IN
WASTEWATER EFFLUENT FROM A FISH FARM IN GALICIA (N.W. SPAIN)

RICARDO JARA-JARA, MARCELINA ABAD, ANTONIO J. PAZOS,
MARIA LUZ PEREZ-PARALLE, AND JOSE L. SANCHEZ

Departamento de Bioqiiimica y Bioloin'a Molecular.

Institiito de Acuicultura.

Uiiiversidad de Scintiiii;o de Compostela,

1 5706-Santiago de Compostela, Spain

ABSTRACT The use of eftluenl water from a turbot (Psctta iiuninui) farm for ciilturing Vcncnipis [ntlUi.slni juveniles (seed) was
evaluated. Reproductive activity, growth, condition, and survival rate were investigated. After a 2-mo acclimation period, clam seed
(mean live weight = 360 ± 18 mg SD. mean length = 14 ± 1.7 mm SD) were grown from June 30 to November 20 of 1993 in three
different flow-rates of wastewater. Thirty individuals were sampled from each tank biweekly. Stability m the main environmental
parameters of the effluent (temperature, salinity, oxygen, etc.) provided good conditions for seed growth. After the acclimation period,
mortality was less than 18%. Growth-rate coefficients (A^ values) of V. pullastra were correlated positively with increase in the effluent
flow and were significantly higher than in the control tanks {K = .001 ). Shell length (27.4 ± 1.8 mm SD) and. in particular, live weight
(2.907.6 + 530.9 mg SD) were highest in tanks with a flow of 4 vol/h and these tanks also produced the fastest growth rate (K = .0445).
In tanks with flows of 1 vol/h and 2 vol/h. final weights and lengths were 1.823 ± 273.2 mg .SD. 23 ± 1.8 mm .so and 2.361 ± 351.4
mg SD. and 25 ± 1.4 mm SD. respectively. Condition indices of clams followed a similar pattern reaching final values ranging from 18
to 28 and were significantly higher than in the control tanks. High condition index values might be due mostly to the high amount of
food present in the effiuent water throughout the experiment. Stereological and histological techniques were used to determine
gametogenic activity. Successive spawnings and recovery of the gonad, which are characteristic of this species, were noted in clams
in experimental tanks. However, they occurred in a shorter time period. Results suggest that it is possible to rear V. ptillostni seed under
these special culture conditions and that a better balance between volume of effluent used and clam productivity was achieved with
a fiow rate of 2 vol/h.

KEY WORDS: clam, effiuent water, gametogenic cycle, growth, seed. Venenipis piilhisira

INTRODUCTION

Galicia (N.W. Spain) is among the greatest consumers of clams
in the world. Two species are especially popular in Galicia. In
order of economic impoitance, these are Riidilapes decussatus and
Venenipis pullastra. The two species have a similar market value,
but V. pill lustra has the advantage of a faster growth rate (Perez-
Camacho and Cuna 1987), although it is less resistant to tidal
exposure and low salinities. Depletion of natural and introduced
stocks of clams has caused an increased demand for hatchery-
produced seed (Laing et al. 1987. Beiras et al 1993). but the large
amount of food (mainly microalgae) needed to culture postlarval
bivalves for extensive periods beyond settlement makes nursery
operations too costly.

The largest turbot {Psetta maxima) culture industry in the Eu-
ropean Cominunity ( 1300 ton/year. Unidade Estadistica. Xunta de
Galicia. personal communication) is located in Galicia. This in-
dustry discards daily considerable quantities of untreated to sea
water to the sea. Only 20-30% of the nitrogen and phosphorus
added as food to the marine fish ponds is consumed by the fish
(Kroni et al. 1985a. Krom et al. 1985b. Porter et al. 1987). There
is a considerable literature describing the biological treatment of
such effluent by using it to grow algae (Ryther et al. 1972. Ryther
et al. 1975. Cohen and Neori 1991. Shpigel et al. 1993b) or to feed
bivalves (Gordin et al. 1981. .Shpigel and Fridman 1990. Trevor
and Iwama 1991. Shpigel et al. 1993a. Shpigel et al. 1993b). The
main purpose of those studies was to improve the quality of the
effluent and minimize its effect on the environment (Krom et al.
1985a. Krom et al. 1985b. Porter et al. 1987. Shpigel and Blaylock
1991). It is possible that effluent from intense turbot culture op-
erations, with high levels of nutrients, could be used as a food

source for clam seeds, thus reducing costs of the bivalve nursery
and reducing the negative effects of the effluent on the environ-
inent. We have already successfully grown Riiditapes decussatus
seed in effluent froin turbot ponds (Jara-Jara et al. 1997).

In this study, we investigated the possibility of rearing Veneru-
pis pullastra seed in effluent from turbot culture operations. Ac-
climation period, growth rate, gonad development and survival
rate of V. pullastra were investigated.

MATERIALS AND METHODS

Experimental Procedure

The experiment was carried out on a pilot-scale level at a
private turbot farm at Nastos, from April 29 to October 20 1993.
Hatchery-reared Venenipis pullastra seed with a live weight of 9
± 0.8 g SD and length 2.7 ± 0.7 mm so was maintained for an
accliination period in an open flow system at a density of 0.1
kg/ni" in two rectangular fiber glass tanks (4.0 x 1.4 0.4 m) with
a sand substrate. Flow rate of the effluent froin the turbot culture
operation was sufficient to replace the water volume of each tank
twice every hour.

After an acclimation period of approximately 60 days, the seed
(mean live weight 360 ± 18 mg so and about 14 mm shell length),
was placed in 12 rectangular tanks (4.0 x 1.4 x 0.4 m deep) with
a total water volume of about 560 L. The bottom was covered with
10 cm sand. The initial density was 0.5 kg/m". Different flow rates
were used to determine the effect of the flow rate on growth and
to manage the rate of use effluent water effectively. Three flow
rates replicated 3x were tested. In three sets of tanks, the waste-
water flow rate was such to produce one. two and four complete
water exchanges per hour (560, 1,120, and 2,240 L/h. respec-

949

950

Jara-Jara et al.

lively). A fourth set of tanks received only fresh sea water at a rate
of two tank vol/h. and served as control. For the remainder of this
article, flow rates in tanks will be expressed us 1 x h. 2 x h, and
4 X h.

Biweekly between June 30 and November 20 1993, 30 indi-
viduals from each tank (90 clams per flow rate) were sampled
randomly and placed in filtered sea water at 17 °C for 24 h. Total
shell length and height of each individual were measured to the
nearest 0.1 mm with vernier callipers and after drying each clam
on ab.sorbent paper for 10 min to remove surface water, total
weight of each clain was measured to the nearest 0. 1 g using an
electronic balance (±0.1 mg). Soft parts were separated from the
shell and both were dried to constant weight at 80 °C. Condition
index (CI) was calculated by: CI = (mean dry meat weight/mean
dry shell weight) x 100.

Relative daily growth rates (K) were calculated using the equa-
tion:

K = (InW, - lnW|)/t, - t,

where: W, = the initial mean total weight. W, = the final mean
total weight, t, - t, = the elapsed time in days. The coefficient K.
multiplied by 100. yields the % change per day.

Statistical analysis of the results was performed using a one-
way ANOVA and the differences were tested by Duncan's mul-
tiple range test.

Water Quality

Water temperature, salinity, and oxygen concentration were
measured daily (from April to November 1993) in the experimen-
tal tanks. Temperature was measured with a thermometer
(±0.1 °C). salinity with an ATGO model S/MILL salino-meter.
and oxygen level was always maintained above 7-8 mg/L by in-
jecting liquid oxygen constantly into the turbot culture tanks. Chlo-
rophyll (I concentration of effluent from turbot culture ponds was
determined every ]5 days from April 1993 to October 1993. One-
to three-liter aliquots were concentrated on GF/C filters and chlo-
rophyll a was extracted with 90% acetone and assayed according
to the Strickland and Parsons (1972) method.

The turbot were fed daily with a pelleti/ed diet (EWOS turbot
4.5 mm and 12% humidity) containing 49Vf protein. 14% lipid. 5%
carbohydrate, 12% ash, 1.5% fiber, 6% vitamin premix. 2% cal-
cium, 1% phosphorus, 0.2% sodium, with a digestible energy of
the 17.4 Kj/g. Relative values (kg of component/tonne of fish) of
organic particulate matter, total nitrogen, and phosphorus in the
effluent from April to October 1993 were 467.5, 24.7, and 3.3
respectively.

Histological and Slireoloaical Techniques

Biweekly. 20 random individuals from tanks 2 x h were dis-
sected and fixed in Bouin-Hollande's solution (Gabe 1968). After
dehydration, the tissue was embedded in paraffin wax (Merck m.p.
56-58 °C). Sections 6-|j.m thick, separated by at least 100 |a.m
(Heffeman and Walker 1989). were cut and stained in Cleveland-
Wolfe dye (Gabe 1968).

Ouantitative slercological methods were used loilelermine si/e
distribution of oocytes for each sampling date. Methods were those
described by Morvan and Ansell (1988), Paulel and Boucher
( 1991), and Pazos et al. (1996). Video print at 2()0x magnification
were obtained from 1 2 randomly selected fields from each histo-
logical section of an ovary. All oocyte profiles were markeil with

a black felt tip pen. The size of each oocyte was obtained by video
camera and processed by Visilog 3.21 image analyzer (Noesis.
France). These analog video signals were converted to a binary
format using upper and lower gray-level thresholds set by the
operator (Heffernan et al. 1989).

To obtain the oocyte size distribution. 90 to 200 complete
oocyte profiles of each female were measured, according to Mor-
van and Ansell (1988) and Laruelle et al. (1994). Statistical errors
are minimized by taking sufficient measurements. The image ana-
lyzer \was used to determine the profile areas and a theoretical
diameter (D,) calculated, based on the assumption that each section
is a circle: D, = (4s/'7r)''-. A frequency distribution of the profile
diameters in the 5-(i.m size class was obtained for each animal. The
Saltykov (1958) algorithm, a non-parametrical method applicable
to polymodal distributions was used to obtain the real oocyte-size
distribution from the profile diameter distribution (Williams 1981,
Morvan and Ansell 1988, Paulet and Boucher, 1991).

Oocyte size-frequency data for a number of animals or samples
can be considered as a (r x c) contingency table where /■ is the
number of individuals (or samples) and c is the number of size
classes (Grant and Tyler 1983). The statistic G was computed. G
is distributed similarly to X" with ()■- l)(c- 1 ) degrees of freedom
(v). If a significant value is obtained, it can then be concluded there
is heterogeneity within the samples. It is possible to determine
which size classes and which individuals (or samples) contribute
most to the value of G by examining adjusted residuals in a con-
tingency table (Haberman 1973, Grant and Tyler 1983). A positive
residual indicates that the frequency of oocytes in that size class is
greater than expected. ANOVA and the Student-Newman-Keuls
(SNK) 'a posteriori' procedure were used to test for significant
differences between means (Sokal and Rohlf 1981 ). Normality of
variables was determined using the Kolmogorow-Smirnof test
(Sokal and Rohlf 1981) and homogeneity of variance with the
Bartlett test (Sokal and Rohlf 1981). Statistical analyses were per-
formed using the SPSS statistical package.

RESULTS

Water Quality

Figure 1 shows water temperature and chlorophyll a levels.
Temperatures ranged from 1 1 °C to 20.3 °C. Chlorophyll a levels
varied between 2.8 and 5.0 (Jig/L. Two phytoplankton blooms were
recorded during the experimental period, one in June and another
in September. Salinity was stable at 35-36%<i.

Survival, Griiwlh Rale, and Condition Index

Mortality was 24% during the acclimation period (mainly in the
smaller seed) but less than 18% throughout the course of the ex-
periment. No significant effects of experimental conditions on sur-
vival were observed. During the acclimation period the seed un-
derwent rapid growth (Fig. 2A) reaching an average live weight of
360.1 ± 127.2 mg sii (n = 100) and length o{ 14.3 ± 1.7 mm so
in 60 days.

Growth rate of the seed in the effluent water was significantly
faster (Table 1. Fig. 2A) and shoued better condition indices than
clams grown in sea water (Fig. 2B). Duncan's test showed there
were significant dilfercnces (/' < .05) in clam growth rate between
different flow rates.

Length and weight increases were lowest in the control tanks.
(A' OOi. n ^)()). In the 1 x h tanks, the final weight and

Growth of V. i'ullastra in Fish Farm Effluent

951

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500

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Figure 1. Biweekly mean values for chlorophyll a concentrations (n =
3) and maximum and minimum daily temperatures in the wastewater
from turbot culture.

length of the seed (an average of 1 .823 ± 273.2 nig sd and 23 ± 1 .9
mm SD) {K = .0102. n = 90) were greater than that in control
tanks. In the 2 x h tanks, clam weight and length was 2,361 ± 35 1 .4
and 24.8 ± 1.4 mm sd on October 20. Shell length (27.4 ± 1.8 mm
SD) and particularly live weight (2.907 ± 530.9 mg sd) of V. put-
lastra was higher in the 4 x h tanks and the fastest growth rate was
also obtained in these tanks {K = .0445, n = 90).

CIs in the experimental tanks (1 x h. 2 x h. and 4 x h), ranging
from 18 to 28 (Fig. 2B), were significantly higher than in the
control tanks where the CI fell drastically in the first two weeks
from 25 to 10, reaching the maximum value of 16 in September.

30
28 -
26
24

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Figure 2. Mean live weight ± .sd (A) and condition index ± so (B) of
Venerupis pullastra seed (n = 90) reared in turbot culture wastewater
and control tanks.

Gonad Development and Stereology

As expected, microscopic examination of histological prepara-
tions showed that sex ratios did not diverge significantly from a
1:1 ratio (Perez-Camacho 1980), except for the August 1 1 sample
in which only 5 females were found. Gonadal sections showed
females were in different developmental stages at each sampling
and that oocytes were in different stages of maturation in indi-
vidual females. Males showed a much higher synchronization of
development and gametes were in the same phase of development
in each male. Gametogenic activity was evident throughout the
experimental period and it appears several spawnings occurred
during this period.

Figure 3 shows the distribution of oocyte size-frequency during
the experimental period. Greatest mean oocyte diameter was found
in the August 1 1 sample, where the 45-50 |jim and 50-55 ^i,m
classes formed 50% of the total oocytes measured. In the previous

952

Jara-Jara et al.

TABLE 1.

Growth of Venerupis pullastra reared in a turbot culture effluent at
different flow rates

DMW ± SD

Length ± SD

Height ± SD

Date

(mg)

(mm)

(mm)

Control

30 June

36.2 ± 1.8

14.3 ± 1.7

8.4+ 1.0

14 July

34.4 ± 0.4

15.0 ±0.9

9.1 ± 1.0

28 July

37.2 ± 1.2

15.4 ±0.9

9.3 ±0.9

1 1 August

39.7 + 0.6

15.4 ± 1.1

9.4 ± 1.0

25 August

60.9 ±2.1

15.5 ±0.9

9.4+ 1.0

8 September

64.2 ± 0.9

15.9 ± 1.0

9.7 ± I.O

21 September

69.6 ± 0.6

16.3+ I.I

10.0 ± 1.1

20 October

79.9 ±2.1

17.5 ± 1.3

10.8 ± 1.2

Ixh

30 June

36.2 ±1.8

14.3 + 1.7

8.4 ± 1.7

14 July

40,4 ± 2.7

14.6+ 1.7

8.9 ± 1.0

28 July

41.6 ±3.8

16.2 ± 1.2

9.8 ±0.8

1 1 August

51.5± 1.4

16.7 ±2.2

10.4 ± 1.3

25 August

68.9 ± 2.5

I8.9± 1.9

1 1 .5 ± 1 . 1

8 September

76.5 ± 2.0

19.9 ±2.4

12.1 ± 1.5

21 September

92.4 ± 3.7

20.8 + 2.3

12.7 ± 1.4

20 October

126.6 + 2.9

23.0 ± 1.9

14.3 ± 1.2

2xh

30 June

36.2 ±1.8

14.3 ± 1.7

8.4 ± 1.0

14 July

46.7 ± 8.3

I6.4± I.I

10.0 ±0.7

28 July

80.8 + 5.3

18.9 ± 1.1

1 1 .6 ± 0.9

1 1 August

92.3 ±15.1

20.6 ± 1.9

12.8 ± 1.2

25 August

96.2 ± 9.7

20.6 ± 1 .4

12.6± 1.0

8 September

124.8 ± 13.5

22.5 ± 1 .5

13.8+ 1.1

21 September

127.6 ±8.7

22.6 ± 1.8

13.9 ± 1.2

20 October

180.7 ±7.9

24.8 ± 1.4

15.3 ± 1.0

4xh

30 June

36.2 ±1.8

14.3+ 1.7

8.4 ± 1.0

14 July

63.2 ±4.9

17.1 ± 1.5

10.5 ± 1.0

28 July

103.7 ± 11.1

20.1 ±1.1

12.3 ± 1.0

1 1 August

115.5 ±7.9

21.9 ± 1.3

13.4 ±0.7

25 August

138.8 ±6.5

22.4 ± 1.0

13.5 ±0.7

8 September

161.5+ 10.3

23.7 ± I.I

14.5 + 0.7

21 September

168.6 ±3.5

24.2 ± 1.0

15.1 ±0.9

20 October

191.1 ±2.1

27.4 ± I.X

15.9 ± 1.0

Values are mean ± SD (n = 90).
DMW = Dry meat weight.

sample (July 28), mature eggs were not found, indicating a rapid
redevelopment following spawning.

The dynamics of oocyte cohort maturation can be studied from
the position of positive residuals in the contingency table (Grant
and Tyler 1983). The contingency tabled (r x c) of adjusted re-
siduals for oocyte size-frequency is shown in Table 2, where /■ was
eight samples and c- 1 1 oocyte size classes. Maturation of the
gonad involved a displacement of the positive residuals to greater
size classes (35-.S() |xni) and spawning to smaller classes (0-35
fj.m).

A complclc spawning took place between the July 14 and 28
samples, after which the gonad underwent rapid recovery, as
shown by the large number of oocytes with diameters between
35-50 |xm found in the August 1 I sample (Fig. 3, Table 2). This
indicates that a second spawning, possibly a partial spawning, was
imminent. Although most of the oocytes on August 25 were in the
S-IO p.m and 10-15 |xm size classes, mature eggs {35—40 |xm)
were also present \n the follicles. .Subsequently, a period of go-

nadal redevelopment occurred, although it was slower than the one
that took place between July 14 and 28. A third spawning, al-
though small, occurred between August 25 and September 8 pos-
sibly as a result that this of oocytes of the larger size classes in the
previous sample that were not released because they were not
mature. Between September 8 and 21. another gonadal recovery
was observed that was confirmed by the presence of oocytes of the
0-5 (Jim class and others in the 30^0 jxm class. Finally, a fourth
spawning occurred between September 21 and October 20. This
spawning was larger than the previous one and had positive re-
sidual values coinciding with the larger oocyte size classes (Table
2). Data presented suggest the existence of multiple spawning
cycles that is characteristic of this molluscan species.

The degree of synchrony between the stage of oocyte matura-
tion in a sample is indicated by the values of the mean oocyte
diameter variation coefficient (CV = 100* (.s/.v)). In general the
higher synchronization occurred in the earlier gonad development
stages and the greatest values of the variation coefficient in the
spawning periods.

DISCUSSION

Venerupis pullastra acclimated well to effluent water from tur-
bot culture operations and had better growth than those held in the
control tanks. An important objective of this study was to deter-
mine the effect of effluent flow rate on the growth. Spencer ( 1988)
demonstrated the influence that flow rate has on the growth of
juveniles oysters in experimental outdoor pumped upwelling sys-
tems. This author reported that the effect of flow rate on growth
depends on the food concentration in the water. For commercial
scale culture of V. pullastra, a balance needs to be maintained
between sufficient flow and available food in the effluent. Insuf-
ficient water exchange could cause an excess of organic inatter that
would adversely affect water quality (low oxygen levels, increased
ammonium concentration, etc.). which could induce stress on the
clams.

Cultivation density is an important parameter to be considered
when evaluating growth rate of a population in a particular site
(Spencer et al. 1991). Final density attained in the present work
was 5.5 kg/m" (flow 4 x h). which is greater than that found in
extensive culture of this species in the natural environment where
2.5 kg/m" is not surpassed (Walne 1976. Cervitio et al. 1993.
Robert et al. 1993. Pech et al. 1993). Our results in the 4 x h tanks
showed that this stocking density (5.5 kg/m") produced growth
rates similar to those reported under natural conditions by several
authors for the same species (Perez-Camacho 1980).

Two other important parameters that influence growth and ga-
metogenic development in bivalve molluscs are temperature and
available food (Sastry 1979. Bodoy et al. 1980. Maitre-Allain
1982. Beninger and Lucas 1984. Wilson and Simmons 1985. Laing
et al. 1987. Ruiz el al. 1992 ).

Several authors (Mann and Glomb 1978. Mann 1979. Wilbur
and llilbish 1989. Albetitosa et al. 1994) have .studied the effect of
lempcrature on the growth in bivalves. Albentosa et al. (1994)
showed the optimum temperature for growth of Venerupis pullas-
tra spat was about 20 "C when sufficient food is available. When
the temperature rose to 25 "C. ingestion decreased, ammonia ex-
cretion and respiration rate increased, and the smallest individuals
were more sensitive to these changes. Shpigel and Fridman ( 1990)
studied growth and gonad cycle in the clam Ruditapes philippi-
naniiii held m eflliicnt water from an intense fish cultivation in the

Growth of V. pullastra in Fish Farm Effluent

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20 October

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OOCYTE DIAMETERS (urn) OOCYTE DIAMETERS (Mm)

Figure 3. Distribution of oocyte size-frequencies (classified in size ranges of 5 fim) at each sampling of Veiterupis pullastra seed reared in 2 x h
wastewater flow.

954

Jara-Jara et al.

TABLE 2.
Contingency table of adjusted residuals for oocyte size-frequency data in Venerupis pullastra reared in a turbot culture effluent.

Oocyte diameter size

class (^m)

Date

0-5

5-10

10-15

15-20

20-25

25-30

30-35

35-40

40-45

45-50

50-55

30 June

-14.54

-7.47

-4.15

4.32

6.33

4.69

4.43

7.65

1.39

-3.10

-2.06

14 July

-22.66

-5.30

-10.78

-0.56

4.57

12.59

24.16

6.79

-3.82

-7.56

-2.48

28 July

80.46

-8.88

-13.09

-11.96

-11.71

-11.46

-11.06

-9.64

-7.05

-4.63

-1.65

1 1 August

-10.93

-7.38

-8.71

-9.94

-4.91

-3.97

0.99

9.82

31.80

31.23

3.52

25 August

-17.52

44.85

27.57

1.47

-5.92

-11.23

-12.92

-11.22

-10.37

-7.29

-2.60

8 September

21.93

-15.19

-7.30

0.26

4.74

5.92

-6.75

-4.33

-2.30

-3.67

0.72

21 September

-13.98

-9.61

-5.92

3.44

2.38

-2.37

2.17

11.81

8.72

12.79

8.03

20 October

-9.72

3.33

20.02

8.77

-0.62

-0.98

-6.75

-9.41

-7.17

-5.11

-1.82

Po.sitive residuals are in bold type.

Gulf of Eilat (Aqaba, Israel) and showed the importance of tem-
perature in evaluating culture under such conditions. They showed
that the high temperatures (>27 "O and salinities of 40 ppt. which
occur in the Gulf of Eilat in July and August, produced high
mortality rates and a decrease in growth of Ruditapes philippi-
minim. However, the lower temperatures and salinities found in
this study are more compatible for culture of this species. As a
consequence there was better growth, lower mortality rates, and
normal development of the gonadal cycle.

Although oxygen levels were greater in the tanks with higher
effluent flows, the injected oxygen in the fish tanks ensured that
good levels reached the clams and were not a limiting factor for
growth.

Differences in growth may be attributable to differences in
available food in the experimental tanks. The high ingestion rate of
Venerupis pullastra seed (Beiras et al. 1993) might explain this.
Poor results found in the August 25 sample for the 2 x h and
4 X h tanks are likely to have been due to the intermittent water
supply for 5 days caused by pump failures. This would cause a
lower effluent flow and a reduced amount of food. This reduction
in growth was not found in the 1 x h tanks where increases in both
length and weight were recorded. There are two possible explana-
tions for this difference: smaller size of the individuals in the
1 X h tanks (mean live weight of 91 1.3 ± 258.0 mg so and mean
length of 18.9 ± 1.8 mm so) and a 20% mortality that occurred in
the smaller aniinals in these tanks that would produce an increase
in mean length and weight. The mortality may have been due to
lack of food and more importantly to high levels of metabolites
(Albentosa et al. 1994) in these tanks due to lower flow rates.

Results of the analysis of variance for weight, length and height
of the seed indicated that significant differences existed (P < .05)
between tanks 2 x h and 4 x h, but that growth in 4 x h tanks was
only 18% greater than the 2 x h tanks although it received twice
the amount of effluent.

Results showed thai condition index increased with higher
Hows of effluent. Condition index in animals depends primarily on
the amount of food available in the environment (Lucas and
Beninger 1985). Condition index of clams in the 1 x h tanks
declined at the beginning of the experiment. This may have re-
sulted as a consequence of the acclimation period where the seed
was held in a higher water How (1,120 l./h). Condition indices of
clams held in tanks with Hows 2 x h and 4 x h were much greater
than those of clams in the control tanks and showed a progressive
increase in value, except between August 1 I and 25 for reasons
already given. In general, condition indices of seed held in effluent

water were superior to those of seed held in the natural environ-
ment. Brown and Hartwick (1988) studied the effect of tempera-
ture, salinity and food on Crassostrea gigas culture and found that
thickness of the shell is directly related to food abundance. When
food was scarce, growth and dry weight of the soft parts were low
with respect to dry weight of the shell and the internal volume was
reduced thus increasing shell thickness.

Many investigators have studied the influence of quantity of
available food on gametogenesis in inarine bivalves (Sastry 1975,
Bodoy et al. 1980, Himmelman 1980, Maitre-Allain 1981,
Beninger and Lucas 1984, Wilson and Simmons 1985, Ruiz et al.
1992). Velez and Epifanio (1981) reported that the experimental
manipulations of food quantity changed the reproductive cycle of
the mussel, Penui periia. Newell et al. (1982) studied growth and
reproduction of Mytiliis ediilis from different locations with the
same temperature characteristics and showed that individuals ex-
hibited different reproductive patterns that were due to differences
in trophic levels (measured as chlorophyll a), which could greatly
delay the reproductive cycle at the different sites.

Perez-Camacho (1980) showed that Venerupis pullastra had a
characteristic rapid growth in a relatively short period of time,
which probably explains why this species reaches sexual maturity
at such an early age compared to other similar species such as
Ruditapes decussatus (Figueras 1957).

When food is abundant, storage of energy reserves is simulta-
neous with fast maturation of the gonad, spawning, and a rapid
regeneration of the gonad to maturity. In our experimental tanks,
there was an abundant and constant supply of food and the sea-
sonal rapid recovery of the gonad found in the August 1 1 sample
after the first spawning occurred between July 14 and 28.

Finally, the gonadal cycle of clams held in the effluent from
fish ponds developed without apparent disruption. Total matura-
tion and apparent normal development of gametes was observed:
thus a complete life cycle of Venerupis pullastra may be attained
by holding them in effluent fish ponds. We believe this was pos-
sible because of the favourable and stable parameters of the efflu-
ent (temperature, salinity, oxygen, chlorophyll a. etc.) that oc-
curred throughout the evperimcnt.

ACKNOWI.KDGMENTS

This research was supported, in part, by a grant from Ministerio
dc Industria, Comcrcio y Turismo, Spain (CDTI-7()9/91 ) and by
fellowship to R. Jara-Jara from Instituto de Cooperacion
Ibcroamericana. Scientific contribution no. 001/2000 of the Insti-
tuto de Acuicullura, Universidad de Santiago de Compostela.

Growth of V. fuliastra in Fish Farm Efficient

955

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Ji'iirikil ot Shfllt'ish Rcsciirh. Vol. 14, No. 2, 957-%2, 2000.

BALANCING TRADEOFFS BETWEEN PREDATOR PROTECTION AND ASSOCIATED

GROWTH PENALTIES IN AQUACULTURE OF NORTHERN QUAHOGS, MERCENARIA

MERCENARIA (LINNAEUS, 1758): A COMPARISON OF TWO COMMON

GROW-OUT METHODS

JONATHAN H. GRABOWSKI,' SEAN P. POWERS,' AND
MARK HOOPER-

'University of North Carolina at Chapel Hill

Institute of Marine Sciences

Morehead City. North Carolina 28557
'Hooper Family Seafoods

Smyrna. North Carolina 28579

ABSTRACT Poor survivorship of Mercenaria mercenaria seed clams is the chief obstacle hindering economically successful
grow-out operations of hard clams. While much effort has been invested in decreasing such mortality, increasing protection of clams
often results in a reduction in their growth. We examined how the mortality and growth of seed clams during the first year of culture
differed between two common grow-out methods: tented, nylon-mesh bags and mesh-covered bottom areas. We also determined how
initial seed size modified these relationships. Specifically, three initial seed sizes (SL = 10.9, 13.0, and 14.8 mm) were grown
separately in 1.2 x 1.2 m (9.4-mm) mesh bags tented with a 30-cm-long PVC stake in the center. At the same time, two initial seed
sizes (SL = 12.8 and 14.9 mm) were grown in 1.2 x 1.2 m bottom beds covered with 7.0-mm polypropylene mesh. All treatments
were stocked with 700 seed clams. Clam survival in tented bags was enhanced by increasing initial planting size. Small seed (10.9 mm)
had a mean survival rate of 76.69^ and large seed ( 14.8 mm) had a mean survival rate of 93.1'J. Survival was greater in nylon bags
than mesh-covered bottom beds when similar initial seed sizes were used. When data for medium ( 13.0 mm) and large (14.8 mm) initial
seed sizes were pooled, the mean survival in tented bags was 90.1%. Survival in the mesh-covered bottom-beds for similar initial clam
sizes (12.8 mm and 14.9 mm) was 71.7%. Clam growth (in SL) was 21.7 % greater within mesh-covered bottom beds than in nylon
bags. Increasing initial seed size enhanced clam growth slightly in both grow-out methods. This study demonstrates a method to
increase survival using tented nylon-bags for the first year of grow-out as compared to mesh-covered bottom-beds; however, there is
a growth penalty associated with this increased survival. The project also suggests a minimum initial seed size (>12 mm SL) that should
be attained from a nursery system before planting in tented bags or bottom beds because greater survivorship associated with larger
initial clam size more than compensated for the increased cost of planting larger seed clains.

KEY WORDS: Merceniirin mercenaria. quahog, aquacultutre, survivorship, growth penalty, flow, bottom culture

INTRODUCTION

There has been substantial interest and growth in hard clam
(Mercenaria mercenaria) aquaculture in several states along the
Atlantic and Gulf Coasts over the last three decades. Despite in-
creased awareness of the potential of aquaculture and reports of
success in clam aquaculture in some states (notably Florida and
Virginia), growers in many areas, including North Carolina, have
not fully realized the potential of the industry (Diaby 1997). Per-
haps the greatest inhibitor to the establishment of an economically
viable culture industry is controlling clam mortality caused by
predatory crabs (Carriker 1959, Menzel et al. 1976, Whetstone and
Eversole 1978, Castagna and Kraeuter 1981. Walker 1984, Gib-
bons and Castagna 1983. Peterson et al. 1995, Marelli and Arnold
1996. Kraueter et al. 1998, Smith and Langdon 1998). Although
several techniques have been propcsed to decrease predation. it
still remains a critical factor in determining the success of clam-
aquaculture operations (Fernandez et al. 1999). While the success
of many of these predator-exclusion techniques has varied, the
methods that have been successful at increasing clam survivorship
are generally associated with reduced clam growth (i.e. a growth
penalty). Unfortunately, several studies have failed to examine the
relationship between the efficacy of predator-exclusion methods
and any associated growth penalty of using such devices (e.g.
Castagna and Kraueter 1977, Gibbons and Castagna 1985, but see
Peterson et al. 1995, Kraeuter et al. 1998).

Of the methods proposed to decrease predation on seed clams.

three of the most common are the use of mesh netting to cover
clam beds (e.g. Manzi et al. 1981, Kemp 1991 ), the use of gravel
or shell hash (e.g. Castagna and Kraeuter 1977, Summerson et al.
1995), and more recently, the use of nylon-mesh bags (Kraueter et
al. 1998, Fernandez et al. 1999). Summerson et al. (1995) docu-
mented a substantial increase in survivorship with the addition of
gravel substrate to clam beds compared to beds without gravel. Yet
the percentage of marketable clams after 36 mo was 20% to 25%
higher in beds without gravel substrate compared to beds with
gravel at the same planting density. The mechanism by which the
introduction of gravel reduces growth has yet to be fully explained;
however, disruption of the fluid boundary layer (Weissburg and
Zimmer-Faust 1993) may be partially responsible. Reduced
growth may also be a problem when using mesh netting or cages
if fouling algae and animals settle onto the mesh (as is the case in
many aquaculture areas). Fouling algae reduce the flow of water
and consequently the delivery of food to animals under the mesh
(Wildish and Kristmanson 1984, Paul and Davies 1986). Encru.st-
ing animals that settle on the mesh may also directly compete with
clams for food (Peterson 1979, Fernandez et al. 1999). Growth
reductions may be further exacerbated by planting seed at densities
that are too high because food depletion of bivalves at higher
densities has been shown to be problematic (Peterson and Black
1987, Summerson et al. 1995). Planting seed at high densities can
also result in reduced survivorship as a consequence of density-
dependent predation by crabs (Eggleston et al. 1992). Clams

957

958

Grabowski et al.

grown in mesh bags have shown reduced mortality in seed grow-
out trials (Kraeuter et al. 1998, Fernandez et al. 1999); however,
comparisons of survivorship and growth with alternative grow-out
methods have yet to be examined.

Other techniques that have received attention include the use of
rafts, cages, (Manzi et al. 1981 ), grow-out trays or racks (Eldridge
et al. 1976, Eldridge et al. 1979), and biological controls. Biologi-
cal controls tested include: ( 1 ) the introduction of a predator that
consumes crabs, but not clams (Castagna and Kraeuter 1981, Jory
et al. 1984, Bisker and Castagna 1989) and (2) increasing the size
of seed clams at planting (Kraeuter and Castagna 1985, Peterson
1990, Peterson et al. 1995). In a previous demonstration project,
we found that initial seed size appeared to affect the eventual
survival (Hooper unpubl. data). These findings are in general
agreement with others who have suggested survival differences
related to initial seed sizes (Kraeuter and Castagna 1985, Peterson
et al. 1995). If initial seed size is directly related to survival, the
nursery phase of clam aquaculture could be adjusted to ensure
maximum survival.

In this paper we report the results of grow-out experiments that
examined the importance of initial seed size and the efficacy of
tented, nylon-mesh bags in bottom culture of M. mercenaria. The
wide use of mesh netting to cover bottom areas warranted the
comparison of this "industry standard" with the nylon-bag tech-
nique. Specifically, this paper examines whether there are differ-
ences in terms of survival, individual growth, or total yield be-
tween bottom grow-out of clams using tented, nylon-mesh bags
(nylon bags) and traditional mesh-covered bottom areas (bottom
beds). Further, we investigate how these relationship change with
size of the initial seed clams. We predicted that any increase in
survival would outweigh any growth penalty when seed clams
were grown in nylon bags as compared to bottom beds.

classes were significantly different among the three size treatments
(F,y = 542.5; />< 0.0001).

In October 1998, we also placed seed clams in bottom beds ( 1 .2
X 1 .2 m) and covered the beds with 7-mm polypropylene mesh that
was held in place with a 1 .3-cm rebar frame staked at the corners.
For this experiment, we used two size classes of seed clams in
separate plots (medium, mean SH = 12.8 mm and large, mean SL
= 14.9 mm) and scattered the clams under the mesh at the same
densities as in the nylon bags (700 clams). There were five bottom
beds of each initial size (10 bottom beds total). Random samples
of 100 clams were measured for SL from two of the five bottom
beds of each initial size (medium and large). A two-factor
ANOVA confirmed that the two size classes differed in initial SL
(F, g = 359.5; P < 0.001 ), but within each initial seed size (me-
dium and large), mean SL did not differ between bottom beds and

nylon bags (F,

.03; P = 0.34). Thus our overall design

allowed for comparisons between the two different methods (nylon
bags and bottom beds) and two initial seed sizes (medium and
large). The clams were planted in shallow water (<1 m MLW) and
on a substrate of firm sand. The 30 nylon bags and 10 bottom beds
were interdispersed by assigning random positions within a sub-
plot of North Carolina shellfish lease 9102, located close to the
premises of Hooper Family Seafood.

In October of 1999, all nylon bags and bottom beds were har-
vested. Live clams in each treatment were counted and a random
sample of 100 clams was measured for SL. Additionally, a volu-
metric measurement was taken for each treatment. This involved
determining the displaced water volume of 50 randomly selected,
live clams from each graded size class for all bags in these treat-
ments in order to estimate the entire volume of each replicate.

Statistical Analyses

MATERIALS AND METHODS

Experimental Design

In August 1998, seed clams (4-6 mm) were obtained from
Atlantic Farms, Inc., South Carolina and placed into a nursery
system on the premises of Hooper Family Seafood, Smyrna, Car-
teret County, North Carolina. By October 1998 the clam seed had
grown sufficiently to be graded into three distinct size classes for
our experimental purposes (small, mean SL = 10.9, medium.
mean SL = 13.0, and large, mean SL = 14.8 with SL being the
maximum measurement along the anterior-posterior axis). Differ-
ences in clam seed size at the end of the nursery phase were
attributed to slight variations in flow within the raceways during
the previous two months.

In October 1998, three sets of 10 nylon bags of mesh si/e 9.4
mm (stretch) and measuring 1 .2 x 1 .2 m (4 x 4 ft) were filled with
700 .seed clams. Our experimental density was similar to the den-
sity recommended in Fernandez et al. (1999). Each of the three sets
of nylon bags corresponded to one of three size classes of seed
clams (small, medium, and large). After filling, each nylon bag
was sealed with a cable tie, slaked down on each corner, and raised
in the center with a 30-cm-long PVC stake which projected 20 cm
above the substrate surface. In January of 1999, ihe center stake of
each nylon bag was removed. A random sample of 100 clams was
measured for SL from four of Ihe 10 nylon bags in each size
treatmcnl (small, medium, and large) at the beginning of the ex-
perimenl. A one-faclor ANOVA confirmed thai the initial si/e

Data were analyzed using either one- or two-factor ANOVA
for clam survivorship and growth. A two-factor ANOVA was
conducted to as.sess whether grow-out technique (bottom beds ver-
sus nylon bags), initial seed size (restricted to medium versus large
for this analysis), or their interaction affected survivorship. Our
experimental design prevented comparison of all three size classes
in the two-factor ANOVA because our bottom bed trealmeni did
not include the small seed clam size class. In order to determine if
initial seed size affected the survivorship of .seed clams among all
three size classes (small, medium, and large) within nylon bags, we
performed an additional one-factor ANOVA.

Similar analyses (a two-factor ANOVA for the effect of seed
size and grow-out technique, as well as a one-factor ANOVA for
the effect of seed size within the nylon bags) were also conducted
for the following growth parameters: total clam volume, mean
individual clam volume, and mean individual shell length. The
data sets in the two-factor ANOVA's assessing the effect of grow-
out technique and the interaction between grow-out technique and
initial size on clam survival and growth were unbalanced (/i = 5
for bottom beds versus ;; = 10 for nylon bags). To overcome this
polenlial problem with our analysis, we balanced the data sets
according to methods given in Underwood (1997). Our conclusion
and the ANOVA tables presented in this paper are based on the
balanced data sets. Prior to any of these analyses, data were tested
for homogeneity of variances using Cochran's lesl. In no case was
transformation necessary and analysis proceeded with the original
data. Post hoc contrasts were performed on all significant main
effects delected by the ANOVAs or within each group if an in-

Tradeoffs in Hard Clam Aquaculture

959

teraction was sigiiilicant using Fisher's PLSD test (Day and Quinn
1989).

RESULTS

TABLE L

Results of the two-factor ANOVA testing whether clam survivorship

varied in response to grow-out method, initial seed size (SL), or

their interaction.

Survivorship

Survivorship differed between grow-out methods. Survivorship
also differed among initial seed sizes within the nylon bag treat-
ment, but only when small seed clams were included in the analy-
.sis. For clams grown in nylon bags, a significant trend of increas-
ing survivorship with larger initial size was detected (Fig. 1; one-
factor ANOVA F2.27 = 3.50; P = 0.044). Post hoc tests
demonstrated that survival was significantly higher in bags seeded
with large clams compared to bags seeded with small clams (P =
0.014 for Fisher's PLSD): survivorship of medium-sized clams in
nylon bags seeded did not differ between either the large or small
clams {P > 0.05 for both comparisons). Comparison of the method
of grow-out and initial size (medium and large) demonstrated a
significant effect of grow-out method {P < 0.001 ). but no signifi-
cant effect of seed size {P = 0.636) or interaction between seed
size and grow-out method (P = 0.355: Table 1 = results of
two-factor ANOVA). Survivorship was higher in nylon bags
(90.1%) than in bottom beds (71.7%; Fig. 2).

Growth

Within the nylon bags initial size of seed clams had no effect on
mean final SL (F-,,^ = 2.10; P = 0.14) or individual volume
(F, ,7 = 1.37; P = 0.27), but did have an effect on total volume
of all surviving clams (F, ,7 = 5.05: P = 0.01 ). Individual SL of
surviving clams ranged from 32.6 to 31.5 mm (mean SL for large
= 32.6 mm, medium = 32.3 mm, and small = 31.5 mm).

For the two-factor ANOVA, which tested the effect of both
grow-out method and initial clam size, final SL, individual vol-
ume, and total volume of clams differed between grow-out meth-
ods (F < 0.05; Table 2). While individual volume and total volume
did not differ with initial seed size (P > 0.05), the effect of initial
seed size (P = 0.038) was significant for final SL. No significant
interactions between initial seed-size and grow-out method were
detected; however, there was a marginally significant trend {P =

100
90

Factor

df

SS

80
70

g 60

3 50
CO

i 40
^ 30 -

20 1

10

Large

(14 8 mm)

Small
(10 9 mm)

Initial Clam Size (SL)

Figure. 1. Percent clam survival in nylon bags after 1 y of grow-out:
large, 93.0% ; medium, 87.2%; and small, 76.6%. Error bars denote +1
SE (II = 10 for each seed size).

Bed vs. tent

1

1541.70

1 1 .73

0.0086

Seed size

1

69.74

0.44

0.5670

Bed vs. tent x seed size

1

137.90

0.87

0.4668

Residual

16

227.';.95

0.060) for the interaction when final SL was examined. Individual
clams attained greater SL (Fig. 3) and had higher individual vol-
ume (Fig. 4) in the bottom beds than in the nylon bags. Total
volume of all surviving clams was also higher in bottom beds
compared to nylon bags (Fig. 5). Final SL was the only variable
that demonstrated a significant effect of initial seed size. Overall,
clam SL was significantly higher in bags (mean 32.6 mm) or beds
(mean 40.9 mm) seeded with larger seed clams compared to bags
(mean 32.3 mm) or beds (mean 38.1 mm) seeded with medium
seed clams (Fig 3). This pattern appeared more pronounced in the
bottom bed treatment than in the nylon-bag treatment: the P value
for the interaction was marginally significant at the P = 0.05
level.

DISCUSSION

Our study compared two of the most common grow-out meth-
ods in aquaculture of hard clams: tented, nylon-mesh bags, used
primarily along the lower Atlantic Coast, and mesh-covered bot-
tom beds, common in Mid-Atlantic States. While survivorship was
considerably higher for clams grown in the nylon bags (-hi 8.4 %)
compared to survivorship of clams grown in bottom beds, our
overall survivorship in the first year of grow-out was still high with
either method (>70%). This high survivorship was most likely a
function of the large initial size of our seed clams (>10.9 mm).
Survivorship increased with further increases in seed size of clams.
While survivorship of large (14.8-14.9 mm) and medium-sized
clams (12.8-13.0 mm) did not differ between grow-out methods,
small seed clams (10.9 mm) had significantly higher mortality than
large seed clams. Similar relationships with initial size of seed

100

in

E

Bottom Beds Nylon Bags

Treatment
Figure, 2. Percent clam survival in bottom beds (71.7%) versus nylon
bags (90.1%) after 1 y of grow-out. Error bars denote +1 SE (n = 10
for clams grown in bottom beds and n = 20 for clams grown in nylon
bags).

960

Grabowski et al.

TABLE 2.

Results of the two-way ANOVAs testing whether final individual clam SL, individual volume, and total volume varies in response to grow

out method, initial seed size (SL), or their interaction.

Final Individual Clam SL

Individual Volume

Total Volume

df

SS

F

P

SS

F

P

SS

F

P

Bed vs. tent

1

252.49

126.53

<.0001

104.55

60.74

<.0001

8861290.72

8.85

0.0127

Seed size

1

10.66

5.37

0.0376

3.82

2.16

0.1718

1742160.53

1.56

0.2859

Bed vs. tent x seed si/e

1

S.61

4M

0.0596

3.33

1.96

0.2065

321309.25

0.37

0.6586

Residual

16

3.1.37

27.79

16543835.07

clams and survivorship have been shown by others (Walker 1984,
Kraeuter and Castagna 1985, Peterson et al. 1995. Marelh and
Arnold 1996). The increase in profit as a result of higher survi-
vorship of large seed clams more than coinpensates for the addi-
tional cost of achieving larger clams in raceways before planting
(assuming 95% survivorship after year 1 for all clam sizes). There-
fore, evidence from our study indicates that the optimal size for
planting in North Carolina is between 12.8 and 14.8 mm (SL). In
order to compare our findings with other studies which report clam
planting sizes in SH (with SH being the maximum measurement
from the umbo to the ventral margin), we estimated the ratio of
SH/SL at .875 from empirical data and converted our optimal
range to SH (-11.2-13.0 mm). This approximate size range is
smaller than the 15- to 20-mm (SH) range reported by Menzel et
al. (1976), Whetstone and Eversole ( 1978), Eldridge et al. (1979),
and Walker (1984), but substantially larger than the 8-mm (SH)
size that Marelli and Arnold (1996) reported as the size where
clam predation becomes insignificant in Florida's Indian River
lagoon. Optimal planting size is influenced by factors such as the
local predator community and growth rates (which will vary with
food availability and food delivery rates), limiting the appropri-
ateness of one particular seed size for multiple regions. Further
research on the inlluence of these factors on seed survival coupled
with a greater understanding of the local dynamics of a system

Initial Clam Size

■ Large (14.9mm)
n Medium (12.9 mm)

Bottom Bed Nylon Bag

Treatment
Figure. 3. Final clam shell length (mlllimelers) lor large and niediuni
seed clams in hotloni beds and in nvlon hags alter I > of grow-out.
Error bars denote +1 SF (;i = 10 for each size class within nylon bags
and 5 I'or each one within hiiltom hedsl.

should enhance predictive models about the profitability of clam
aquaculture ventures for any specific region.

Differences in growth between the two grow-out techniques
were opposite of the survivorship results (higher growth occurred
in bottom beds, which had lower survivorship), a pattern that has
been reported in other studies (e.g. Summerson et al. 1995). Over-
all, growth was 21.7% higher in bottom beds than in the nylon
bags. After I y, large seed clams had a final SL of 41 ±2 mm and
medium size clams had a final SL of 38 ± 2 mm in bottom beds,
whereas in the tented bags, large seed clams had a final SL of 33
± 1 mm and medium-sized seed clams had a final SL of 32 ± 1
mm. Final differences between large and medium-sized seed clams
in bottom beds reflected the initial size differential of the two
groups (-2.1 mm). The initial difference between size in seed
clams grown in tented bags was virtually absent at the end of 1 y
of growth. These results suggest that growth rate for large and
medium-sized seed clams was reduced in comparison to small seed
size, u pattern that was not evident in the bottom beds.

Given that the patterns in growth and survivorship were oppo-
site, the question of whether differences in growth can be ex-
plained by density dependent factors merits attention. Fernandez et
al. ( 1999) examined the effect of density on growth, measured in
terms of shell length, for clams grown in bags similar to the nylon
bags we used in this experiment. Their results showed no effect of
density when seed clams were placed in bags at densities between

Bottom Bed

Treatment

Nylon Bag

Figure. 4. Individual clam miIimiic after 1 j of grow-out in bottom bed
clams (11.74 niL/surviving clam) and tented hag clams (7.194 niL/
surviving clam). F.rror bars denote +1 SF (n = 10 for clams grown in
hotliim beds and n = 2(1 for clams grown in nylon bags).

Tradkoffs in Hard Clam Aquaculture

961

7000

Bottom Bed

Nylon Bag

Treatment
Figure. 5. The mean volume of clams after 1 y of grow-out in bottom
beds (5810.7 mL) and in tented bags (4550.1 niL). Error bars denote
+1 SE (H = 10 for clams grown in bottom beds and n = 20 for clams
grown in nylon bags).

750 and 1.250 clams/bag. densities higher than those used in our
study. Further support for our conclusion that the differences in
growth between grow-out methods were not derived from differ-
ences in density is provided by the examination of our volume
data. Individual volume of clams (total volume divided by the
number of clams surviving) also showed a significant effect of
grow-out method with individual volume higher in bottom beds
than in bags. Total volume of all clams within each bag or bed, a
parameter that incorporates both survivorship and growth, also
differed between grow-out method. If growth were reduced be-
cause of density-dependent resource depletion, we would expect
both methods to yield similar total volumes of clams (i.e. if re-
source depletion was a factor, decreases in the number of clams
should allow surviving clams to grow faster); however, total vol-
ume was greater in the bottom bed grow-out.

Differences in clam growth between the two methods were
probably a function of variation in water flow patterns resulting
from differences in the physical structure of the methods. Changes
in water tlow alter the tlux of food particles (the product of hori-
zontal advection and concentration of food) over an area usually
resulting in differential growth patterns (Wildish and Kristmanson
1997). Differences in mesh size could potentially affect the supply
of food; however, our results were opposite what would be pre-
dicted under this scenario: mesh size was larger for bags (9.4-mm
opening) than bottom mesh (7.0-mm opening). The profile caused
by the tenting of the bags would create a strong reduction in flow
within the mesh area. Compared to the bottom beds, which would
have fairly uniform, more laminar flows, the tented bag would
create an area of decreased tlow under the bag canopy. Such a
difference in flow regime could result in differences in growth
between the two methods. Although tenting the bags may drive the

pattern of growth differences, tenting was shown to substantially
decrease predation in previous trials (Hooper unpubl. data). In
these trials, tenting the bags seemed to reduce crab access to clams
under the canopy. The survivorship that we report with tenting
(77%-93%) is slightly higher than that reported by Fernandez et al.
(1999) (75%-879'f).

A comparison of the economic feasibility of these two grow-
out methods for clam aquaculture should consider differences in
cost between grow-out methods in addition to the economic im-
plications of the tradeoff between clam growth and survival. The
material and labor costs of growing clams in bags are approxi-
mately $0,014 per clam higher than the cost per clam in beds.
Therefore, bags must increase clam survivorship by 1 1.6% in ny-
lon bags to compensate for the higher cost of growing clams in this
method (assuming a 5% discount rate, clam price of $0.15 per
clam. 957^ survivorship after first year of grow-out. and 3-year
grow-out phase). If increased clam growth in bottom beds during
the first year of grow-out results in clams achieving legal size more
rapidly (i.e. greater percentage of legal clams after 2 and 3 y),
nylon bags must increase clam survivorship more than 11.6% to
compensate for the economic consequences of the growth penalty
during the first year of grow-out. In our study, projections of
expected profits suggest that the nylon bag method is more prof-
itable by $0,016 per planted clam than growing clams in bottom
beds even after adjusting the expected percentage of legal clams in
subsequent years to account for the growth penalty exhibited
within nylon bags.

Certain environmental and economic factors should also influ-
ence a grower's decision when selecting a grow-out method: when
growing conditions favor high clam growth rates and/or predation
intensity is very high, enhancing survivorship could increase a
grower's return on their investments. On the other hand, when
clams are marketed by weight rather than by count, when risk of
clam loss to theft or from hurricane (a particular concern for south
Atlantic states) damage is high, and/or when interest rates are high,
using a method capable of growing clams more rapidly could
increase a grower's profit margin. Studies determining whether
growing larger clams in the first year of grow-out necessarily
results in achieving a higher proportion of legal clams after 2 and
3 y of culture are needed to more accurately assess the economic
consequences of this tradeoff between growth and survival exhib-
ited by the two methods.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the assistance of B. Wood-
ward, M. Dolan, and D. Kiinbro in the field. The manuscript
benefited from comments provided by C.H. Peterson, S. E. Shum-
way. and two anonymous reviewers. Support for this research was
provided by the North Carolina Fisheries Resource Grant Program
administered by the North Carolina Sea Grant College Program.

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THE REPRODUCTION CYCLE OF TRIDACNA SQUAMOSA AND TRIDACNA MAXIMA IN
RENGIS ISLAND (TIOMAN ISLAND), MALAYSIA

SHAU-HWAI TAN AND ZULFIGAR YASIN

Muka Head Marine Research Station
Centre For Marine and Coastal Studies
Universiti Sains Malaysia
11 HOG Penan ji. Malaysia

ABSTRACT Hypodermic extraction of gonadal materials was taken from Tridacna squamosa and TriJacna maxima populations
on Rengis Island (south east of Malaysia in the South China Sea) at approximately monthly intervals between March 1996 and April
1997 to investigate the seasonality of reproduction. A spawning sea.son extending over several months was indicated for both species.
The influence of environmental factors such as temperature and salinity on the reproductive cycle of these two species are discussed.

KEY WORDS:

Reproductive cycle, Tridacmi sqiiaiiKisa. Tridacna maxima

INTRODUCTION

The populations of giant clams (Family: Tridacnidae) in the
Indo-West Pacific is declining due to the combined effects of
pollution, environmental degradation, as well as harvesting for
commercial and subsistence purposes (Munro 1983). The distri-
bution and abundance of giant clams in Malaysia have been sur-
veyed and documented in Redang Island, Terengganu (Mohamed-
Pauzi et al. 1994), Pemanggil Island, Johore (Zulfigar and Tan
1995, Zulfigar and Tan 1996a, Zulfigar and Tan 1996b), and Ti-
oman Island, Pahang (Tan et al. 1998). Low densities of giant
clams are found in Malaysia coastal waters and the stocks are
gradually decreasing (Zulfigar and Tan 1993). An estimate of 2
clams/ 1 00 nr T. squamosa was found in Redang Island and Kapas
Island and 8 clam.s/100 m" T. squamosa was found in Perhentian
Island (Mohamed-Pauzi et al. 1994). Tan et al. (1998) have re-
ported an average of 6 clams/ 1 00 m" T. squamosa. 13 clams/ 1 00
m- T. maxima, and 21 clams/100 m" T. crocea in Tioman Island.
Only three species of giant clams were reported on Tioman Island.
which are T. squamosa. T. maxima, and T. crocea, whereas Hip-
popus hippopus species can only be found on the Johore Islands
located south of Tioman Island. T. squamosa and T. maxima are
considered endangered while stocks of T. crocea are more abun-
dant. In light of this, restocking of T. squamosa and T. maxima is
needed. Detailed knowledge of the gametogenic cycle will provide
necessary data for the successful production of seeds via hatchery
techniques. Reproduction studies involving examination of gonad
samples from natural populations of giant clams are very limited in
Malaysia, The objective of this study is to characterize the repro-
ductive cycles of T. squamosa (Lamarck 1819) and T. maxima
(Roding 1798) at Rengis Island.

MATERIALS AND METHODS

Rengis Island is a small island to the west of Tioman Island, in
the southeastern part of Peninsular Malaysia (Fig. I). Tioman Is-
land has been gazette as a Marine Park by the Federal Government
of Malaysia since 1 984. Very few scientific or management studies
have been carried out on the island. Rengis Island is one of the
very few islands on the Peninsular Malaysia with relatively good
stock of giant clams (Tan et al. 1988). The island is surrounded by
fringing reef, which provide a suitable habitat for the giant clams.

Giant clams are functional hermaphrodites (Wada 1952), first
reaching sexual maturity as males, then later developing ovaries
which produce eggs and function simultaneously with the testes.

The study on the reproduction cycle of T. squamosa and T.
maxima was conducted from March 1996 to July 1997. Sampling
was done using SCUBA. The gonad biopsy technique described by
Braley ( 1984; Fig. 2) was used in this study. A 20-cm hypodermic
needle with a plastic plunger was used to extract approximately I
to 2 g of gonadal tissue from the clams. The needle was inserted
vertically into the gonad through the mantle, entering the gonad
several centimeters anterior to the exhalent siphon to one side of
the medial axis of the clam. The first 30 animals encountered from
each species were sampled each month. The sizes of the clams
sampled ranged from 35 to 65 cm in shell length

The extracted gonadal material was examined immediately us-
ing a light microscope. Egg size was determined under a com-
pound microscope, equipped with a 50 x 2-micron graticule. Go-
nadal stages for eggs were classified based on a modification of the
scheme described by Braley (1988; Table 1 ).

Figure 1. The study location at Rengis Island.

963

964

Tan and Yasin

B

Adductor and pedal
Retractor (anterior) muscles

Excurrent siphon

Gonad /
visceral
mass

ANTERIOR

Inner and outer
(dorsal) demibranchs

Ventral

Figure 2. Tridacnid clam, dorsal view (A) and side viev» (with right
valve removed, B). The arrow shows the area from which biopsies of
gonadal tissue were taken (modified from Braley 1988).

RESULTS

The results indicated that the regressive stage was predominant
most of the year for both T. squamosa and T. maxima. The hypo-
dermic extraction technique tended to extract regressed gametes
from within follicles in the gonad, thus biasing the outcome to-
wards the regressive stage (Braley 1988). However, other clear
examples of developing, ripe, and resting stages were found in
individual clams, indicating that the hypodermic extraction tech-
nique does give a true picture of the state of the gonad.

Tridacna squamosa

Figure 3 shows the stages of egg development for T. squamosa
over a 17-mo period. It is notable that the regressive stage was
predominant throughout the study period, including August
through November, when ripe gonads were at the peak. The per-
centage of ripe animals recorded is low throughout the study pe-
riod, with the highest being 209'f . Developing and partially devel-
oped biopsy samples were present throughout the year. The num-
ber of clams with developing eggs was the highest in June 1996,
just before the ripe gonads were observed in August 1996. Clams
with resting eggs were present at all sampling occasions.

Tridacna maxima

Figure 4 summarizes the stages of egg development for T.
maxima: the results are similar to T. squamosa. The clams with
regressive eggs predominate throughout the study period, followed

df =,te c» tj" «* <,» a* «» «« =r =f a" ^ ^ ^ <^ J^
J.*" ^O'' ,S»- ^»- ^'" ^•>* *<S tf=- -■>- Nf^ ^'^ •,«' J.*' >•> -■.•"■ •,■>'■ ■>*

MONTH

Figure 3. Stages of egg development for T. squamosaover a 17-mo
period on Rengis Island.

by the clams with resting eggs. Ripe biopsy samples were recorded
throughout most of the study period. A significant proportion of
clams with ripe gonads was observed from April to July 1996 and
from April to June 1997. The highest percentage of the clams with
ripe gonads was ?>b7c in May 1996, followed by 2.5% in May 1997.
The percentage of clams with developing gonads was the highest
in April 1996 and April 1997, just before the appearance of a high
percentage of ripe gonads in May 1996 and 1997. The clams with
resting eggs were present at all sampling periods except in June
1996.

Temperature and Salinity

The water temperature was very stable, and ranged from 29 °C
to 33 °C all year round with daily fluctuations to less than 3 °C
(Fig. 5). The temperature increased from 31 °C to 32 °C in early
March to 33 °C in April. Maximum temperature variation occurred
in the beginning of the dry season during the months of April to
June due to precipitation and evaporation, as well as minimum
turbulence and mixing. The temperature was slightly lower and
fluctuated between 29 °C and 31 °C during the wet season from
late September to March.

The salinity at Rengis Island at a 5-m depth was also very
stable and ranged from 33 ppt (wet season) to 34 ppt (dry season)
throughout the study period (Fig. 5). There was no significant
variation in salinity at Rengis Island because the island is unaf-
fected by runoffs from large rivers along the peninsular coast.

DISCUSSION

Our study indicates that both 7". squamosa and T. ma.xima ex-
hibit reproduction periodicity on Rengis Island. However, the pe-
riod rellecting the highest percentage of clams with ripe gonads

Stage

Developing
Ripe

Regressive (posi spawnin};)
Rfslini;

TABLE 1.
Characteristics of eggs at different stages of maturity after Braley (1988).

Description

Egg diameter up to 1 10 p.ni: generally various sizes in a progressive stale; some developing eggs have a

distinct peduncle, which is the area of attachment to the follicle wall; and the chorion layer is intact
Egg diameter 1 10 p.m or larger; vitellogenesis complete (ova cytoplasm filled with yolk), easily ruptured:

ova have intact chorion layers, occasionally ruptured out of the chorion; and large numbers of ripe ova

somewhal polygonal in shape due to the dense packing inside ovary follicles
Eggs of various sizes, but degenerative; chorion layers often damaged or soniclimes not presenl; and

phagocytic amoebocytes presenl In moderate lo large numbers rcsorbing residual eggs
Lack of any recognisable eggs; and residual gonadal nialcnal and phagocytic aniocbocylos were noiniall\

presenl

The Reproduction Cycle of Tridacna squamosa and Tridacna maxima

965

^%^

(A)

^-^ ^ s- «* =.'>

J.*' ^■J- J.*- s-" >^'' >•>* -'" o'' -°- .o"^ ^''^ ^'' J.*' ^"S" J.'" s<>' ^'^

MONTH

Figure 4. Stages of egg development for T. maxima over a 17-nio
period on Rengis Island.

was different between the two species. The clams with ripe gonads
for T. scjiiamosa were present from August to November 1996. and
for T. mauma it was from April to July 1996 and April to June
1977. The differences in the spawning season between the two
species may be related to their spatial distributions on the reef. T.
maxima usually occurs at water depth of 0.5 m (below Chart
Datum) to 1.5 m. whereas T. squamosa usually occurs at depth of
2.5 to 6.0 m (Tan et al. 1998).

A comparison of the reproduction cycle of both species of
tridacnids showed that seasonality is more prominent in T. maxima
with spawning peak in April to June for both 1996 and 1997.

The season with a high percentage of ripe gonads for T. squa-
mosa recorded in this study (August to November 1996) is similar
to the spawning period of T. squamosa kept in the hatchery in
Prachuab Khiri Khan. Thailand (Nugranad et al. 1997). The results
obtained in this study are also similar to the reproduction study of
T. squamosa in Redang Island, Malaysia (also located on the east
coast of Peninsular Malaysia), where the highest percentage of
clams with ripe gonads was recorded in July and August (Adib et
al. 1993). Another study in Republic of Belau, Western Caroline
Islands found that the peak months of T. squamosa with ripe eggs
were in January through March and August through December
(Fitt and Trench 1981).

At Rengis Island, the temperature and salinity were very stable.
T. squamosa spawned at the beginning of the wet season when the
temperature declined from 33 °C to 32 °C and at reduced salinity
from 34 to 33 ppt. T. maxima spawned at the beginning of the dry
sea.son when temperature and salinity were slightly elevated. How-
ever, the changes in temperature and salinity were not significant
and do not seem to have played an important role in the reproduc-

," <,«> <,<= ,fc c*' <,*> ,*> <,«> 0,* <,*> c'^ ,f- J^ ^ c^ cj^ 4^

' .»* .^» ^-^ > .j/i „t"- .o" -c' .»<■ ,g> ^■^ -o^ .^ .^<- s*

■*■' ^^ ^»' b*

' cf" & «" <f i»" <»" *»• ^<!■ ^»^ i*" S»'

(B)

Figure
Island

tf c" cf <,"<,» <f> <,*> c" cf> (,*> c^ ej^ d^ 4" 4^ 4" 4"
♦»' vS'**'* b-»* i-^ ^>^^ o'^o" <,'' b»* <'"*»' ^'^V*'^ b-* V*

5. Montlily temperature (A) and salinity (B) changes on Rengis
in the South China Sea from March 1996 to July 1997.

tion cycle of giant clams on Rengis Island. Specific environmental
cues may be required before the release of eggs occurs (Braley
1988). Cues such as high phytoplankton blooms or rise in tem-
perature may occur in certain years only, resulting in sporadic
spawning. Clams that do not release their eggs during the repro-
ductive .season could hold the eggs in various states of regression
for several months. In the tropical oyster. Saccostrea cucuUata. the
residual gonadal material may be reabsorbed (Braley 1982). Fur-
ther studies are necessary to determine the specific environmental
cues for the giant clams here.

ACKNOWLEDGMENTS

We gratefully acknowledge the Pulau Tioman Marine Park
Authorities for providing boat services and accommodation in Pu-
lau Tioman during the study period. Special thanks to Universiti
Sains Malaysia's Reef Research Group for their assistance and
dedication to the giant clam project.

LITERATURE CITED

Adib, H.. .\. Pauzi & O. Noordin. 1993. Pembiakan aruhan kima Tiiclciciui
squamosa (Bivalvia: Tridacnidae) di Taman Laut Pulau Redang,
Terengganu, Malaysia. MFRDMD/SEAFDEC Jabatan Perikanan Ma-
laysia, 14 pp.

Braley. R. D. 1982. Reproductive periodicity in the indigenous oyster
Saccostrea cucullata in Sasa Bay. Apra Harbor. Guam. Mar. Biol.
69(.^):165-I73.

Braley, R. D. 1984. Reproduction in giant clams Tridacna gii^as and T.
derasa in situ on the North Central Great Barrier Reef and Papua New
Guinea. Coral Reefs 3(4):22 1-227.

Braley. R. D. 1988. Reproductive condition and sea.son of the giant clams
Tridacna gigas and T. derasa utilising a gonad biopsy technique, pp.
98-103. In: J. W. Copland and J. S. Lucas (eds.). Giant Clams in Asia
and the Pacific. Monograph No. 9. Australian Centre for Inlernational
Agriculture Research. Canberra.

Copland. J. W. & J. S. Lucas. 1988. Giant Clams in Asia and the Pacific.
Monograph No. 9. Australian Centre for International Agriculture Re-
search. Canberra, 274 pp.

Fitt. W. K. & R. K. Trench. 1981. Spawning, development and acquisition
of zooxanthellae by Tridacna squamosa (Mollusca: Bivalvia). Biol.
Bull. I61{2);2l3-23.'i.

Mohamed-Pauzi. A.. A. H. Mohamed. A. Ahmad & Y. Abdul-Aziz. 1994.
A preliminary survey of giant clams in Malaysia. Proc. Fish. Res. Conf.
DOF Mai. IV:487^93.

Munro. J. L. 1983. Giant clams: food for the future? Int. Cent. Living
Aqua. Resources Manag. Newslett. 6(1 ):3^.

Nugranad, J.. T. Traithong. T. Poomtong & S. Sahavacharin. 1997. Hatch-
ery seed production of the Huted giant clam (Tridacna squanwsa
Lamarck 1819) and ocean nursery of the juveniles for restocking in

966 Tan and Yasin

Koh Tao. Thailand. Phuket Mar. Biol. Cent. Spec. Piihl. 17(11:101- Molecular. Biotechnological. and Conventional Approaches. Novem-

108. ber 19-23. 1995. Kuching. Sarawak. Malaysia.

Tan, S.H., Y. Zulfigar. S. Ibrahim & Y. Abdul- A/i/. 1998. Status ot giant Zulfigar, Y. & S. H. Tan. 1996a. Giant clams: a preliminary survey on their

clams in Pulau Tioman, Malaysia. Malaxiin Ncii. J. 58(3 & 4):2()5-216. distribution, abundance and reproductive condition in Pulau Pemang-

Wada. S. K. 1952. Protandric functional hermaphroditism in tridacnid gil. Presented at World Aquaculture '96. January 29-February 2. 1996.

clams. Ocea/ii)i;r. Maf-. Tokyo 4:23-30. Bangkok. Thailand.

Zulfigar. Y. & S. H. Tan. 1995. Giant clams; a preliminary survey on their Zulfigar. Y. & S. H. Tan. 1996 b. Extinction of giant clams in Johore

distribution, abundance and reproductive condition. Presented at the Islands. Presented at Malaysia Marine Park Council Meeting. Decein-

International Symposium and Workshop on Conservation Biology: ber 5-6. 1996. Pulau Besar, Melaka. Malaysia.

Jiiiiimil (It Shellfish Ri-.ccinh. Vol. \9. No. 2. 967-977, 2(K)0.

THE NEW LESSEPSIAN ENTRY BRACHIDONTES PHARAONIS (FISCHER P., 1870) (BIVALVIA,
MYTILIDAE) IN THE WESTERN MEDITERRANEAN: A PHYSIOLOGICAL ANALYSIS UNDER

VARYING NATURAL CONDITIONS

G. SARA, C. ROMANO. M. CARUSO, AND A. MAZZOLA

Marine Biology and Sea Resource Research Group

Animal Biology Department

University of Palermo

Via Archirafl 18. 90123 Palermo. Italy

ABSTRACT The feeding behavior of Brachidontes pharaimis (Mollusca, Bivalvia). a new Lessepsian entry in the western Medi-
terranean, hving in a coohng vat of a saltworks system in western Sicily, was asses.sed by estimating its physiological rates throughout
a 6 month-long study (May 1998 to March 1999). Clearance, filtration, ingestion, and food absorption rates were estimated using the
biodeposition method and the results correlated to variations in temperature, salinity, and quality and quantity of available food.
Measured seston concentrations were on average 81.5 ± 95.5 mg L"'. its labile fraction (estimated as the sum of particulate lipids,
carbohydrates and proteins) was on average 0.55 ± 0.07 (j.g L"'. representing only 15'7f of the total organics. Phytoplankton biomass.
as suspended chlorophyll-a, was on average 0.88 ± 0.4 |xg L"'. Mean weight standardised rates of Brachidoines pbaraonis were
clearance rate 1.64 ±0.82 1 h"'. filtration rate 110+ 107 mg h"' of total suspended material, and egestion rate 0.60 + 0.16 mg material
h"'. There was a mean selection efficiency of 0.50 ± 0.22. Ingested organic matter varied between about 2 mg h"' and 270 mg h"'.
and food absorption efficiency ranged between 0. 1 and 0.99. The Brachidontes feeding process seems to be regulated at the initial
filtration stage, and most of the control determines the quantity of absorbed ration. Clearance rate is maintained independent of changes
in water temperalure and salinity but reflects fluctuations in Ihe quantity and quality of available food. Varying the rate of pseudofaeces
production regulated ingestion rate, although this mechanism be fairly inefficient as a response to local environmental conditions.
Absorption efficiencies may be sensitive to the balance of biochemical components. The physiological plasticity of S. phuraoiiis as
expressed in this study is believed to have played a major role in its ability to reach the western Mediterranean.

KEY WORDS: Lessepsian, Brmhidomes pharaonis. bivalve mollusc, feeding behaviour, shallow environment, Mediterranean sea

INTRODUCTION

The common Indo-Pucific mussel Brachidontes pharaonis
(Fischer P., 1870; = Brachidontes variabilis Krauss 1S481 re-
cently has appeared in the western Mediterranean, reaching the
Sicilian coasts through the Suez Canal (Safriel et al. 1980). This
hyperhaline mussel is a Lessepsian "new entry" (For 1971) which,
with a few other species [e.g.. Portumis pelagicus (Linne 1758)
and Cerithiwn scabridiim (Philippi 1848)]. has been able to cross
the Red Sea Barrier (Safriel and Ritte 1977) and reach the tem-
perature eastern Mediterranean.

B. pharaonis was never found in the Mediterranean before the
opening of the Suez Canal ( 1869). and its penetration and conse-
quent diffusion has been rapid, due possibly to human phoresys.
The species was first recorded in the Eastern Mediterranean 7
years after the opening of the Suez Canal (Fuchs 1878), and ap-
proximately 60 years passed before B. pharaonis was recorded
along the coasts of Lebanon (Safriel et al. 1980). B. pharaonis has
been able to cross the barrier of the Levantine Basin and has
successfully colonized the western Mediterranean basin. It has
now colonized several parts of western Mediterranean coasts (Di
Geronimo 1971. Gianguzza et al. 1997) and is becoming a poten-
tial resource and space competitor (Safriel and Sasson-Frostig
1988) with its Mediterranean ecological equivalent Mytilaster
minimus (Poli). B. pharaonis has been documented in cooling vats
of a saltworks system in western Sicily (Gianguzza et al. 1997.
Vitturi et al. in press), where it has extensively colonized hard
substrates.

Nevertheless, little is known about small mytilids (Brachidon-
tes or Mytilaster). which have a low importance for aquaculture (as
inedible species) but which represent key species in the recycling
of matter and energy in some aquatic ecosystems (Riisgard 1988.
Dame 1996).

To our knowledge, there are no previous studies on trophic
preferences, adaptability, and physiological plasticity in these bi-
valve species. Very few data exist on the Bracliid<mtes genus
(Stern and Achituv 1978). except for some information on B. ex-
ustus (Riisgard 1988). and there are no data on the feeding activity
and physiological features of Brachidontes pharaonis. Morton et
al. (1988) reported only that B. pharaonis. like many other bivalve
molluscs, is an active suspension feeder that filters and sorts par-
ticles (seston) from the seawater.

Analysis of physiological features is considered among the best
tools for investigating plasticity in mollusc bivalves (sensu Bayne
1998). especially when experiments are carried out in situ with
natural food concentrations. The main aims of the present article
are thus to describe for the first time some features of the feeding
behaviour of B. pharaonis estimated using the biodeposition
method (Iglesias et al. 1992 and following related literature) and to
measure food consumption, rejection, ingestion, and absorption
rates throughout an annual cycle in the field. Consequently, the
physiological responses of this species are determined under a
wide range of ambient conditions of temperature, salinity, and
food supply.

Description of Ihe Study Area

This study was carried out between May 1998 and March 1999
in a cooling vat (60.000 m"; 48.000 m': 1 m average depth) of a
saltworks system adjacent to the Stagnone di Marsala shallow
sound in western Sicily (37°52' NORD; 12°28' EAST), B. phara-
onis has extensively colonized the submerged hard substrates on
the saltworks bottom. The soft substrates in the saltworks mainly
have been colonized by Cymodocea nodosa, whereas the hard
substrates are covered with a number of species of macroalgae
such as Lcuirencia papillosa. Padina pavonica. and Acetahularia
acetabulum.

967

968

Sara et al.

MATERIALS AND METHODS
Experimental Design

On the day before the fihration experiments, specimens were
collected randomly from the saltworks. Individuals from a 30 ±
1.5-mm size class were selected, cleaned of epibionts and byssus.
and then placed overnight in small plastic net-bags in the saltworks
water to permit re-acclimatization after the manipulation. In the
early morning of the following day, 14 organisms were carefully
collected from the acclimatized groups and put into 14 small ex-
perimental rectangular chambers (20 x 5 x 5 cm: available volume
about 500 niL) with a V-shape bottom. After the valves of the
mussels had opened and as soon as their filtration activity was
stabilized, the experiments were started. Two chambers without
mussels were used as controls. Filtration measurements were car-
ried out in continuous flow, with water from the saltworks con-
tinuously pumped into a header-tank (about 50 L). The saltworks
water was agitated well by a magnetic stirrer in the tank to avoid
particle settling. The continuous tlow was regulated at about 180
mL h"' (±20 mL), and this flow was maintained by means of a
peristaltic pump placed between the header-tank and the experi-
mental chambers. At this flow rate, little or no sedimentation oc-
curred. Any sedimentation of suspended particles was accounted
for by the control chamber (with no animals). After approximately
1 h of active filtration under continuous flow conditions, water
samples were collected at the end of the experimental chambers.
On completion of the clearance experiments, the organisms were
kept for a further 2-3 h in their respective chambers. Separately,
faeces and pseudofaeces were carefully collected. This material
was filtered onto pre-weighed and ashed GF/F filters, washed with
ammonium formate to remove seawater salts, and processed, as
described later, to determine the total mass and organic content of
the biodeposits.

These experimental procedures allowed clearance rates to be
calculated, by applying the following formula:

CR = f* |(Ci - Co)/Ci| (Labarta et al. 1997, Widdows and
Staff 1997)

where CR = the clearance rate in liters h '

f = the flow rate of water through the chambers (I h"')

Ci = the concentration of labile particulate organic matter
(LPOM: mg L~') in the in-flowing water (determined from the
mean of the concentration of LPOM in the water in the tank and
in the control chamber)

Co = the concentration of LPOM (mg L"') in the out-tlowing
water from the experimental chamber.

The labile fraction of particulate organic matter (LPOM. mg L"')
was calculated from the sum of particulate lipids, proteins and
carbohydrates (Widdows et al. 1979. Fichc/ 1991. Navarro et al.
1993. Saract al. 199H).

In order to confirm the estimates obtained using (his method,
clearance rates were also estimated using the biodeposition method
(Iglesias et al. 1992) as follows: CR = (mg inorganic matter
produced both as true faeces and pseudofaeces per hour) -^ (mg
inorganic matter per liter in saltworks water). All clearance rates
were corrected to a I g dry weight standard-sized individual using
the weight exponent b ( = 0.53) reported in Labarta et al. (1997).
Results obtained using both methods described were compared and
averaged. Production rates of pseudofaeces (namely rejection

rates: RRs. mg h"') were calculated both for inorganic (IRR. mg
h ') and organic (ORR. mg h"') suspended matter. Also, produc-
tion rates of true faeces (ER, mg h"') were calculated for both
inorganic (lER, mg h"') and organic suspended matter (OER, mg
h"'). Assuming that absorption of inorganic matter through the
digestive system was negligible (Iglesias et al. 1992, Iglesias et al.
1998). the rate of inorganic matter filtration (IFR. mg h"') was
calculated by summing IRR and lER. Thus clearance rates (CR, L
h"') were estimated as CR = IFR/ISM where ISM (mg L"') was
total suspended inorganic matter (Iglesias et al. 1996. Wong and
Cheung 1999). By multiplying clearance rates by the total sus-
pended matter (TSM. mg L^') and by its organic fraction (OSM,
mg L^') total (FRts^.,. matter per unit time per mussel, mg TSM
h"') and organic matter filtration rates (FRosm- mg h"') were
estimated. Ingestion rates of total (IRysM- mg TSM h ' ) and or-
ganic matter (IRosm- mg OSM h"') were extrapolated as follows:
'Rtsm = FRtsm - RR and IRos^, = FRq^^, - ORR, respec-
tively. Absorption food rates (AR, mg h~') were extrapolated as
follows: AR = IRqsm - OER. while absorption efficiencies (AE)
as AE = AE/IRf,j;„. Preingestive .selection efficiencies for the
total filtered organic matter (SEosm) were also estimated accord-
ing to Navarro et al. (1992).

Chemical Analyses

In the laboratory, samples of water collected in the saltworks
and from the experimental filtration chambers, faeces and pseu-
dofaeces were filtered onto pre-washed. precombusted (450 °C. 4
h) and pre-weighed Whatman GF/F filters to determine organic
and inorganic matter contents.

All measurements of total organic content in the food (OSM),
faeces (FOM), and pseudofaeces (PsOM) were obtained using the
difference on ignition method. Filters were weighed after desicca-
tion ( 105 °C, 24 h) using a Sartorius A200 (accuracy ± I (xg) and
the inorganic fractions of total matter were calculated as the weight
of the material remaining after combustion at 450 °C for4 h. OSM,
FOM, and PsOM were calculated from the difference between the
weights obtained after desiccation and those obtained after com-
bustion.

For the water samples, the particulate carbohydrate concentra-
tions (P-CHO, mg L"'). particulate proteins (P-PRT. mg L~'). and
particulate lipids (P-LIP, mg L ' ) were detemiined using methods
reported in Sara et al. (1998).

LPOM as defined above was also converted into carbon
equivalents (using 0.75, 0.40, and 0.49 |xg C |jig"' conversion
factors for particulate lipids, carbohydrates, and proteins, respec-
tively) and used as an expression of the biopolymeric fraction of
particulate organic carbon (BPC) (Sara et al. 1998). Suspended
chlorophyll-a (CHL-a) and phaeopigment (PHAEO) concentra-
tions were measured according to Lorenzen and Jeffrey (1980).
Pigments were extracted with 90'7r acetone and phaeopigments
determined after aciditication with 0.1 N HCI.

The ISM/OSM, LPOM/TSM. and LPOM/OSM ratios (Wid-
dows et al. 1979. Navarro el al. 1993. Navarro and Thompson
1995. Sarii et al. 1998), the C-CHL;i/BPC ratio (the concentration
of CHL-a converted into carbon units using 52 as (he conversion
factor. Nival el al. 1972) and the P-PRT/P-CHO ratio (Navarro and
Thompson 1995, Sara et al. 1998) were used as tools tor gathering
information about the nutritional value of the particulate organic
matter. Temperature (T. Cl and salinity (SAL) were measured al
each sampling olllic saltworks water using a Hydrolab (Inc. Hous-

A Physiological Analysis of the Bhachidontes pharaonis

969

ton. TX. USA) mulliprohe. Salinity signals from the probe were
tested monthly using AgNO, titration.

The relationships between physical, chemical, and trophic vari-
ables with physiological changes were asses.sed using regression
and Spearman correlation (rj tools (Sokal and Rohlf 1981). Sta-
tistica package (Release 5.1: StatSoft. Inc.) was used to perform
statistics.

RESULTS

Physical Conditions and Seslon Characteristics in the Saltworks

The food composition available to su.spension feeders in the
study area as a function of different experimental periods is sum-
marized in Table I. The average temperature was 18.6 ± 7.4 °C.
ranging between the minimum measured in December (9.5 °C) and
the maximum in August (30 °C). The saltworks waters were con-
sistently hyperhaline, showing SAL ranging between 40 and 53
(average 47 ± 4.3). Average seston concentrations throughout the
experimental periods were 81.5 + 95.5 mg L~', ranging between an
average minimum condition of 10.2 ± 2.7 mg L^' (average of
December. January, and June values) and an average maximum
condition of 153 ± 86.7 mg L~' (average of March, August, and
October values). In the low TSM range, the inorganic fraction
greatly exceeded the organic fraction (78'7f ISM vs 22'7f OSM),
while the situation was completely inverted in the high TSM range,
when the organic fraction was more abundant than the inorganic
one (97'7r OSM v.s. 3% ISM).

TABLE L

Mean values of chemical, physical and trophic variables measured
during experimental periods.

Month

Variables

Jan

Mar

Jun

Aug

Oct

Dec

T. X

n.^

15.1

25.0

30.0

20.0

9.5

SAL

58.0

45.0

48.4

53.0

39.6

46.0

CHL-la. (ig L-'

0.7

1.7

1.0

0.6

0.8

0.5

PHAEO. (jLg L '

0.3

0.9

0.4

0.4

0.3

0.8

TSM. mg L-'

10.0

66.6

13.0

240.0

152.0

7.6

OSM. mg L-'

3.0

63.8

2.7

235.0

146.0

1.3

ISM. mg L"'

7.0

2.7

10.3

4.8

6.3

6.4

ISM/OSM. fraction

2.4

0.0

3.8

0.0

0.0

5.0

OSM/TSM, fraction

0.3

1.0

0.2

1.0

1.0

0.2

LIP. mg L-'

0.1

0.1

0.1

0.1

0.1

0.1

PRT. mg L"'

0.3

0.3

0.3

0.2

0.4

0.2

CHO. mg L-'

0.2

0.1

0.2

0.2

0.2

0.3

PRT/CHO fraction

1.3

2.6

1.4

1.0

1.9

0.6

LPOM. mg L-'

0.6

0.5

0.5

0.6

0.6

0.6

BPC. mg C L-'

0.3

0.2

0.2

0.3

0.3

0.3

C-CHLa/BPC. '7c

10.2

33.2

16.1

9.8

10.6

7.5

LPOM/TSM. 7c

5.. 5

0.7

4.0

0.2

0.4

8.3

Abbreviations: T. water temperature; SAL. water salinity; CHL-a. sus-
pended chlorophyll a; PHAEO. suspended phaeopigments; TSM. total sus-
pended matter; OSM. total suspended organic matter; ISM. total suspended
inorganic matter; LIP. lipid concentration in the particulate; PRT. protein
concentration in the particulate; CHO. carbohydrate concentration in the
particulate: LPOM. labile particulate organic matter as the sum of LIP.
PRT and CHO; BPC. biopolymeric fraction of particulate organic carbon
calculated by converting LPOM into carbon equivalents — see text for LIP,
PRT. and CHO conversion factors; C-CHLa. carbon suspended chloro-
phyll-a. See text for explanation of ratios.

Phytoplankton biomass was quite low, with mean CHL-a con-
centrations of 0.88 ± 0.4 |jLg L~' with a maximum measured in
March (1.7 |jLg L"') and a minimum in December (0.5 jjig L"').
Phaeopigments represented on average 32% of total chloropig-
ments. Chloropigments were almost twofold higher in the high
TSM range compared to in the low range (1.1 vs 0.6 jjig L^'l.

Mean labile fraction concentration (LPOM) of suspended or-
ganic matter was 0.55 ± 0.07 |jLg L" ' and throughout the experi-
mental periods represented about 15% of OSM and only 3.2% of
TSM. Two conditions to be identified: at low TSM values LPOM
represented 29% of OSM, while at high TSM values LPOM rep-
resented only 0.45%. Particulate lipids were about 0.07 ± 0.01 mg
L^', representing 13% of LPOM, particulate proteins were 0.27 ±
0.05 mg L~', representing approximately 49% of LPOM, while
carbohydrates were 0.21 ± 0.08 mg L'', representing about 37% of
LPOM. There were no evident differences in lipid concentrations
between conditions of low and high TSM concentrations, while
proteins were higher (54% LPOM) at high TSM values compared
to at low TSM values and. vice versa, carbohydrates were higher
in the low TSM range (43%-). In the low TSM range, proteins and
carbohydrates showed approximately the same concentrations in
LPOM producing a P-PRT/P-CHO ratio of about I. while proteins
exceeded carbohydrates twofold in the high TSM range (P-PRT/
P-CHO ratio about 2). The unicellular autotrophic fraction repre-
sented about 15% of the labile fraction, ranging from 11% in the
low TSM range to 18% of LPOM in the high TSM range.

Clearance, Filtration, and Ingestion Rates

The physiological variables estimated in B. pharaonis through-
out an annual period are summarized in Table 2. Measurements of
clearance rate performed using the trace-substance method were
compared with values obtained with the biodeposition method.
The agreement between the two kinds of estimates is demonstrated
by the following equation: CR,r^„r = 0.46 ± 0.71 * CR^.^,: r =
0.95 (±0.4); II = 20; P < 0.05 where CR„3^^ was clearance rates
measured as the depletion of particle concentration and CR,,,,, was
clearance rates measured with the biodeposition method (CR =
IFR/ISM: Cranford and Grant 1990, Iglesias et al. 1992, Hawkins
et al. 1996). In order to obtain a more precise estimate of feeding
response, the average curve of both (trace-substance and biodepo-
sition) was used.

Weight standardized clearance rates were 1.64 ± 0.82 g L^',
ranging between 0.80 ± 0.15 g L"' in June and 3.02 ± 0.1 1 g L"'
in December. Clearance rate appeared to be inversely related to
seasonal temperature (Eq. 1; Table 3). High clearance rates (3.02
and 2.1 L h"') occurred at low temperatures (9 and 12 °C) and
minimum CRs at high temperatures (0.80 and 1.1 L h"' at 25 and
30 °C, respectively).

The results of a Spearman correlation analysis between CR and
physiological and dietary factors are summarized in Table 4. CR is
independent of changes in SAL, while some dietary variables may
explain the variability of fi. pharaonis clearance rates. In particu-
lar. CR was negatively correlated with TSM, OSM, CHL-a, par-
ticulate proteins, PRT/CHO (Fig. lb), and CCHL/BPC ratios and
positively with ISM/OSM, phaeopigments, particulate lipids, and
carbohydrates and LPOM (Fig. Ic).

During the experimental period. B. pharaonis filtered on aver-
age 1 1 0 ± 1 07 mg h" ' of total suspended material ( mg TSM h ~ ' ),
with a maximum in August and October (276 and 262 mg TSM
h"', respectively) and a minimum (10.4 mg TSM h"') in June.

970

Sara et al.

TABLE 2.
Averaged physiological (n = 84) values estimated in Brachidontes pharaonis throughout an annual period in the Stagnone saltworks.

^"UWst

FRrsM

FR(),SM

IRR

ORR

RR

SE„.sM

'Rl.SM

'RoSM

OER

ar

AE

Jan

2.07

20.57

6.10

0.36

0.09

0.45

0.31

20.12

6.01

0.15

5.86

0.97

Mar

1.09

72.47

69.50

0.11

0.67

0.78

0.10

71.70

68.83

0.46

68.37

0.99

Jun

0.80

10.37

2.16

0.68

0.11

0.79

0.33

9.57

2.05

0.43

1.62

0.79

Aug

1.15

276.16

270.68

0.28

0.22

0.50

0.55

275.67

270.46

0.10

270.36

0.99

Oct

1.73

262.76

251.81

0.44

0.15

0.59

0.74

262.17

251.66

11.63

240.03

0.95

Dec

3.02

23.03

3.86

0.38

0.01

0.46

0.89

22.57

3.85

3.60

0.25

0.06

Mean

1.64

110.89

100.68

0.37

0.21

0.60

0.49

110.. 30

100.48

2.73

97.75

0.79

±SE

0.82

124.77

127.07

0.19

0.24

0.16

0.30

124.80

1 27.04

4.56

124.97

0.37

Annual means and standard errors for means are also reported.

Abbreviations: (CRdwm- L g h"' = weight standardised clearance rate; FR^sm- mg TSM h"' = total suspended matter filtration rates; FR^sm. mg h"'
= suspended organic matter filtration rates; IRR, mg h~' = inorganic content of pseudofaeces; ORR, mg h"' = organic content of pseudofaeces; RR,
mg h"' = rejection rates; SEosm = '"'^l filtered organic matter selection efficiencies; IRtsm- "ig TSM h"' = ingestion rate of total suspended matter;
IRosM- mg TSM h"' = ingestion rate of total suspended organic matter: OER, mg h^' = organic content of true faeces: AR. mg h^' = food absorption
rates; AE. fraction = absorption efifciencies).

FRt.sm produced a significant relationship with TSM, which are
plotted in Figure 2a and described in Eq. 2 (Table 4). Accordingly.
FRysM increases as a function of TSM up to about 100 nig TSM
L"'. after which FR,sm reaches a plateau. Filtration rate was also
a decreasing function (Fig. 2b) of labile organic content of sus-
pended material (LPOM/TSM ratio; Table 4; Eq. 3).

Rejection rate was on average 0.60 ±0.16 mg egested material
h '. The proportion of inorganic egested material was on average
65% throughout the study period. Rejection rate (Fig. 3) reached
minimum values in December and January (approx. 0.4 mg h"')
when TSM concentrations were lower (average 8.8 ± 1.6 mg L"')
and ISM/OSM ratios were on average 3.5. RR reached its maxi-
mum values in June and March when TSM was below 100 mg L"',
after which RR decreased sharply.

Selection efficiency (SE„sm) ranged between 0.1 (March) and
0.90 (December) (average 0.50 ± 0.22). SEosm did not show a
significant relationship with ISM, while the relationship with
LPOM was described by Eq. 4 (Table 3).

Although SEqsm was not measured directly as a function of

TABLE 3.

Relationships between environmental and physiological variables in
the Sicilian saltworks Brachidontes pharaonis.

n.

Equation

randP

1

CR = 4.09 -(l.l3xT

=

-0.92; P = 0.01)

2

FRt.sm = -61 +2.1 xTSM -
0.03 X TSM-

=

0.95; ?< 0.05)

3

FR-,-sM = 17.5 + 495 xexp'-""'""™'"^"

=

-0.91; P <0.0f,)

4

SE,«M = -1.75 + 4.1 xLPOM

=

0.96: P < 0.05)

5

SE„sM = 0.92 - 0.49 X CHl.-a

=

-0.78: /'< 0.1)

6

SE„sM = -0.20 + 3.10 xP-CHO

=

0.78; P<0.1)

7

SE„sM = 0.87 - 0.26 X P-PRT/P-CHO

=

-0.72; P < 0.05)

8

IRtsm = 7.8+ 1.3 X TSM

=

0.96; P < 0.05)

9

AE = 0.95 - 37.4 X exp «"""

=

0.93: P < 0.05)

10

AE = 1.6- 3.97 xP-CHO

=

-0.82: P < 0.05)

11

AR = 2.9+ 1.3xOSM

=

0.98; P <0.05)

12

AR = -1.24 + 0.98 xFR„s.M

=

0.99; P < 0.05 1

13

AR = -4.37 + 18l2xP-PRT

=

0.87: P<0.()5)

each food substrate (Urrutia et al. 1996), we tentatively fitted
SEqsm versus the different dietary features. CHL-a. carbohy-
drates, and the P-PRT/P-CHO ratio in the labile particulate organic
matter produced significant linear relationships with SE„sm-
which are plotted in Figure 4 and described by Eq. 5-7 (Table 3).
According to the above relationships, SEqsm decreased as
CHL-a and P-PRT/P-CHO increased, but increased as a function
of P-CHO.

TABLE 4.

Spearman correlation analysis relating standardized clearance rates

and physical and dietary measured variables throughout the

experimental period.

CR vs.

Rs

SAL

OSM

ISM

TSM

ISM/OSM

CHL-a

PHAEO

LIP

PRT

CHO

P-PRT/-P-CHO

LPOM

LPOM/TSM

C-CHL;i/BPC

LPOM/OSM

0.08
-0.34

0.16
-0.49

0.44
-0.58

0.31

0.43
-0.25

0.50
-0.50

0.59

0.49
-0.58

0..34

0.47 (ns)
0.001 (**)
0.14 (ns)
0.00(***)
0.00(***)
0.00(***)
0.002 (**)
0.00(***)
0.02 (*)
0.00(***)
0.00 (***)
0.00(***)
0.00(***)
0.00 (***)
0.001 (**)

See text for acronyms and explanation, in = equation number; R
correlation coclTicicnl: P = pnihabilily level).

|„ = 84: {*) = P£ 0.05; I**) = PS 0.01: (***) = Ps 0.001; (ns) =
non-significant difference {P s 0.05)].

Abbreviations: SAL, water salinity; CHL-a, suspended chlorophyll a;
PHAEO, suspended phacopigments: TSM, total suspended matter; OSM,
total suspended organic matter; ISM. total suspended inorganic matter:
LIP, lipid concentration in the particulate; PRT, protein concentration in
the paniculate: CHO. carbohydrate concentration in the particulate;
LPOM, labile paniculate organic matter as the sum of LIP, PRT and CHO;
BPC. biopolymeric fraction of particulate organic carbon calculated by
convening LPOM into carbon equivalents — see text for LIP, PRT, and
CHO conversion factors: C-CHLa, carbon suspended chlorophyll-a). See
text for cxplanalion of ratios.

A Physiological Analysls of thl Brachidontes pharaonis

971

3-

1-

— I —
LS

20
Temperature, ''C

—I —
25

30

0.5

1.0 1.5 2.0

P-PRT/P-CHO. faction

c

3-

DecJ /

is

Jani y^

' Oct

i3
D
1 -

Mar

yi

1
Aug

0-

Jun

' 1

1

— 1 ' 1

0.40

0.45

0.50

0.55

0.60

0.65

LPOM,n^r'

Figure 1. Relationship between a) temperature (T. C); b) labile par-
ticulate organic matter concentrations (LPOM. mg L"'); c) protein by
(do you mean 'over' i.e., divided by?) carbohydrate ratio (fraction)
and clearance rate (L h"') as measured throughout the study period.

Total ingestion by the mussels was estimated by subtracting
total rejection from total filtered suspended material (IR = FR -
RR; Wong and Cheung 1999). The average ingestion rate during
the experimental periods was 110.3 ± 124 mg TSM h"', with a
maximum in August (276 mg TSM h"' ). and a minimum in June
( 10 mg TSM h"' ). The relationship between rates of seston inges-
tion (as IRtsm) and TSM is described by Eq. 8 (Table 3).

300-

a)

Dei^n ,,
'^un

-^>lar

^jy^ I

200-
100-

0-

■ 1 ' 1 '

Aug

— T ■ • 1

-50

0

50

100 150

200

250 30

400 -.

TSM.mgl'

300-

Aug

1

f

b)

200-

\

100-

0 J

\ Mar

Jun Jan

Dec

LPOMA"SM, fraclion

Figure 2. Changes in filtration rates (FR,,sm. mg h ') as a function of
a) total suspended material (TSM, mg L"') and b) labile particulate
organic matter concentrations by total suspended material ratio
(LPOM/TSM, fraction).

Ingested organic matter (IRqs

SM, mg h

I ) varied between about

mg OSM h"' (June) and 270 mg OSM h"' (August). Ingested
organic matter presented the same kind of relationships observed
between IRysM and TSM.

Absorption efficiency ranged between 0.1 (December) and 0.99
(March and August). AE increased as a function of ingested or-
ganic material (IRqsm; F'g- 5'- showing a plateau after approxi-
mately 7 mg ingested OSM and as a function of nitrogen content
in the available food (P-PRT; Eq. 9; Table 3). Also, AE showed a
negative correlation with particulate carbohydrates, described by
Eq. 10 (Table 3). Absorbed organic material was at a minimum in
December (0.1 8 mg OSM h"') and June (1.6 mg OSM h"'). anda
maximum in August and October (270 and 240 mg OSM h"'),
respectively). Absorption rate was a positive linear function of the
amount of seston organics (OSM) and of filtered organic matter
(FRosM^Eq. 11-12: Table 3).

The rate of absorbed organic matter seemed to reach a maxi-
mum at 0.2 mg P-CHO L"', after which a large decrease was
observed when P-CHO reached higher concentration values. AR

0&.

■Jun

"Mar

0.7.

0.6.

"Ocl

05-
0.4-

De(^,
^Jan

"Aug
' 1 '

50

250

TSM.mgl '
Figure 3. Changes in rejection rates (RR. mg h"') as a function of total
suspended material (TSM, mg L"').

972

Sara et al.

1)9.

DA

1 "■'■

'Oct

a)

} OS.
5 04.
^ OJ.

i'. 0,1 .

Aug

Jan Juj

M^

on.

Fined line [kO.1

1.0 .

0.9.

Dea

Oil.
07.

Ocl

b)

0.6.

Au^

OS.

04.

OJ.

Jurr

•Jan

05.

0,1 -
00.

•Mar

1 ■ F '■

- fined hne jkOIG
1 ■ 1 ' 1

CHLra,|igl

020 025

P-CHO. mg 1 '

09.

IIB

Dec

0.7.
0.6.
05.

Au|

Ctel

c)

OA.

OJ.

Jar» •'""

02.
0.1 .

Mar

Ofl-

Fm

e<lli«

ikO.1

ID 15 2^1

PRTCHO ratio

0.9.

Do?

O.R.

0.7.

0^' d)

0,6.
OJ.

Aug

0,4.

OJ.
02.

Ju»

j'an

0,1.

M0

OjO.

.0,1-

• 1 —

^

1 • —

fined hneixOPS

050 055 0-60 0.65

LPOM.mgT

Figure 4. Changes in selection efficiency (SE) as a function of a) chlorophyll-a (ng L '); b) particulate carbohydrate (mg L '); c) particulate
protein by carbohydrate ratio (fraction): and d) labile particulate organic matter concentrations (LPOM, mg L"').

seemed to assume a different relationship to particulate proteins
(Eq. 13; Table 3).

DISCUSSION

The physiological process of B. pharaonis measured in situ
using the biodeposition method provided a series of complex re-
lationships. Although B. pharaonis represents one of the most
common species along Red Sea, Pacific, and Indian coasts (Mor-
ton 1988), its physiological processes and feeding behavior have
never been measured, either in the laboratory or in ambient con-
ditions. Consequently, the general physiological responses of B.
pharaonis are compared here with those of other species which
have been investigated more closely.

The complexity of feeding behavior in B. pluiraonis appears to

■

1 11-

•jan

"Mar

Ocl-

•Aug

11,8.

■Jun

1

•3

ll,ft.

\t

E-
5
<

(1,4-

1)11

.D.C

u HI 11)11 i.^ii :iiii 251) .1011

Ingcslud lUfianif material (IK,^^,. mg h ')

Figure 5. Relationship between ingested organic material and absorp-
tion efficiency as measured throughout the study period.

be correlated with their complex and varying environmental con-
ditions. Bivalve molluscs are a "species mirror" that describe well
the conditions of their colonised environments. B. pharaonis is
possibly a good example of a descriptor species because, like the
other bivalves, it possesses compensatory mechanisms to regulate
its behaviour in response to fluctuations in environmental and
trophic factors.

The western Mediterranean saltworks environments, like other
similar environments (Pusceddu et al. 1999, Sara et al. 1999), are
characterized by shallowness and partial enclosure, which deter-
mine much of the variability in their physical, chemical, and
trophical features. A good example is given by water temperature.
The temperature of the Mediterranean never falls below 12-14 °C
(except in the Adriatic) and above all never exceeds about 26.5 °C
(Margalef 1985). Mediterranean benthic communities therefore
experience a moderate temperature range throughout the year. In
contrast, in the Sicilian saltworks temperature ranges widely, be-
tween 9-10 "C in winter and 2S-3() "C in summer— a range that is
rarely found in other Mediteiranean coastal areas (Margalef 19S5).
These wide variations can be correlated with atmospheric tempera-
ture changes and various meteorological phenomena which, in
small enclosed shallow basins such as our study area, can strongly
influence the usual physical and chemical dynamics (Sarii et al.
1999). Throughout the year, B. pluiraonis experienced physical
conditions that were often close to the survival limits for Medi-
terranean bivalve molluscs (Sara et al. 1998). However, it has been
documented that B. pluiraonis is a typical intertidal and hyperha-
line species (Por 1971 ). which can live in very different and quite
extreme conditions (Stern and Achituv 1978, Morton I9S8).

The average clearance rate (1.64 ± 0.82 L h ') measured in
adults of H. pluinumis falls into the general range measured for
many other epilaunal bivalve molluscs (Widdows el al. 1979, Ri-
isgard 1988, Navarro et al. 1991, NavaiTO et al. 1992, Urrutia et al.
1996. Wong and Cheung 1999) and was quite similar to that mea-

A Physiological Analysis ok the Brachidontes pharaqnis

973

sured in other experimental conditions for adults of B. e.xusnis
(range 1.2-1.7 L h~'; Riisgard 1988).

The first response of B. pharaonis to temperature changes
seems to be to regulate its clearance rate. The documented re-
sponse of bivalves to temperature changes is a general reduction in
clearance rates at low and high temperatures (Bayne 1976, J0r-
gensen 1990, Hawkins and Bayne 1992). This has been observed
in many bivalve molluscs (Schulte 197?, Aldridge et al. 1995,
Hawkins et al. 1998). However, as a general trend, our Bmclii-
dontes data did not fit with this documented response as its clear-
ance rate showed higher values (2.54 ± 0.67 L h~': average of
December and January values) at the lowest temperatures (10 and
12 °C) and lower CR values with an increase in temperature (up to
25 °C). It is difficult to explain the peak in clearance rate measured
in January and December only as a function of temperature. It may
be a species-specific response to thermal stress or changes in the
endogenous reproductive phases. No literature data regarding simi-
lar behaviour in bivalve molluscs exist, and the presence of this
species at the low temperatures observed in winter in the study
area has not been demonstrated previously. That this species may
be better adapted to high than low temperatures (Stem and Achituv
19781 could be explained by the fact that B. pharaonis is enzy-
matically well equipped for bodies of water in which salinity is
high and the temperature variable (Stern and Achituv 1978). Fur-
ther research is needed to resolve the relationship between tem-
perature, CR, and the role of the endogenous reproductive cycles
in B. pharaonis.

The hyperhaline environment did not seem to produce any
effect on clearance, as demonstrated by its independence of salin-
ity. Nevertheless, it has been reported (Bayne 1976, Widdows
1985) that in some conditions salinity can be a regulating factor for
clearance rates in several bivalve molluscs, often producing a de-
crease in clearance rate or, in any case, a certain dependency.
However, literature findings refer to acclimatization in typical
coastal or estuarine bivalve molluscs, which are generally adapted
to sea salinity and are thus less comparable with Brachidontes.
which are generally well adapted to high salinity (For 1972, Mor-
ton 1988).

The clearance rate of western Mediterranean B. pharaonis does
not appear to be fundamentally affected by physical factor (i.e.,
water temperature and salinity) but more likely by fluctuations in
the feeding environment. The food supply in the saltworks envi-
ronment investigated seemed to be limited by resuspension from
sediment. The main effect of sediment resuspension is usually an
increase in the inorganic fraction of the suspended bulk (i.e., silt
material) (Flegey et al. 1992. Sara et al. 1999, Wong and Cheung
1999). Instead, in the particular conditions of the basin studied
(i.e., shallow and enclo.sed), wind-driven resuspension events (in
March. August, and October) led to a marked increase in sedimen-
tary organic matter, producing an unusually high dominance of
organic fraction over inorganic (ISM/OSM ratio < O.I). Although
similar results have been observed rarely in coastal environments
around the world, the presence of a large amount of rich organic
sedimentary detritus composed of seagrass wrack particles and
filter feeder biodeposition products may justify organic contents
with values that were higher than those for pure phytoplankton
cultures.

The nature of the relationships between food acquisition pro-
cesses in bivalve molluscs and the quantity and quality features of
available food has been widely and well documented in the current
literature (Widdows et al. 1979, Iglesias et al. 1992, Bayne et al.

199-^, Navarro et al. 199.1, Navarro and Thompson 1995, Hawkins
et al. 1996, Iglesias et al. 1996, Urrutia et al. 1996, Navarro and
Widdows 1997, Hawkins et al. 1998, Iglesias et al. 1998, Wong
and Cheung 1999). Most of the above-cited articles have shown a
general decline in clearance rate with an increa.se in the quality and
quantity of available suspended food (Winter 1978, Bayne et al.
1989, 1993). The present results are consistent with the findings in
the literature. Indeed, our data show a general negative dependence
of CR on total suspended matter and in particular on its total
organic fraction (OSM) (Table 4). Furthermore, the food acquisi-
tion process in Brachidontes was also dependent on food quality,
as highlighted by the relationships between CR and the dietary
parameters (Table 4). This relationship has often been tested in
laboratory experiments using phytoplankton-based diets. Such ex-
periments have clearly highlighted that most bivalves reduce their
clearance rate as a function of an increase in phytoplankton bio-
mass (Winter 1978, Bayne et al. 1989). When the nutritional value
of the phytoplankton diet was reduced using inorganic particles to
simulate an artificial dilution effect (Iglesias et al. 1992, Bayne et
al. 1993), the immediate physiological response was an enhance-
ment of clearance rates. Similar findings have always led research-
ers to conclude that bivalves respond to food quality. However,
these observations cannot be taken as general findings applicable
to ambient conditions (Incze et al. 1981, Wildish and Kristmanson
1997) and the necessity to gather information directly from the
field has often been highlighted (Bayne et al. 1989, Wildish and
Kristmanson 1997).

The CRs obtained in this study were negatively correlated with
particulate proteins, suspended cholorophyll-a and consequently
with the P-PRT/P-CHO and C-CHL-a/BPC ratios in the particu-
late, suggesting that B. pliaraonis reduces its clearance activity as
a direct response to an increase in the labile fraction of the avail-
able food. In contrast, the positive correlation between CR and
particulate carbohydrates and lipids could indicate that in the pres-
ence of these classes of compounds B. pliaraonis increases its rate
of clearance to enhance its food uptake. This picture would fit well
with the particular feeding environment of B. pharaonis, in which
the main vegetal component is Cymodocea nodosa detritus, which
is, by definition (Mann 1988, Velimirov 1991), highly refractory
and thus less available to benthic filter feeders (Mann 1988, Va-
liela 1988). Although this kind of relationship has been hypoth-
esized widely (Bayne 1976, Widdows et al. 1979, Bayne et al.
1993, Dame 1996), it has not been documented clearly.

A diluted effect produced by inorganic material on bivalve
clearance rate (see reviews by Hawkins and Bayne 1992 and Dame
1996) has been well verified on many occasions. Consequently,
clearance rate increases in correspondence with increments in
ISM. However, CRs of saltworks B. pharaonis were "statistically
independent" of ISM concentrations. Clearance reached its maxi-
mum in December and January while under high ISM/OSM ratio
conditions but al the lowest temperatures (10 and 12 °C). Such
high values cannot be explained by the influence of temperature on
CRs but may be explained by the dilution concept widely proposed
in the literature (Widdows et al. 1979). However, in order to ex-
plain the absence of a well-defined statistical relationship between
CRs and inorganic material concentration, we could also hypoth-
esize that in other experimental periods characterized by a large
quantity of suspended organics (>60 mg OSM L"'). inorganic
matter was not sufficient to reduce the food value. Consequently.
B. pharaonis did not need to offset the inorganic seston dilution
effect by increasing its clearance rate (Dame 1996).

974

Sara et al.

B. pharannis specimens were able to filter a wide range of total
suspended matter (0-280 mg TSM h"'), showing a great capacity
to respond to wide changes in food supply. Although our mea-
surements were performed over a wide TSM range. B. pharaonis
filtering activity was related to seston concentration, like most
suspension feeders investigated. The mass of filtered seston in-
creased exponentially up to a certain TSM threshold, after which
it reached a plateau followed by a possible decline (Dame 1996).
Similar behaviour has been widely observed in situ experiments
carried out with a seston concentration of up to 100 mg L"' in
Perna viridis (Hawkins et al. 1998), Mytilus chilensis (Navarro
and Winter 1982), Crassostrea belcheri (Hawkins et al. 1998),
Cerastoderma edule. Mytilus ediilis. and C. gigas (Hawkins et al.
1998). Our data would indicate that filtration depends also on the
available food quantity (LPOM/TSM, Fig. 2b). When there was a
high concentration of LPOM in the saltworks the filtration rate
levelled off, whereas when the available organic matter was low
the total mass filtered increased. Therefore it is hypothesised that
when available food increa.ses B. pharaonis levels off its filtration
activity because it fulfils its energy requirements with sufficient
organic-rich particles.

Our data demonstrate that B. pharaonis may not control inges-
tion rates by varying the rate of pseudofaeces production, and such
a mechanism seems to be quite inefficient compared to other bi-
valve molluscs. Indeed, the average rejection rate by B. pharaonis
(RR = 0.60 ± 0.16 mg h"') was lower than those documented
for Mytilus edulis (1.2 mg h"', Bayne et al. 199.^), Perna viridis
(0.2-9 nig h ', Wong and Cheung 1999), Cerastoderma edule
(2.9 mg h^'; Iglesias et al. 1992) and several tropical bivalves
(Hawkins et al. 1998). In most bivalves the control of ingestion
level by pseudofaeces production has already been observed (Fos-
ter-Smith 197,'^, Cranford and Gordon 1992, Iglesias et al. 1992,
Bayne et al. 1993, Urrutia et al. 1996, Arifin and Bendell-Young
1997, Hawkins et al. 1998, Wong and Cheung 1999) and is con-
sidered to be an overflow mechanism. Pseudofaeces production
together with the modulation of clearance rate (Winter 1978) can
maintain bivalve ingestion rates constant (Iglesias et al. 1992) in
order to reduce the "detrimental effect" of food dilution by inor-
ganic material. The material rejected by the saltworks B. pharaonis
was mainly represented by inorganic material (65%). However,
only about 2.2% of filtered seston (77% and 23% of inorganic and
organic material, respectively) was rejected as pseudofaeces, indi-
cating that the feeding conditions of the saltworks did not induce
a high level of rejection. As confirmation of this, the rejection rates
are lower than those reported by Iglesias et al. ( 1992) for C. edule,
which rejected material representing approximately 31% of fil-
tered materials (approximately 87% and 13%' inorganic and or-
ganic material respectively).

However, assuming that ingestion rates were extremely high as
a specific response to very high seston organic availability (seston
organic content >9()%), it would seem that in B. phanumis. there
was a lack of ingestion regulation, which, under these trophic
conditions would render digestive processes impossible. Without
considering physiological compensatory mechanism that have yet
to be found in a coloni/cr mollusc such as B. pharaonis. the
ingestion rate measured in the saltworks conditions would imply
mean gut passage times of about 30-00 min. Such lime lengths
would generally be incompatible with the breakdown and absorp-
tion of food in the mussel gut. We einisage that such discrepancies
may be explained by the slight overcstimation of ingestion rates
due to the use of experimental methodologies (i.e.. biodeposition
method; Iglesias el al. 1998), which rarely have been tested in

similar environments (with huge concentrations of particulate or-
ganics), coupled to the great capacity of B. pharaonis to adapt to
very unusual variability in environmental conditions. On the other
hand, the presence of active populations of B. pharaonis has also
been documented in Mediterranean ultra-oligotrophic sea waters
(|CHL-al « I (i-g L '; salinity 37: Di Geronimo 1971), and such
a finding agrees with the huge physiological plasticity of this spe-
cies.

Under our study conditions, B. pharaonis was able to perform
sorting of filtered material with an efficiency (SEosm = O-^)
similar to that reported for other bivalves (Kiorboe et al. 1981,
Iglesias et al. 1992, Hawkins et al. 1996, Navarro and Widdows
1997, Hawkins et al. 1998, Wong and Cheung 1999). Although our
experiments were not designed to test specifically the selection
ability of the pallial organs in B. pharaonis. it is apparent that this
species is able to sort between refractory and labile food (see Fig.
3). The general picture of selection efficiency in bivalves is that
they have an ability to discriminate organic from inorganic mate-
rial but generally not refractory from labile organics. Nevertheless,
the general tendency of B. pharaonis is to increase SE^sm when
P-CHO in the available food increases (see Fig. 4b and 4c), pro-
ducing a dilution of the particulate protein nutritional value with
CHL-a not exceeding 1 (i.g L~'. In contrast, when inorganic ma-
terial did not exceed 4 mg L~', selection efficiency decreased. The
ability of the pallial organs to sort particles of different organic
content and to respond to food quality has already been demon-
strated in other bivalves. Thus we believe our results may be
explained by (1) a species-specific response (Ward and Mac-
Donald 1996, Wong and Cheung 1999) reflecting the huge plas-
ticity of B. pharaonis and its ability to colonize a wide variety of
tropical, subtropical, and temperature environments; and (2) a par-
ticular strategy activated at a local level by B. pharaonis in order
to minimise the huge variability in the feeding environment.

Absorption efficiency in Brachidontes is a complex process
that needs further investigation through specifically designed ex-
periments. It has been documented that most bivalve ab.sorption
efficiency values are generally low, ranging from 0.4 to 0.6. In the
present study, the average AE value of B, pharaonis (0.79) was
comparable to those reported for other tropical bivalves (Hawkins
et al. 1998) but higher than those for Mytilus edulis and cockles
(Bayne et al. 1989, 1993, Navarro and Widdows 1997, Wong and
Cheung 1999). Also, AE depended positi\ely on ingested organic
matter (IRosn,), reaching higher values when IR^sm exceeded
6-10 mg h~' and remaining fairly constant above this level.

This type of dependence agrees with that observed in Perna
viridis and other tropical bivalves (Hawkins ct al. 1998, Wong and
Cheung 1999), but it differs from those obtained in Mytilus edulis
(Bayne et al. 1989) and C. edule (Iglesias et al. 1992, Urrutia et al.
1996). Moreover, AEs of Brachidontes were apparently dependent
on the quantity and quality of ingested matter. Significant positive
relationships were observed between AEs and particulate proteins,
while particulate carbohydrates showed a negative correlation. In-
deed, it seemed that absorption efficiency reached higher values
when proteins exceeded approximately 0.23 mg L ', while they
decreased when carbohydrates increased. This suggests that B.
pharaonis may prelcrcnlially absorb proteins with respect to car-
bohydrates and lipids. This hypothetical order oi' absorption effi-
ciencies contradicts the findings of Bayne el al. (1993), who de-
scribed higher efficiencies for carbohydrates than for proteins and
lipids. However, it agrees with the results of Hawkins and Bayne
( 198.5). who recorded the same rank order of absorption efficien-
cies lor biochemical components of the diet.

A Physiological Analysis of thl Brachidontes pharaonis

975

This B. pharaonis rank order of absorption efficiencies agrees
with our unpublished data on suspended bacteria in the saltworl<s
(Sara, La Rosa and Mazzola. unpubhshed data). We found con-
centrations of attached-bacteria fraction in the saltworl<s water
particulate of up to 1 x 10'' [two orders higher ( 10'* vs. 10^)] than
those found in Mediterranean fish farming impacted coastal waters
and sediments (Mazzola et al. 1999. La Rosa et al. in press). This
would clearly justify the preferential absorption of proteins by B.
pharaonis. In addition, the marked dominance of the attached-
bacteria fraction in the particulate also indicates that B. pharaonis
may be able to actively exploit nitrogen from bacteria, as sug-
gested by Langdon and Newell (1990). justifying estimated ab-
sorption efficiencies which are so high and so rarely documented
in the current literature.

Our tmdings agree well with the good sorting ability observed
in B. pharaonis. The hypothesized ability to sort labile (mainly
organic compounds originating from bacteria?) from refractory
material at the level of the pallial organs may allow Brachidontes
to maintain constant ingestion rates by channelling more labile
(i.e.. more digestible) material into the gut. Absorption rate fol-
lowed the same trend and both the quantity and quality of ingested
organic matter was affected. The correlation of absorption rate
with filtration activity demonstrates that B. pharaonis control of
feeding begins with food acquisition processes. Similar findings
have been documented in many bivalves, above all in those envi-
ronments that are limited by high variability in food supply (Igle-
sias et al. 1992, Bayne et al. 1993, Navarro and Widdows 1997,
Wong and Cheung 1999).

CONCLUSIONS

The mechanisms controlling food acquisition processes in B.
pharaonis appear to reflect the particular feeding environment in
which it lives. The complex feeding process of B. pharaonis is
regulated by the first stages of feeding (clearance and filtration),
and most of the control reflects the quantity of food absorbed. In
this paper we have clarified the following points:

1. Clearance rate does not appear to be fundamentally regu-
lated by physical factors (i.e.. water temperature and salin-
ity), but may reflect fluctuations in the feeding environment
and, more closely, the quantity and quality of food available.

This finding is consistent with the idea of a trophic regula-
tion of feeding processes in bivalves (Bayne, 1998). How-
ever, it contradicts Jorgensen's idea that food uptake in bi-
valves is an autonomous process which is not regulated at
the organism level according to nutritional needs (J0rgensen
1990, Clausen and Riisgard 1996). Further investigation is
needed to resolve and fully understand this apparent dis-
crepancy.

2. Brachidontes may control ingestion rate by varying the rate
of pseudofaeces production, although our data demonstrate
that this may not be a very efficient response under the
prevailing environmental conditions. However it is able to
perform sorting of filtered material. These mechanisms may
be used by this species to maintain the ingestion rate con-
stant, as this rate may be affected by the "dilution effect" of
both inorganic material and refractory matter in the avail-
able food.

3. Absorption efficiencies may be sensitive to the balance of
biochemical components in the diet (on average composed
of 50% P-PRT, 38% P-CHO and 12% P-LIP). Accordingly,
we can infer that Brachidontes absorbed organic matter with
different efficiencies for proteins, carbohydrates and lipids.

In conclusion, although these findings are currently being in-
vestigated further in a set of specifically designed experiments, in
the meantime we can say that the Mediterranean B. pharaonis
seeins to be a "complex machine" that operates as a function of a
complex synergy of trophic, chemical and physical factors. Such a
fact may be the key to understanding the huge plasticity of this
organism, which is able to colonize a great variety of habitats at
different latitudes around the world.

ACKNOWLEDGMENTS

We thank Dr. John Widdows (Plymouth. UK) for his sugges-
tions, which were invaluable for improving the experimental de-
sign, and for his precious comments on reading an early version of
the manuscript. This work was funded by the Ministero Politiche
Agricole (MiPA, Italy) and the Ministero delLUniversita Ricerca
Scientifica e Tecnologica (MURST, Italy). All literature cited in
this article can be freely consulted by the scientific community
through the corresponding author.

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of Stress on Marine Organisms. John Wiley and Sons Ltd.. New York. quality. J. E.xp. Mar Biol. Ecol. 236:191-207.

Joimuil ofSlu-llthh Research. Vol. 14. No. 2. 974-4X2. 2000.

KARYOTYPES OF THREE SPECIES OF CORBICVIA (BIVALVIA: VENEROIDA) IN KOREA

GAB-MAN PARK,' TAI-SOON YONG,' * KYUNG-IL IM,' AND
EE-YUNG CHUNG^

^Department of Piirasitolofiy and Institute of Tropical Medicine,
Yonsei University College uf Medicine, Seoul 120-752

-Department of Marine Living Resources,
College of Ocean Science and Technology.
Kunscm National University, Kunsan 573-701, Korea

ABSTRACT The chromosome numbers of three species of Korean Corhicula are investigated here: C. fluminea 54, C. papyracea
54 and C colowu, ^8. In C flammec, and C painrcuea. the mitotic chromosomes from 18 sets of three chromosomes each showed
that these two species are triploids. In C. colorcm. the mitotic chromosomes from 19 groups with two chromosomes each mdicated
that it is a diploid. C. fluminea and C papxnuea have one set of metacentric chromosomes, five sets of submetacentrics chromosomes.
and 12 sets of subtelocentric chromosomes. C. colomui has been considered a subspecies of C. papxracea, but its karyotype mdicates
that it is a distinct species since it is undoubtedly reproductively isolated from the other two Korean species studied.

KEY WORDS: Diploid, triploid. Corhicula fluminea. C papyracea. C. colorata, echinostome, Korea

INTRODUCTION

The family Corbiculidae is a group of relatively small Asian
clams belonging to the bivalve order Veneroida. They live mostly
in freshwater, although several species inhabit brackish water.
They are a human food item over much of their natural range, and
in some areas the extent of their consumption makes them a sig-
nificant commercial crop. In its native range, the clam is the in-
termediate host for Echinostonui lindoensis trematodes. Since it is
sometimes eaten raw or partially cooked, there is often a high local
incidence of echinostomiasis in Asia (Sandground and Bonne
1940. Benthem Jutting 1953, Sinclair 1971, Brusca and Brusca
1990). Corhicula is often used as food in Korea, and there is no
evidence of such a trematode cycle being established. In North
America where Corhicula have been introduced by human agency
and have then spread widely, corbiculid clams are little eaten, but
instead have a negative environmental and commercial impact
(Britton and Morton 1982, Cherry et al. 1986, Johnson et al. 1986,
Macphee 1986, Page et al. 1986, Potter and Liden 1986, Smithson
1986). The pest aspect of Corhicula in North America centers on
its high reproductive capacity, high growth rate, free-living juve-
nile stage, and great powers of dispersal (McMahon 1983).

Hermaphroditic freshwater clams in the genus Corhicula have
been reported to produce non-reductional spermatozoa in Japanese
and Taiwanese species compared with somatic DNA content (Ko-
maru et al. 1997). Okamoto and Arimoto (1986) suggested the
possibility that C. lecma reproduces by gynogenesis, i.e.. repro-
duction by parthenogenesis requiring stimulation by a spermato-
zoan for the activation of the egg. In Korea the freshwater clam C.
fluminea has been reported to have a special mode of reproduction:
it is ovoviviparous. hermaphroditic, and broods its larvae in the
inner and/or outer demibranchs (Kwon et al. 1986).

The chromosome numbers of three bivalve species belonging
to the Corbiculidae family have been reported previously: C. leana
(Nadamitsu and Kanai 1978. Okamoto and Arimoto 1986), C.
japonica and C. sandai (Okamoto and Arimoto 1986). The occur-
rence of triploidy in C. learnt has been reported (Okamoto and
Arimoto 1986).

In Korea, six species of Corhicula are recognized (Kwon et al.
1993). (They did not subdivide Corhicula into subgenera). Of

*Corresponding author. E-mail; tsyong212@yumc.yonsei.ac.kr

these six species, we studied the karyotypes of C. fluminea, C.
papyracea. and C papyracea colorata. These taxa live in fresh-
water and are all hermaphroditic.

MATERIALS AND METHODS

The specimens used in this study were collected in Korea dur-
ing March 1998 and February 1999. and examined shortly after
collection. Twenty-five specimens of C. papyracea colorata were
collected in the Chungpyung Dam reservoir; 20 specimens of C
fluminea and 22 of C. papyracea were collected in Lake Uiam in
Chunchon. Chromosome preparations were made from gonadal
tissues by the air-dry method of Kligerman and Bloom (1977) with
minor modification (Park 1994). Gonadal tissues were treated with
0.1 mL of 0.05% colchicine solution and set aside for 20 to 24 h
in a moist chamber at room temperature. The treated tissues were
dissected and minced with needles in a hypotonic 0.01% NaCl
solution. Separated cells were collected by centrifugation at 1,000
rpm for 10 minutes. These cells were fixed in freshly mixed modi-
fied Camoy's fixative (three parts methanol and one part glacial
acetic acid). The supernatant was replaced by fresh fixative. The
centrifugation (1.000 rpm, 10 minutes) was repeated two more
times. A drop of the cell suspension was then pipetted by a mi-
crohematocrit capillary tube and dropped onto a clean slideglass
pre-cooled at 4 °C. The cells left on the slide were air-dried and
then stained for 10 min with 4% Giemsa (Gurr R66) solution made
up in 0.1 M phosphate buffer. pH 7.0. The prepared slides were
observed under an Olympus (VANOX) microscope with a lOOx
(n.a. 1.25) oil immersion objective and a lOx ocular.

Morphological features of the chromosomes used to compare
karyotypes were the total lengths and the relative lengths ot the
chromosomes, as well as the positions of their centromeres (pri-
mary constrictions). Nomenclature of chromosome morphological
types follows Levan et al. (1964).

Voucher specimens of the Corhicula species used in this in-
vestigation have been placed in the Department of Parasitology,
Yonsei University College of Medicine, Korea.

RESULTS

Corhicula fluminea (Muller)

Chromosomes in 28 cells were observed. This species has 54
chromosomes, which can be grouped into 1 8 sets of three homo-

979

980

Park et al.

logues (Fig. 1 ). These 18 sets can be divided into one metacentric
group, five submetacentric groups and 12 subtelo- or telocentric
groups (Fig. 1). The mean total length of all chromosomes was
44.3 ± 2.80 |xm. Table 1 shows mean measurements of chromo-
somes in six cells. Meiotic chromosomes in this species were not
observed.

Corbicula papyracea (Heude)

A total of 54 chromosomes was observed in 28 cells. These
chromosomes were from 18 sets with three homologues each.
These chromosomes can be divided into one group of three meta-
centric chromosomes, five submetacentric groups of three chro-
mosomes, and 12 groups of three with subtelocentric chromo-
somes (Fig. 2). The mean total length of the chromosomes was

42.6 ±0.16 |a.m. The maximum length of chromosome number 1
was 3.8 |jLm. Table 2 shows the mean lengths and relative lengths
of each chromosome as examined in five cells. Meiotic chronio-
.somes in this species were not observed.

Corbicula colorata (von Martens)

In 25 cells, 38 chromosomes were observed. The karyotype of
this species consists of one pair of metacentric chromosomes and
18 pairs of subtelocentric chroinosomes (Fig. 3). The mean total
chromosome length based on the measurements of five cells was

56.7 ±0.19 (xm (Table 3). The presence of eggs, sperm, and
hermaphroditic in this species was observed.

DISCUSSION

The Korean clams C. flitminea and C. papyracea have 54 chro-
mo.somes. When the karyotypes of these two species are analyzed

TABLE 1.

Relative lengths and total lengths (micrometers) of chromosomes of
C. fluminea.*

Chromosome

RL±SE

TL±SE

Type

1

8.58 ± 1.04

3.8 + 0.37

M

2

6.09 ± 0.80

2.7 ±0.21

SM

3

5.41 ±0.76

2.4 ±0.16

SM

4

5.41 ±0.72

2.4 ±0.1 5

SM

5

5.41 ± 0.67

2.4 ±0.12

SM

6

5,41 ±0.67

2.4 ±0.1 2

SM

7

5.64 ± 0.43

2.5 ±0.1 9

ST

8

5.64 ± 0.39

2.5 ±0.17

ST

9

5.64 ± 0.39

2.5 ±0.17

ST

10

5.64 ± 0.34

2.5 ±0.14

ST

11

5.41 ±0.84

2.4 ±0.19

ST

12

5.41 ±0.79

2.4 ±0.17

ST

13

5.19 + 0.73

2.3 ±0.1 6

ST

14

5.19 ±0.66

2.3 ±0.10

ST

15

4.97 ± 0.39

2.2 + 0.12

ST

16

4.97 ± 0.35

2.2 ± 0.09

ST

17

4.97 ± 0.35

2.2 ± 0.09

ST

18

4.97 ± 0.33

2.2 ± 0.08

ST

* Based on measurement of six karyotyped cells. RL ± SE, relative length
of the chromosome, percentage of the total length of the autosomes in
diploid: TL. total length of the autosomes in diploid; PM. metacentric; SM,
submetacentric; ST, subtelocentric.

the chroino.somes fall into 18 sets of three chromosomes each.
Therefore, it seems obvious that C. fluminea and C papyracea are

triploid species.

A Japanese Corbicula, C. leana. also had 54 chromosomes that
could be classified into 18 sets of homologues, and they indicated

5 im

B

m

Mi Mil Mm (I

III ii» ««* iM ffi«^« «ti

10

12

^1 iti »•% ilf Aft* >tkmm

13

14

15

16

17

18

Figure I. \. Mcliipliase chromosome of C. fluminea; H, Karyotype
constructed from \.

A 4$ 5 ;mi

B III IH ilU Ml MA AM

11

13

13

IT

18

14 1.5 16

Figure 2. A, Metaphase chromosome of C. papyracea; B, Karyotype
shown in A.

Karyotypes of Thref. Species of Corbicvia

981

TABLE 2.

Relative lengths and total lengths micrometers of chromosomes of
('. papyracca.*

TABLE 3.

Relative lengths and total lengths (micrometers) of chromosomes of
f '. colorata.*

Chromosome

RL ± SE

TL ± SE

Type

Chromosome

RL ± SE

TL±SE

Type

1

S.92 ± 0.77

3.8 ± 0.36

M

1

6.00 ± 0.64

3.4 + 0.41

M

T

6.10 ±0.68

2.6 ± 0.23

SM

2

5.64 ± 0.37

3.2 ± 0.24

ST

3

5.63 ± 0.38

2.4 ±0.17

SM

3

5.64 ± 0.35

3.2 ±0.21

ST

4

5.63 ± 0.36

2.4 ±0.16

SM

4

5.64 ± 0.35

3.2 ±0.21

ST

5

5.40 ±0.61

2.3 ±0.1 7

SM

5

5.64 ± 0.33

3.2 ± 0.20

ST

6

5.40 ±0.61

2.3 ±0.1 7

SM

6

5.64 ± 0.33

3.2 ± 0.20

ST

7

6.10 ±0.62

2.6 ± 0.20

ST

7

5.47 ± 0.44

3.1 ±0.17

ST

8

5.40 ± 0.63

2.3 ±0.1 8

ST

8

5.47 ± 0.41

3.1 ±0.16

ST

9

5.40 ±0.61

2.3 ±0.1 7

ST

9

5.47 ±0.41

3.1 ±0.16

ST

10

5.40 ±0.61

2.3 ±0.1 7

ST

10

5.47 ±0.41

3.1 ±0.16

ST

11

5.40 ± 0.59

2.3 ±0.16

ST

11

5.29 ± 0.29

3.0 ± 0.20

ST

12

5.40 ± 0.55

2.3 ±0.14

ST

12

5.29 ± 0.26

3.0 ±0.17

ST

13

5.16 ±0.49

2.2 ±0.1 3

ST

13

4.94 ± 0.27

2.8 ±0.14

ST

14

4.93 ± 0.46

2.1 ±0.11

ST

14

4.94 ± 0.23

2.8 ±0.1 3

ST

15

4.93 ± 0.43

2.1 ±0.09

ST

15

4.94 ± 0.23

2.8 ±0.1 3

ST

16

4.93 ± 0.40

2.1 ±0.07

ST

16

4.94 ±0.19

2.8±0.10

ST

17

4.93 ± 0.40

2.1 ±0.07

ST

17

4.76 ±0.32

2.7 ± 0.20

ST

18

4.93 ± 0.37

2.1 ±0.05

ST

18

4.41 ±0.17

2.5 ±0.18

ST

* R'lf^H i~\r\ mtiitiir

^mmit*. rif t^\ 1^ l-ir\,'nl\

r\fi\ i-^»IIv

19

4.41 ±0.15

2.5 ±0.1 5

ST

that the Japanese species is a triploid. (Okamoto and Arimoto
1986). The second Japanese species had 38 chromosomes, and the
third species had 36 chromosomes (Table 4). They concluded that
the 36-chromosome species is ancestral to the other two: the 38-
chromosome species arose by aneuploidy. and the 54-chromosome
species arose by a triploid mechanism. Also, they assumed that the
triploid species reproduces by parthenogenesis, the way other trip-
loid animals reproduce (Okamoto and Arimoto 1986). Komaru et

c «

^\

> ^f c

t .

^

;^^

5 im

BiV Htf^ AA tkm mm aa

#i»i^f» ^#

^m ^A

9 10 U 12

15 16 n 18 19

Figure 3. A, Metaphase chromosome of C. papyracea colorata: B.
Karyotype constructed from A.

13

14

* Ba.sed on measurements of five karyotyped cells.

al. (1997) reported that hemaphroditic species C. leana and C.
fluminia produce non-reductional spermatozoa compared with
chromosome number and DNA content of somatic cells and sper-
matozoa. On the other hand, the dioecious C. sandai has been
reported produce reductional spermatozoa. Consequently, they as-
sumed that triploid C. leana and diploid C. fluniinea reproduce by
gynogenesis. In this study, chromosome numbers of C. fliiminea
counted 54. We assume that triploid C. fluminea in Korea repro-
duce by parthenogenesis, as Komaru et al. (1997) observed in the
C. leana.

In comparing the karyotypes of the Korean and Japanese spe-
cies, the Japanese triploid (C leana) has one more subterminal-
terminal chromo.somal homologue and one less submedian homo-
logue. However, because of the minimal difference, including con-
siderable similarity of the chromosomes classified as submedian or
subterminal-terminal, this apparent difference between the Korean
and Japanese karyotypes may be artificial. The same can be said of
the Korean and Japanese diploid species. The Japanese 38-
chromosome and 36-chromosome species have a submedian chro-
mosome, whereas the Korean species (C. colorata) apparently
does not.

Perhaps more significant than the apparent differences in the
chromosomal morphologies between the Korean and Japanese spe-
cies are their similarities (Table 4 and Figs. 1-3), which raises the
question of taxonomy. The genus Corhiciila is well known for its
intra- and interpopulational morphological variability, and the re-
sulting over-naming of the "species" has led to much confusion
that persists to the present time. Just which nominal species are
good biological species needs to be determined. An especially
valuable first step in such an endeavor is a cytogenetic one, as
demonstrated here.

Finally, it should be pointed out that chromosomes do not
support the taxonomic placement of C. colorata as a subspecies of
C. papyracea. The diploid C. colorata is obviously reproductively
isolated from the triploid C. papyracea and thereby the former
cannot be considered a subspecies of the latter.

982

Park et al.

TABLE 4.
Karyotypes in seven species of genus Corbicula.

Species

Karyotype

Somatic Chromosomes

M

SM

ST-T

References

C. flumineu
C. papyrucea
C. leana
C. colorala
C. japfniicii
C. scinclui
"C. leana"

?4 (3n)
54 (3n)
54 (3n)
38 (2n)
38 (2n)
36 (2n)
24

12
12
13
18
17
16

Present study

Present study

Okamoto and Arinioto 1986

Present study

Okamoto and Arimoto 1986

Okamoto and Arimoto 1986

Nadamitsu and Kanai 1978

LITERATURE CITED

Benthem Jutting, W. S. S. 1953. Systematic studies on the non-marine

mollusca of the Indo-Australian Archipelago. IV. Critical revision of

the freshwater bivalves of Java. Treubki 22:19-73.
Brusca, R. C. & G.J. Brusca. 1990. Invertebrates, pp. 279-313. Sinauer

Associates, Sunderland, MA.
Britton, J. C. & B. Morton. 1982. A dissection guide, field and laboratory

manual for the introduced bivalve Corhiciihi fluminea. Malacological

Rev 14(suppl.): 1-82.
Cherry, D. S., R. L. Roy. R. A. Lechleitner, P. A. Dunhardt, G. T. Peters &

J. Cairns, Jr. 1986. Corbicula fouling and control measures at the Celco

Plant. Virginia, pp. 69-81 In: Proceedings of the Second International

Corbicula Symposium, Special Edition 2.
Johnson, K. I., C. H. Henager, T. L. Page & P. F. Hayes. 1986. Engineering

factors influencing Corbicula fouling in nuclear service water systems.

pp. 47-52 In: Proceedings of the Second International Corbicula Sym-
posium, Special Edition 2.
Kligerman, A. D. & S. E. Bloom. 1977. Rapid chromosome preparation

from solid tissues of fishes. J. Fish. Res. Board Canada 34:266-269.
Komaru A., K. Konishi. I. Nakayama, T. Kobayashi, H. Sakai & K. Kawa-

mura. 1997. Hermaphroditic freshwater clams in the genus Corbicula

produce non-reductional spermatozoa with somatic DNA content. Biol.

Bull. 193:320-323.
Kwon, O. K., J. S. Lee & G. M. Park. 1986. A study of the embryonic

development and the egg deposition of Corbicula fluminia (Palaeohet-

erodonta: Corbiculidae). Korean J. Malacol. 2:26-29.
Kwon, O. K., G. M. Park & J. S. Lee. 1993. Coloured Shells of Korea, pp

1-445. Academy Publishing Company, Seoul, Korea.
Leven, A., K. Fredga & A. A. Sandberg. 1964. Nomenclature for centro-

nieric position on chromosomes. Hercdiias 52:201-220.
Macphee, D. D. 19X6. A mechanical strainer design for Corbicula fouling

prevention in the service water system at Arkansas nuclear one, unit 2.
pp. 59-61. In: Proceedings of the Second International Corbicula Sym-
posium, Special Edition 2.

McMahon R. F. 1983. Ecology of an invasive pest bivalve, Corbicula. Pp.
505-561. //).■ The Mollusca, Vol. 6. Ecology, Academic Press, San
Diego.

Nadanisitu. S. & T. Kanai. 1978. On the chromosomes of three species on
two families of freshwater Bivalvia. Bull. Hiroshima Woman 's Univ.
10:1-5.

Okamoto, A. & B. Arimoto. 1986. Chromosome of Corbicula japonica. C.
sandai and C. (Corbiculina) leana (Bivalvia: Corbiculidae). Venus 45:
194-202.

Page, T. L., D. A. Neilzel, M. A. Simmons & P. F. Hayes. 1986. Biofoul-
ing of power plant service systems by Corbicula. pp. 41—45 In: Pro-
ceedings of the Second Inlernalional Corbicula Symposium, Special
Edition 2.

Park, G. M. 1994. Cytotaxonomic studies of freshwater gastropods in Ko-
rea. Malacological Rev. 27:23^1.

Potter. J. M. & L. H. Liden. 1986. Corbicula control at the Potomac river
steam electric station Alexandria, Virginia, pp. 53-58. In: Proceedings
of (he Second International Corbicula Symposium. Special Edition 2.

Sandground, J. H. & C. Bonne. 1940. Ecliinoslcmui lindoensis n. sp. A new
parasite of man in the Celebes with an account of its life history and
epidemiology. Am. J. Trap. Med. 20:51 1-535.

Smithson, J. A. 1986. Development of a Corbicula control treatment at the
Baldwin power station, pp. 63-67. In: Proceedings of the Second In-
ternational Corbicula Symposium, Special Edition 2.

Sinclair R. M. 1971. Annotated bibliography on the exotic bivalve Cor-
bicula in North America, 1900-1971. Slerkiana 43:1 1-18.

Jimrmil of Slu-iljish Kc.scurch. Vol. \9. No. 2. yS3-990. 2000.

EVALUATION OF POTENTIAL ANESTHETICS FOR THE FRESHWATER
MUSSEL ELUPTIO COMPLANATA

WILLIAM A. LELLIS,' TIMOTHY A. PLERHOPLES," AND
KIMBERLY A. LELLIS'

U.S. Geological Survey

Biological Resources Division

Northern Appalachian Research Laboratory

R.D. 4, Box 63

Wellsboro, Pennsylvaitia 16901

ABSTRACT A .series of experiments was conducted to develop a safe, rapid, and reliable method to relax and anesthetize freshwater
mussels for collection of biological samples and assessment of reproductive status. Various concentrations and combinations of eight
potential anesthetic agents were administered by bath, slow drip, or injection into the foot or incurrent aperture of 10-40 Elliptio
complanalu per treatment group. Mussels were considered relaxed when the fool extended 2 cm beyond the shell and anesthetized when
the valves gaped, and the animal became impervious to touch. Buffered MS-222 (pH 7) produced relaxation within 60 min at
concentrations greater than 50 ppm and anesthesia within 3 h at 3(10-1, 000 ppm. Mussels exposed to unbuffered MS-222 at concen-
trations greater than 250 ppm (pH .^— t) ceased siphoning and closed tightly. Phenoxyethanol at 1.5-3.0% produced anesthesia within
20-50 min but had associated mortality. The effective dose of phenoxyethanol could be reduced to 0.25% with no mortality if mussels
were first relaxed with MS-222. Injection of 0.5-5.0 mg succinylcholine chloride into the foot produced rapid immobilization that
lasted 20-30 min. Dichlorvos at concentrations of 25-50 ppm induced anesthesia in 3-5 h, but mussels were extremely slow to recover.
Clove oil at 0.25-1.00 mL/L anesthetized 65-95% of the mussels tested but proved difficult to work with in confined spaces.
Magnesium chloride, potassium chloride, and menthol crystals had no apparent affect on Elliptio complanata. This study identified
several anesthetic agents for freshwater mussels, each differing in induction time, duration of effect, and degree of foot relaxation. We
recommend 500 ppm buffered MS-222 for general laboratory use on Elliptio complanata because of ease of handling and safety for
both humans and animals.

KEY WORDS: freshwater mussels, Elliptio complanata. anesthetics, relaxants

INTRODUCTION

The introduction and rapid spread of the zebra mussel Dieis-
sena polymorpha throughout North America has lead to extensive
mortality among native unionid mussels in many freshwater sys-
tems (Schloesser et al. 1996). Death of the mussels is believed to
be caused by either colonization of exposed valves by Dreissena.
habitat alteration, or depletion of food resources from the water
column (Strayer and Smith 1996). No effective means of control or
eradication for zebra mussels has yet been devised. One con.ser-
vation strategy being considered to preserve populations of the
most threatened native species is to remove them from Dreissena-
infested waters physically and place them into uninfected refugia
(Cope and Waller 1995), Monitoring of the chosen refugia for
adequacy in maintaining health, metabolic condition, and repro-
duction of the relocated mus.sels often requires visual examination
of mussel soft body parts and collection of tissue and physiological
fluids. Samples are usually obtained by mechanically prying the
valves apart with a reversing plier, which can fracture the shell,
damage the mantle, exhaust the mussel, and tear the adductor. The
additional stress of satnple collection potentially could reduce the
mussels' tolerance to the refugia environment.

An alternate means of obtaining tissue samples is to relax or
anesthetize the mussels with a chemical agent. This has been ac-
complished with such marine bivalves as oysters (Namba et al,
1995, Norton et al. 1996), scallops (Heasman et al. 1995). and

'Corresponding author. E-mail: lelliswm@usgs.gov

-Current address; 234 Delevan Avenue, Corning, NY 148.30.

'Current address: Box 1381, Geuysburg College, Gettysburg, PA 17325.

giant clams (Rosewater 1963) using benzocaine, chloral hydrate,
clove oil, magnesium chloride, menthol, MS-222, 2-phenoxyetha-
nol, propylene phenoxetol, and sodium pentobarbitone. Other mol-
lusks, including sea hares (Beeman 1968), land snails (Chung
1985), pond snails (Girdlestone et al, 1989), and abalone (White et
al. 1996) have been successfully relaxed using entlurane, halo-
thane, isoflurane. magnesium sulfate, and succinylcholine chlo-
ride. Although not intended as an anesthetic agent, the organo-
phosphate dichlorvos, used for treatment of sea lice in Atlantic
salmon net pens, has been reported to relax the adductor muscle of
marine bivalves for as long as 42 h after the end of exposure (Le
Bris et al. 1995).

Studies with freshwater unionids indicate that combinations of
pentobarbitol, urethane, clove oil, MS-222. nembutal, and menthol
can induce muscle relaxation or general anesthesia (Araujo et al,
1995, Coney 1993, Smith 1996). These techniques, however, were
developed to relax mus.sels into a lifelike position before death for
subsequent dissection or fixation, and thus recovery potential was
not considered. The objective of our study was to identify a
method to anesthetize freshwater mussels in a relaxed position
(i.e.. foot extended, shell gaped, unresponsive to touch) in a mini-
mum of time (less than 4 h) using agents that were safe to handle,
obtainable without a permit, inexpensive, and provided full and
unharmed recovery of the subject animal.

MATERIALS AND METHODS

Twenty-nine separate trials were conducted between July 1995
and June 1997 to evaluate the potential of eight chemical agents to
produce nonlethal anesthesia in the freshwater mussel Elliptio
complanata (Lightt'oot 1786). Mature mus.sels ranging in size from

983

984

Lellis et al.

63 to 1 15 mm and 26 to 165 g (mean 95 mm, 100 g) were collected
from Pine Creek, Tioga County. Pennsylvania (41 "44.408' N.
077°25.777' W) and transported in coolers without water to the
U.S. Geological Survey (USGS) Northern Appalachian Research
Laboratory in Wellsboro, Pennsylvania for experimentation. At the
laboratory, mussels were cleaned and measured then sorted into
groups of 10 mussels of equal total mass. Each group of 10 mus-
sels was then randomly allocated into a 132-L glass culture
aquarium containing 8 cm of white sand substrate and assigned a
treatment. The aquaria were each supplied with 1 L/min of
15-17 °C well water and illuminated with overhead fluorescent
lamps set to 14 h light: 10 h dark photoperiod. Water was circulated
within each aquarium by aeration from a 29-cm air diffuser set at
the drain end of the tank. Mussels were fed twice daily a mixture
of cultured Naiiochloropsis sp. (Florida Aqua Farms, Inc., Dade
City, Florida) and benthic detritus vacuumed from a concrete fish
pond. Tanks were cleaned weekly by scrubbing glass, stirring
sand, and draining.

Chemicals tested included MS-222 (Finquel; tricaine niethane-
sulfonate. Argent Chemical Laboratories, Redmond, Washington)
with or without Tris buffer (Sigma 7-9: Sigma Chemical Com-
pany, St. Louis, Missouri), magnesium chloride (MgCK, Fisher
Scientific Company, Fair Lawn, New Jersey), potassium chloride
(KCI, Aldrich Chemical Company, Inc., Milwaukee, Wisconsin),
succinylcholine chloride, 2-phenoxyethanol, clove oil, menthol
(Sigma), and dichlorvos (DDVP: 0.0-diniethyl-2,2-dichlorovinyl
phosphate, AMVAC Chemical Corporation, Los Angeles, Califor-
nia). MS-222, MgClj, and KCI were dissolved in well water before
addition to test tanks. Succinylcholine chloride was dissolved in
distilled water then either added directly to the test tanks or in-
jected into the mussel using a 26-G I2.7-mm hypodermic needle.
Clove oil and 2-phenoxyethanol were shaken vigorously with 250
mL well water before addition to the tanks. Menthol crystals were
powdered and mixed in 100 mL well water before application.
Dichlorvos was dissolved in distilled water to form a 6 mg/niL
stock solution, and the appropriate volume was added to the tanks.

Tests were conducted by transferring mussels from their culture
aquaria into separate treatment aquaria (no substrate) containing
2-30 L well water at the same temperature as the culture tanks.
Mussels were arranged within each treatment aquarium in two
rows of five and numbered 1-10 according to position. Water was
circulated within each aquarium by aeration from a 29-cm air
diffuser set in the center of the lank. After a 60 min acclimation,
treatments were administered to each aquarium as either a bath
(entire dosage applied at one time), by slow drip over a predeter-
mined period, or by injection. Drip treatments were administered
from a l,0()0-mL separation flask suspended over each tank. Num-
ber of replicate tanks of 10 mussels varied from I— f. depending
upon treatment (Tables 1-3). Time to relaxation and/or anesthesia
was recorded for each mussel from the moment of treatment ap-
plication, or from the start of application in the case of drip de-
livery. Some treatments included a pre-application of unbuffered
MS-222 before the primary chemical in an attempt to improve the
effectiveness or lower the required dosage of the primary chemical
agent. In these cases, time to relaxation was recorded from the
MS-222 pretrcatmcnt: whereas, time to anesthesia was recorded
from application of the primary treatment. A mussel was consid-
ered relaxed when the foot extended 2 cm beyond the valve and
anesthetized when the valves gaped, and the animal became im-
pervious to touch. These two events are independent phenomena.

with the goal being to identify a treatment that produced an anes-
thetized animal in a relaxed state.

Mussels were removed from treatment tanks either immedi-
ately upon detection of anesthesia or after a predetermined period
of 1-6 h. Mussels were rinsed in clean water, returned to culture
tanks, and arranged in the same order as when in the treatment
tank.

Recovery time, defined as the ability to maintain valve closure,
was individually recorded from moment of removal from the treat-
ment tank. Mussels were checked 24 h post-treatment for activity
(movement or burrowing) and at 7 days for mortality. Mussels
subjected to 2-phenoxyethanol, clove oil. menthol, and dichlorvos
were also checked for activity 7 days post-treatment (Table 3).
Data were analyzed using the general linear models procedure of
the Statistical Analysis System (SAS 1988). Any variable ex-
pressed as a percentage was arcsine transformed before analysis
(Rohlf and Sokal 1981 ). Differences in treatment means were de-
tected using the Waller/Duncan multiple range test. Orthogonal
polynomials were used to make linear, quadratic, and cubic con-
trasts among treatment effects in the analysis of variance (Rohlf
and Sokal 1981).

RESULTS

Bath solutions of 50-250 ppm unbuffered MS-222 produced
relaxation in 50-85% of test Elliplio compkinata within 39-54 min
of treatment application (Table I). Mussels exposed to MS-222
concentrations below 50 ppm siphoned normally but did not re-
spond to treatment: whereas, those exposed to concentrations
above 250 ppm stopped siphoning and closed valves tightly within
30 min of treatment application. Water pH decreased within the
treatment tanks from pH 7.0 at 50 ppm MS-222 to pH 3.4 at 1,000
ppm MS-222. No bath treatments of unbuffered MS-222 produced
anesthesia within the 4-h trial period. Activity at 24 h varied
greatly within each group of mussels unrelated to specific treat-
ment. There were no mortalities among the 320 mussels exposed
to unbuffered MS-222 bath.

Dripping unbuffered MS-222 to final concentrations of 25-
1,000 ppm over 20-120 min produced relaxation in 0-10()'>r of the
test animals in times ranging from 24-85 min, depending upon
treatment combination (Table 1). As with the bath treatments,
MS-222 drip did not produce anesthesia at any concentration
tested within the time allowed, and 24-h activity varied greatly
among groups. Most mussels exposed to MS-222 concentrations
above 250 ppm were tightly clo.sed by the end of the drip period.
One mortality occurred among the 320 mussels subjected to un-
buffered MS-222 drip, that being at 750 ppm. Injection of 1.2 cc
of 1 .000 ppm unbuffered MS-222 into the incunent aperture after
60 min of 100 ppm MS-222 bath produced relaxation similar to
bath and drip techniques, but no anesthesia.

MS-222 buffered to pH 7.0 with Tris produced relaxation in
65-95% of the mussels tested v\ithin 31-66 min of exposure to
concentrations of 5()-l.()0() ppm (Table 1 ). Unlike unbulTcred MS-
222, mussels exposed to buffered MS-222 at concentrations above
250 ppm continued siphoning normally and reached a state of
relaxed anesthesia within 126-194 min at concentrations between
500-1,000 ppm (Table I, Figure 1 ). Time to anesthesia decreased
(P < 0,01 ), and lime to recovery increased (/' < 0.01 ) with increas-
ing dosage above 500 ppm. Activity at 24 h was lower (P = 0.03)
among anesthetized mussels than those that were treated but did
noi reach anesthesia. There were no mortalities among the 260
mussels exposed lo buffered M.S-222.

Anesthhtics for Freshwater Mussels

985

TABLE I.
Summan of trials conducted to evaluate MS-222 as a potential anesthetic for Elliplio complanala.

Treatment

Level

Application
Method

Mussels

#

Mussels
Relaxed

1%)

Time to
Relax
(min)

Mussels
Anesthetized

Time to

Anesthesia

(min)

Time to

Recover

(min)

Active
at 24h

(%)

Mortality
at 7 days

(%)

Notes

MS-:::

MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222

MS-:::
MS-:::

MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222
MS-222

MS-:::

0 ppni Bulli

5 ppm

! 0 ppm

15 ppm

:0 ppm

50 ppm

75 ppm

100 ppm

:50 ppm

500 ppm

750 ppm

1.000 ppm

0 ppm

:5 ppm

50 ppm

75 ppm

100 ppm

250 ppm

400 ppm

500 ppm

700 ppm

750 ppm

750 ppm

750 ppm

750 ppm

750 ppm

1 ,000 ppm

1 .000 ppm

Balh
Bath
Balh
Bath
Bath
Bath
Balh
Balh
Balh
Balh
Balh

Drip — 20 min
Drip — :0 min
Drip — :o min
Drip — :0 min
Drip — 1:0 min
Drip — 30 mm
Drip — 1:0 min
Drip — 30 min
Drip — 1:0 min
Drip — 30 mm
Drip — 45 min
Drip — 60 min
Drip — 90 min
Drip — 1:0 min
Drip — 30 min
Drip — 1:0 mm

MS-:::/MS-:22 lOO/l.OOOppm Balh/lnjecl

MS-222 Buffered
MS-2:: Buffered

MS-222 Buffered
MS-222 Buffered
MS-22: Buffered
MS-::: Buffered
MS-::: Buffered
MS-:22 Buffered

0 ppm Balh
50 ppm Balh

75 ppm
100 ppm
250 ppm
500 ppm
750 ppm
1 .000 ppm

Bath
Balh
Balh
Balh
Bath
Balh

20

20
20
20
20
40
20
40
40
40
20

:o

:o
:o
:o
:o
:o
:o
:o

20

:o
:o
:o
:o
:o
:o
:o
:o

:o

15

0

0

0

50

85
85

s:

2
0
0

45
45
85
80
85
30
100
30
0
35
70
90
70
95
55
65

75

i:o
4:

40
54
44
39

:o5

n:

85

6:

46

:6

:5

30

:6
:4

2b
36

4:

56

:4

28
39

20

5

19

20

65

50

20

70

44

40

85

43

40

95

31

40

80

5S

40

85

66

40

88

50

0

0

0

70

85

85

194
149
126

35
49

40

90

75
15
45
32
10
32
48
70
35
30

75
90
55
70
50

100

100

30
80
40
60

100
40

70
30

25
30

5
10

pH 7.1; Trial maxima

4 h
pH 7.1
pH7.1
pH 7.1
pH7.0
pH 7.0
pH6.7
pH 6.6
pH 4.5
pH 3.8
pH 3.6
pH 3.4

Trial maxima 4 h

Injecl 1 .2 cc of
1.000 ppm MS-2::
inlo aperture afler
60-min bath

Trial maxima 5 h
Ail Irealmenls

buffered lo pH

7.0 with Tris

Bath and drip solutions of 10-40 g/L MgCK produced some
relaxation but no appreciable anesthesia in ElUptio complaiuita
(Table 2). Mortality occurred at the higher MgCU doses. Mus.sels
subjected to MgCU generally closed very tightly within 60 min of
exposure and produced copious mucus discharge. Relaxation of
the mussels with 100 ppm MS-222 before MgCl, drip did not help
to induce anesthesia. Injection of 30-60 mg of MgCU into the
incurrent aperture produced an anesthetic state in 10-20*^ of the
mussels, but with 10% associated mortality. The anesthetized mus-
sels, howe\er. were not the same individuals that subsequently
died.

Bath solutions of 10-40 g/L KCl had no relaxing or anesthetic
effects on ElUptio compkmata (Table 2). Mussels stopped siphon-
ing upon exposure to KCl and remained tightly closed throughout
the 2-h trial. Mortality (5-20%) occurred at all levels of KCl above
10 g/L.

Bath solutions of 250-1,000 ppm succinylcholine chloiide had

little effect on ElUptio complaiuita (Table 2). Mussels exposed to
bath concentrations above 500 ppm became sluggish in response to
touch, but only one mussel at 750 ppm reached an anesthetic state.
One mussel exposed to 1,000 ppm died within 7 days of treatment.
Injection of 0.5-5.0 mg succinylcholine chloride into the foot pro-
duced anesthesia in 100% of the mussels within 4—5 min of injec-
tion, with recovery in 23-30 min. Mussels gaped and were unre-
sponsive to touch after injection, but feet remained in a constricted
unrelaxed state. Mussels injected with 0.5 mg showed some sen-
sitivity to stimulation, but could not sustain valve closure. Activity
at 24 h decreased (P = 0.02) with increasing dosage. There were
no mortalities among the 160 mussels injected with succinylcho-
line chloride.

Bath solutions of 0.25-3.0% 2-phenoxyethanol induced anes-
thesia but no foot relaxation (Table 3). Percentage of mussels

986

Lellis et al.

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Anesthetics for Freshwater Mussels

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Lellis et al.

Figure 1. Elliptio complanata relaxed and anesthetized witli 750 ppm
buffered MS-222.

and remained lightly closed for the duration of the 3-h trial. There
were no mortalities associated with exposure to menthol in this
trial.

Bath solutions of 0.1-10 ppm dichlorvos had no affect on El-
liptio complanata within a 6-h exposure period (Table 3). Con-
centrations of 25-50 ppm produced anesthesia in 80-100% of the
mussels tested in an average of 196-267 min. Feet were retracted
during anesthesia. Recovery time was prolonged as compared to
other treatments, requiring 24-36 h to regain full responsiveness to
touch. Mussels anesthetized by dichlorvos were less active {P =
0.0 1 ) at 7 days post-treatment than mussels that had been exposed
to dichlorvos but not anesthetized. Most anesthetized mussels re-
quired in excess of 2 weeks to upright themselves and resume
burrowing activity. No mortalities occurred among the 120 mus-
sels exposed to dichlorvos during this study.

DISCUSSION

anesthetized increased (P = 0.003) in a nonlinear fashion (cubic
response P = 0.005) with increasing dosage to an apparent maxi-
mum of about 70% anesthesia at 2.0% 2-phenoxyethanol. Mussels
exposed to 2-phenoxyethanol solutions of 1 .0% or less continued
siphoning throughout the 5-h trial if they did not reach anesthesia;
whereas, mussels exposed to concentrations greater than 1 .0%
closed tightly and cea.sed siphoning if not anesthetized. Time to
anesthesia decreased {P = 0.005) with increasing dosage in a
nonlinear fashion (cubic response P = 0.001 ) to an apparent mini-
mum at approximately 1.50% 2-phenoxyethanol. Dosage level did
not affect time to recovery (P = 0.30) nor 24-h activity {P =
0.16). Mortality (5% of mussels tested) occurred at 2-phenoxy-
ethanol doses greater than 1 .50%. Mussels exposed to 3.0% 2-phe-
noxyethanol produced copious mucus discharge after being re-
turned to fresh water.

Exposure of mussels to a 60-min bath of 100 ppm MS-222
before 2-phenoxyethanol treatment increased {P < 0.0001) anes-
thetic rate among mussels subjected to low concentrations of
2-phenoxyethanol (Table 3). Time to anesthesia (P = 0.02) and
time to recovery (P = 0.04) were also decreased among these
treatment groups by preconditioning with MS-222. However, MS-
222 also decreased the dosage level of 2-phenoxyethanol at which
mortality occurred to about 1.0%. Bath application of 2-phenoxy-
ethanol was more effective than drip application in inducing an-
esthesia. Although anesthetized mussels generally constricted their
feet while actively exposed to 2-phenoxyethanol, feet became re-
laxed and extended within a few minutes of return to fresh water.

Bath solutions of 0.25-1.00 mL/L clove oil induced anesthesia
in 65-95% of Elliptio complanata tested without significant foot
relaxation (Table 3). Although anesthetic rate was similar among
treatment levels (P= 0.20), lime to anesthesia decreased (qua-
dratic response P = 0.006), and time to recovery increased (linear
response P = 0.01 ) with increasing dosage level. Similar to 2-phe-
noxyethanol, mussels became anesthetized in a constricted posi-
tion, but relaxed and extended feet upon transfer to fresh water.
Exposure of mussels to 100 ppm MS-222 for 60 min before clove
oil addition did not increase anesthetic rate at 0.25 mL/L or 0.50
mL/L dosage levels (P = 0.29), but did reduce time to anesthesia
(P = 0.03). There were no mortalities among the 160 mussels
exposed to clove oil during these trials.

Powdered menthol crystal had no anesthetic or relaxing effects
on Elliptio complanata at 125-500 mg/L dosage levels (Table 3).
Mussels ceased siphoning upon initial exposure to menthol crystal

The goal of this study was to identify a nondestructive method
to anesthetize unionid bivalves in a relaxed position to allow col-
lection of biological samples and examination of internal anatomy.
Relaxation was defined as extension of the foot al least 2 cm
beyond the valves and anesthesia as the gaping of valves with
unresponsiveness to touch. These two events are separate and un-
connected, because a relaxed mussel may be fully responsive to
touch: whereas, an anesthetized mussel may be in a constricted
position. Because foot extension also occurs with burrowing ac-
tivity, some control groups were recorded as having relaxation
even though no chemical treatment was applied (e.g.. MS-222 0
ppm. Table 1 ). However, foot extension associated with burrowing
can often be distinguished from cheinical relaxation, because bur-
rowing was a more active process and typically took longer to
initiate, usually in excess of 100 min. An exception occurred
within the buffered MS-222 control group in which a single animal
extended its foot after 19 min. The term "anesthesia" often implies
the loss of consciousness or sensitivity, but gaping and lack of
response can also be attributable to muscular paralysis. No dis-
tinction was made between these two causes of gaping in this
study.

Using these defmitions, Elliptio complanata were relaxed by
MS-222 and anesthetized by MS-222, succinylcholine chloride,
2-phenoxyethanol, clove oil, and dichlorvos. MgC^. KCI, and
menthol crystal had no appreciable effect on the mussels. Pretreat-
ment with MS-222 before addition of other primary chemical
agents often decreased time to anesthesia and/or dosage require-
ments for the primary anesthetic and placed the mussels in a re-
laxed position during anesthesia.

MS-222 (tricaine methanesulfonate) is the most commonly
used anesthetic for frnfish in North America (Summerfelt and
Smith 1990). Mechanism of action is presumed to be through
stabilization of cellular membranes in nervous and cardiac tissue,
preventing transient increases in sodium permeability and thus
decreasing excitability (Letcher 1992). Dosage of 60-250 ppm
produces anesthesia in Atlantic halibut, red drum, and goldfish in
3-6 min with recovery in less than 10 min (Malmstrom et al. 1993,
Massee et al. 1995). MS-222 has been used less frequently to
anesthetize bivalves. Coney ( 1993) used 75-100 ppm for 12-36 h,
and Araujo et al. (1995) used 5()0-2.()()0 ppm for 24 h to relax
unionids before lethal fixation. Norton el al. (1996) relaxed the
pearl oyster Pinctaeta albina with 1 ,000 ppm MS-222, but recom-
mended buffering to pH S to prevent excess mucus production and

Anesthetics for Freshwater Mussels

989

reduce recovery time. Heasman et al. ( 1995) could not anesthetize
the scallop Pecten fwnalus with 1.000 ppm MS-222 within 60-min
exposure.

In this study, bulTered MS-222 at concentrations greater than
100 ppm produced foot relaxation in 85% of Elliptio complanara
within 30-60 min, and concentrations above 500 ppm produced
anesthesia within 2-3 h. Mussels further relaxed after transfer to
fresh water, hut reco\ered w ithin 30-60 min. Unbuffered MS-222
produced foot relaxation but not anesthesia, presumably because of
low pH of the higher dosages. The inability of unbuffered MS-222
to produce anesthesia may not be completely attributed to cessa-
tion of siphoning activity, inasmuch as direct injection of 1,000
ppm unbuffered MS-222 into the incurrent aperture al.so failed to
anesthetize the animals (Table I ). Although this may simply indi-
cate that the volume of injected MS-222 was insufficient, it may
also indicate that low pH causes physiologic changes in EUiptio
complancila or chemical changes in MS-222 that affect absorption
or metabolism of the compound.

Magnesium chloride solutions of 30-50 g/L have been used to
induce valve gaping in such marine bivalves as the Pacific oyster
Crassostrea gigas (Whyte and Carswell 1983), the European flat
oyster Ostrea editlis (Culloty and Mulcahy 1992), and the scallop
Pecten fiimams (Heasman et al. 1995), but not the pearl oyster
Pinaada cdhina (Norton et al. 1996). Mechanism of action was
considered to be inhibition of muscular contraction attributable to
displacement of calciuin ions from tissue by magnesium (Whyte
and Carswell 1983). In this study, the freshwater mussel Elliplio
complanata showed some signs of foot relaxation at MgCU con-
centrations exceeding 30 g/L. but remained responsive throughout
the 2-4-h trials.

Beeman (1968) reported that injections of 0.05 mg/g of the
myoneural blocking agent succinylcholine chloride (ester di-
methochloride) produced rapid relaxation of the sea hare Aplysia
californica with recovery within 45-90 min. The drug was dis-
solved in seawater and pH of the solution adjusted to 6.-1—7.0 with
HCl to prevent alkaline hydrolysis of the ester linkage. Chung
(1985) found that injections of 0.012 mg/g succinylcholine chlo-
ride combined with 2.4 mg/g MgCl, produced quick and pro-
nounced anesthesia in the land snail Helix aspersa with no mor-
tality. In the present study, succinylcholine chloride injections of
0,5-5.0 mg/mussel produced rapid anesthesia (< 5 min) of Elliptio
complanata with recovery within 30 min. Inasmuch as average live
animal mass was 100 g, with about 207r of that being soft tissue,
injected dosage was approximately 0.005-0.05 mg/g whole body
and 0.025-0.25 mg/g soft tissue weight. All succinylcholine chlo-
ride treatments had equal affect on the mussels, except that 24-h
activity was depressed at the higher levels. Therefore, smaller
dosages than those used in this study would probably be effective
in anesthetizing Elliptio complanata. MS-222 pretreatment was
used to allow easier access to the injection site, and its affects on
anesthetic rate are believed to have been minimal.

Propylene phenoxetol and a related compound, 2-phenoxyetha-
nol, have been used to anesthetize a variety of gastropods and
bivalves, such as the giant clam Triducna maxima (Rosewater
1963). the abalone Haliotis midae (White et al. 1996). and the
pearl oyster Pinctada albina (Norton et al. 1996). Effective dose of
2-phenoxyethanol was 0.3-0.4% for these species. Heasman et al.
(1995) could not relax the scallop Pecten fumatus with 0.06%
2-phenoxyethanol and Araujo et al. ( 1995) could not relax unionid
mussels with 1.0% 2-phenoxyethanol solutions. In the present
study, Elliptio complanata were anesthetized in less than 30 min

using 2.0% 2-phenoxyethanol with recovery in about 1 h. This
dosage could be reduced to 0.5% or less when combined with an
MS-222 pretreatment. Although the sticky, adhesive consistency
and noxious fumes of 2-phenoxyethanol made the substance dif-
ficult to handle, it produced quicker and deeper anesthesia in El-
liptio c(mtplcmata than MS-222 treatment alone. However, expo-
sure to humans may cause irritation to sensitive tissue and damage
to kidney and liver, and Summerfelt and Smith ( 1990) recommend
discontinued use as a fish anesthetic because of inherent toxic
effects.

Clove oil has been used as a fish anesthetic in Southeast Asia,
because it is inexpensive, readily available, and simple to apply.
Soto and Burhanuddin ( 1995) reported that rabbitfish Siganiis lin-
eatus lost consciousness within 3 min of exposure to 0.1 mL/L
clove oil and recovered within 3 min of transfer to fresh water.
Araujo et al. (1995) found clove oil to be an effective anesthetic for
the freshwater mussel Unio sp, and the clam Pisidium amniciim.
but not for the Asian clam Corbicula fluminea. Norton et al. (1996)
relaxed the pearl oyster Pinctada albina with 1 .5 mL/L clove oil.
In the present study. Elliptio complanata reached anesthesia within
90 min of exposure to 0.125-1.0 mL/L. Lack of dose response in
percentage mussels anesthetized may indicate that lower doses of
clove oil can be used for relaxing Elliptio complanata than were
applied in this study. However, clove oil fumes were found to be
particularly irritating to the eyes and respiratory tract, and experi-
mentation was discontinued. Use of this substance may be limited
to outdoors or to indoors within ventilated hoods.

Menthol has been used as a general anesthetic for invertebrates,
because it is readily available, inexpensive, easily handled, and
gives acceptable results over a wide range of species (Araujo et al.
1995). Smith ( 1996) successfully narcotized freshwater mussels by
subjecting them to powdered menthol solutions for 24 h. Re-
sponse, however, is often unpredictable, and neither Coney (1993)
nor Araujo et al. (1995) had success using menthol to anesthetize
freshwater unionids. Norton et al. (1996) used 250 mg/L menthol
crystal to relax the pearl oyster Pinctada albania. but in the present
experiment Elliptio complanata were unaffected by 1 25-500 mg/L
during 3-h trials. Success with menthol may be related to water
temperature, considering that Runham et al. (1965) reported im-
proved results by transferring animals to hot water.

Dichlorvos is an organophosphate that affects the nervous sys-
tem of animals by inhibiting function of the enzyme acetylcholin-
esterase (Murison et al. 1997). This results in elevated levels of the
neurotransmitter acetylcholine, leading to exhaustion and possibly
death by continuous neuromuscular stimulation. Bath treatments of
1.0 ppm are used in commercial salmon farms to kill such ecto-
parasitic crustaceans as sea lice (MacKinnon 1997). Le Bris et al.
( 1995) found that dichlorvos concentrations of O.I-l.Oppm caused
adductor muscle relaxation in Manila clams {Riiditapes philippi-
nanim) and Japanese oysters (Crassostrea gigas) within 2 h of
exposure. Recovery occurred within 1 2 h after removal from treat-
ment with no latent mortalities. In the present experiment. Elliptio
complanata required much greater dichlorvos concentrations (25-
50 ppm) to initiate gaping within the 6-h allotted exposure time.
Recovery required more than 24 h, and activity levels remained
depressed in excess of 7 days. Thus, dichlorvos would not be
appropriate for field use or when immediate recovery and burrow-
ing are required, but may be useful in laboratory situations where
extended anesthesia is necessary.

In summary, this study identified several compounds useful in
relaxing and/or anesthetizing the freshwater mussel Elliptio com-

990

Lellis et al.

planata. each with differing induction times, recovery rates, ease
of use, and danger to the operator. Our present protocol is to
de-water the mussels for 30-60 min before immersion in 500 ppm
buffered MS-222. Mussels are taken to near, but not full anesthe-
sia, then held open with either a finger or reversing plier during
sample collection and internal examination. Several hundred El-
lipiio complanata were sexed using this technique and held in
captivity for over 1 year with no mortality or apparent affect on
behavior. MS-222 has also been used to anesthetize Alasmidonta
undulata. A. vcuicosa, Lasinigona stibviridis. and Strophitus un-
dulatus, although reaction time is quicker for these species than for
Elliptio complanata. Pyganodon cataracta did not respond to 500
ppm MS-222 and may require a different chemical agent and/or

technique for anesthesia. Thus, anesthetic protocols will likely
need to be developed independently for each new species under
investigation.

ACKNOWLEDGMENTS

We thank Connie Johnson, Chri.stine Lellis, and Gina Totino
for assistance in conducting these studies. Dichlorvos was pro-
vided by William Feiler of AMVAC Chemical Corporation. Timo-
thy Plerhoples was funded by the New York Academy of Sciences
through the Science Research Training Program. Kimberly Lellis
participated in these studies as a U.S. Department of the Interior
Student Volunteer.

LITERATURE CITED

Araujo, R.. J. M. Remon. D. Moreno & M. A. Ramos. 1995. Relaxing
techniques for freshwater mollusks: trials for evaluation of different
methods. Mulacologia 36:29^1.

Beeman. R. D. 1968. The use of succinylcholine and other drugs for
anesthetizing or narcotizing gastropod mollusks. Puhhl. Siciz- Zool.
Ncipoli 36:267-270.

Chung. D. 1985. An anesthetic for internal operations on the land snail
Helix aspersa Muller. VW/ger 27:331-335.

Coney, C. C. 1993. An empirical evaluation of various techniques for
anesthetization and tissue fixation of freshwater Unionoida (Mollusca:
Bivalvia). with a brief history of experimentation in molluscan anes-
thetization. Veliger 36:413-424.

Cope. W. G. & D. L. Waller. 1995. Evaluation of freshwater mussel
relocation as a conservation and management strategy. Reg. Ri\er.s:
Res. Man. 11:147-155.

Culloty, S. C. & M. F. Mulcahy. 1992. An evaluation of anesthetics for
Ostrea ediilis. Aquaculnire 107:249-252.

Girdlestone, D. G., S. G. H. Cruick.shank & W. Winlow. 1989. The actions
of three volatile general anaesthetics on withdrawal responses of the
pond snail Lymiuieii Magiuilis (L.). Coinp. Biochem. Physiol. 92C:39-
43.

Heasman. M. P.. W. A. O'Connor & A. W. J. Frazer. 1995. Induction of
anesthesia in the commercial scallop. Peclen fiimauis Reeve. .Acjuacul-
lure 131:231-238.

Le Bris, H., P. Maffart, G. Bocquene, V. Buchet, F. Galgani & G. Blanc.
1995. Laboratory study on the effect of dichlorvos on two commercial
bivalves. Aqiiaeiillitre 138:139-144.

Letcher. J. 1992. Intracelomic use of tricaine methanesulfonate lor anes-
thesia of bullfrogs {Rami calesheiaiui) and leopard frogs {Raiui pipi-
tvi.vl. Z<io Biol. 11:24.3-251.

MacKinnon. B. M. 1997. Sea lice: a review. World Atimunll. 28(31:5-10.

Malmslrom. T.. R. Salte. H. M. Gjiien & A. Linseth. 1993. A practical
evaluation of metoniidate and M.S-222 as anesthetics for Atlantic hali-
but (Hippoglossiis hippoglossus L.). Aquaeullure 113:331-338.

Mas.see. K. C, M. B. Rust, R, W. Hardy & R. R. Stickney. 1995. The
effectiveness of tricaine. quinaldine sulfate, and metoniidate as anes-
thetics for larval fish. Ac/iuicidnire 134:351-359.

Murison. D. J., D. C. Moore, J. G. McHenery, N. A. Robertson & I. M.

Davies. 1997. Epiphytic invertebrate assemblages and dichlorvos usage
at salmon farms. Aquaculnire 159:53-66.

Namba. K.. M. Kobayashi. S. Aida. K. Uematsu. M. Yoshida. Y. Kondo &
Y. Miyata. 1995. Persistent relaxation of the adductor muscle of oyster
Crassostrea gigas induced by magnesium ion. Fish. Sci. 61:241-244.

Norton, J. H., M. Dashorst. T. M. Lansky & R. J. Mayer. 1996. An evalu-
ation of some relaxants for use with pearl oysters. Aquaeullure 144:
39-52.

Rohlf F. J. & R. R. Sokal. 1981. Statistical tables. W, H. Freeman and
Company. New York. 219 pp.

Rosewater. J. 1963. An effective anesthetic for giant clams and other
mollusks. Turto.x News 4I(12):300-301.

Runham, N. W., K. Isarankura & B. J. Smith. 1965. Methods for narco-
tizing and anesthetizing gastropods. Malacologia 2:231-238.

SAS Institute Inc. 1988. SAS procedures guide release 6.03. SAS Institute,
Cary. NC. 441 pp.

Schloesser. D.W., T. F. Nalepa & G. L. Mackie. 1996. Infestation of
unionid bivalves (Unionidae) in North America. Am. Zool. 36:300-310.

Smith, D.G. 1996. A method for preparing freshwater mussels (Mollusca:
Unionoida) for anatomical study. Am. Malacolog. Bull. 13 1/2:125-
128.

Soto, C. G. & Burhanuddm. 1995. Clove oil as a fish anaesthetic for
measuring length and weight of rahbitfish iSigaiurs lineauis). .Aquaeul-
lure 136:149-152.

Strayer, D. L. & L. C. Smith. 1996. Relationships between zebra nius.sels
(Dreissena polymorpha) and unionid clams during the early stages of
the zebra mussel invasion of the Hudson River. Fresh. Biol. 36:771-
779.

Summerfell. R. C. & L. S. Smith. 1990. Anesthesia, surgery, and related
techniques, pp. 213-272. In: C. B. Schreck and P. B. Movie (eds.).
Methods for Fish Biology. American Fisheries Society. Bethesda. MD.

White. H. I., T. Hechi & B. Potgeiter. 1996. The effect of lour anesthetics
on Haliotis midae and their suitability for application in commercial
abalone culture. Aquaeulture 140:145-151.

Whyte. J. N. C. & B. L. Carswell. 1983. Chemical aid for shucking the
Pacillc oyster. Crassostrea gigas. Can. Tech. Rept. Fish. .Aquat. Sci.
1238:1-38.

Joiinml of Sluilfish Rc\t'iiirh. Veil. 14. N(i. 2, WI-W3. 2()()().

SURVIVAL AND GROWTH OF MUSSELS SUBSEQUENT TO HEMOLYMPH

SAMPLING FOR DNA

JENIA F. YANICK AND DANIEL D. HFIATH*

Biology, College of Science and Management
University of Northern British Cohiinbiu
3333 University Way. Prince George
British Coliinihia. Canada. V2N 4Z9

ABSTRACT With the increasing use of molecular genetic techniques in ecology and evolution, it has become apparent thai methods
of non-destructive DNA sampling must be developed. In this study we collected ?0 blue mussels {Mylihis spp.) in each of three size
categories: small ( H)-2() mm), medium (20-30 mm), and large (30+ mm). Hemolymph was extracted from 25 mussels in each size
category and the remaining 25 mussels served as controls. The hemolymph wa.s extracted and control mu.ssels were monitored for 384
days, during which lime no significant differences in survival or growth were found. We extracted DNA from the hemolymph and
successfully polymerase chain reaction-amplified the ITS and Glu-5' .specie.s-specific markers from 81% and 92% of the samples,
respectively, and determined that all mussels were Mylilus Iros.uilus (Lamarck). The extraction of hemolymph for DNA analysis allows
for molecular investigations of populations or species which are either rare or in limited nutnbers, and for life history investigations
where survival of the organism is necessary.

KEY WORDS: non-destructive. Mylilus. PCR. DNA. survival, growth, hemolymph

INTRODUCTION

Molecular genetic techniques and the genetic characteiization
of individuals have become comtnon in the study of the ecology
and evolution of marine invertebrates, particularly bivalves (Mit-
ten 1994), The benefit of the polymerase chain reaction (PCR) is
that very little DNA is required, thus making analysis possible
when the quantity and/or quality of DNA is limited. PCR-based
species markers have been used within the Mytihis species com-
plex for conservation, ecological, and evolutionary applications
(Heath et al, 1995, 1996, Rawson et al. 1996). Molecular genetic
characterization has also clarified population genetic structure in a
variety of other bivalves (Sarver and Foltz 1993, Manuel et al.
1996, David et al. 1997, Suchanek et al, 1997, Herbinger et al.
1998), as well as aiding in investigations into the ecology and life
history of bivalves with planktonic phases (Tore 1998).

Typically, shellfish are destructively sampled in order to
sample tissue for DNA extraction. Destructive sampling involves
killing the animal to obtain the necessary tissue for genetic analy-
sis (Taberlet et al. 1999), Although this is acceptable for some
studies (Sarver and Foltz 1993, Heath et al, 1995, Hare et al. 1996,
Heath et al. 1996, Suchanek et al. 1997, Herbinger et al. 1998,
Toro 1998), destructive sampling is clearly not acceptable for stud-
ies involving growth or survival measurements, or for investiga-
tions of small or rare populations. For such studies there is a need
for a technique that would allow the collection of DNA without
harming the organism. Non-destructive sampling generally in-
volves capturing the target organism, taking an invasive sample
without killing it. and then releasing it (Taberlet et al. 1999). It is,
therefore, important that any potential technique be tested for even
minor adverse effects on the survival or growth of the target or-
ganism.

Here we describe sampling hemolymph for DNA extraction
from shellfish. Specifically, we sampled hemolymph from three
size categories of blue mussels {Mytihis spp.), extracted DNA, and

*Correspondmg author, current address: Great Lakes Institute lor Environ-
mental Research. University of Windsor, Windsor. ON, Canada N9B 3P4.

amplified fragments using PCR with species-specific markers.
Hemolymph is made up of mostly water, but does contain cells,
including nucleated hemocytes (Morse and Zardus 1997) and is
responsible for the transportation of digestion products throughout
the body (Brusca and Brusca 1990), among other functions. We
followed the survival and growth of hetnolymph-extracted and
control mussel groups for over 1 y to ascertain whether this tech-
nique resulted in decreased survival and/or growth.

MATERIALS AND METHODS

Fifty mussels in each of three size categories were collected
frotn the western coast of Quadra Island, located near Campbell
River, British Columbia, Canada. The mussels were measured with
calipers to the nearest 0.01 min and sorted into small (10-20 mm),
medium (20-30 inm). and large (30-t- mm) size categories. In each
group, hemolymph was extracted (50-200 |jiL) from 25, while the
other 25 (control) mussels were handled, but not sampled. A l-cc
syringe (22-gauge, 1.5-inch needle) was inserted through the rear
hinge joint and hemolymph was extracted until no more fluid
could be removed. The extracted hemolymph was expelled into 1 .0
mL of 95'7f ethanol and stored at rooin temperature. After sam-
pling, mussels were placed in six cages (3 hemolymph-extracted
and 3 control) and hung approximately 1 m below the surface at
the original collection site. The cages were 5 x 5 x 10 cm and were
slotted to enable free water flow through the cages, but excluded
potential predators.

The hemolymph and alcohol were transpoited to the laboratory
where they were centrifuged (13,000 rpm, 15 min), the liquid was
removed, and the pellet dried (LABCONCO Centrivap Concen-
trator) at 60 °C for 8 min. The dried cells were digested overnight
in 200 (jiL of lysis buffer (10 niM Tris-HCI, pH 8.0. 15 iiiM
ethylenediamine tetra acetate, and 0.5'7f sodium dodecyi sulphate)
and 125 jxg of proteinase K at 37 "C. The solution was then
extracted once with an equal volume of phenoLchloroform:
isoamyl alcohol (24:24:1), followed by isopropanol precipitation
(Heath et al. 1995). The extracted DNA was resuspended in 100
|j.L of double-distilled water and was then PCR-amplified follow-
ing the ITS protocol described in Heath et al. (1995) and the

991

992

Yanick and Heath

Glu-5' protocol described in Rawson et al. (1996). Both markers
give species-specific results (Heath et al. 1995. Rawson et al.
1996) and thus we are reasonably certain we amplified target
DNA, and not contaminating DNA. The PCR products were visu-
alized on a 1.8% agarose gel stained with ethidium bromide (Fig.
1 ). Individual mussels were scored for genotype at each marker
locus on the basis of a diagnostic restriction fragment length poly-
morphism (ITS: Heath et al. 1995) or using an automated DNA
sequencer to determine amplified fragment length (GLU-5'; Raw-
son et al. 1996).

Between April 3, 1998 and April 22. 1999. the mussels in this
experiment were measured and the survivors were counted at three
sampling times after transfer to the experimental cages (58, 140,
and 384 days). Student's / test (shell length) and chi-square (sur-
vival) were used for statistical analysis to determine whether dif-
ferences existed between the two groups at day 58 and day 384.
Day 58 comparisons were made to test for short-term effects, while
day 384 comparisons were made for long-term effects.

RESULTS

DNA was successfully PCR-amplified from 61 of the 75
samples (81%) for ITS, and from 69 of the 75 samples (92%) for
GLU (Fig. I ). There was no consistent effect of mussel size (and
hence hemolymph volume) on the success of the PCR amplifica-
tion. All mussels were determined to be Mxtihis trosstilus. The
hemolymph technique was found to have little effect on either
survival or growth (Fig. 2). At day 58 and day 384, the survivor-
ship of the hemolymph-extracted mussels was not found to be
significantly different than the survivorship of the control mussels
in any of the size categories (P > 0.10). At day 58, the control
mussels were slightly larger than the hemolymph-extracted mus-
sels in the small size category (P = 0.018), but there was no
significant difference in either the large or the medium size cat-
egories (P > 0.50). At day 384, the hemolymph-extracted mussels

were larger than the control mussels in the large size category (P
= 0.033), but there was no significant difference in the medium or
small size categories (P > 0.10).

There were also no consistent differences found in .survival or
growth among the mussel size categories. The smallest mussels we
sampled were between 10 and 20 mm and had mortality and
growth similar to the larger size categories.

DISCUSSION

We sampled a wide range of sizes of mussels using a non-
destructive method of DNA sampling and successfully extracted
DNA for PCR purposes from most of the mussels, including those
in the small category. Our PCR success rates were comparable to
those of Heath et al. (1995) who used destructive tissue sampling
methods. The technique described here is a useful tool for field-
work, as it does not require the killing of the organism under study.
Furthermore, we found no consistent effect of hemolymph sam-
pling on either survival or growth of the mussels. Although this is
not surprising for the larger mussels, it is unexpected for the small
animals, as the extraction of a large portion of the organism's body
fiuid would be expected to negatively effect the organism's growth
and/or survival.

Although we used mussels, our technique is applicable to other
bivalve species. For example, Manuel et al. ( 1996) used a similar
technique on scallops {Placopecten magetlanicus): however, they
extracted approximately 5 to 10 times the volume of hemolymph
and did not test for potential growth or survival effects of their
sampling method. Other researchers have reported hemolymph
sampling in bivalves for various purposes, including DNA extrac-
tion (Marsh et al. 1995), hemocyte pathology and function (Moore
etal. 1991; Oliver* Fisher 1995), and ploidy analysis (Komaru et
al. 1988). However, no attempt was made to determine the effect
of that sampling on the viability of the animals. Our study also

ITS

Glu-5'

RLMS RLMS

1000 bp-

500 bp -
300 bp -

Kijiurf I. Aj-arosc gel clec-tri>ph<iri'sis n[ I'CK-ampiilu'd DNA lr:i(;nuiils usiny llic I IS and (ilii-S' ^|)^^.il•^-^(H•l■i(k prinur mIs. Ihc various lanes
are I't'U resiills iisinu 1)N.'\ from a regular exlruction iiK'thocI (Ul and DNA extracted from hemolymph taken from mussels in three size classes
(L, large: M, medium; S, small).

Survival and Growth ok Hkmol'i mph-Samples Mussels

993

Control

Hemolymph Extracted

400

Time (days)

Figure 2. Comparison of mean shell length (±1 SE) and survival for hemolymph-extracted and control mussels from three size classes over a
385-day period, post-treatment. The circles represent the large size class, the squares represent the medium size class, and the triangles represent
the small size class. The Tilled symbols nith the solid lines are the hemolymph-extracted mussels, while the open symbols with the dashed lines
are the control mussels.

showed the syringe-extracted hemolymph consistently provides
PCR-quahty DNA from large numbers of animals sampled under
field conditions. Hemolymph extraction clearly has considerable
potential for studies requiring the non-destructive sampling of
DNA from bivalves, and thus has applications for growth and
survival studies. This technique will also be u.seful for studies of
the ecology and population dynamics of bivalves where destruc-
tive sampling of the organism is either not permitted or not desir-
able for the experimental design.

ACKNOWLEDGMENTS

We would like to thank C. Bryden, R. Hepburn, and S. Henry
for their field assistance. This study was supported by a Natural
Science and Engineering Research Council of Canada grant (to
D.D.H.). Fieldwork was funded by Yellow Island Aquaeulture,
Ltd., the Natural Science and Engineering Research Council of
Canada, and the Science Council of British Columbia supplied
post-graduate funding to J.F.Y.

LITERATURE CITED

Brusca. R. C. & G. J. Brusca. IWO. Invertebrates. Sinauer Associates Inc..
Sunderland. MA.

David. P.. M.-A. Perdieu, A-F. Pemot & P. Jarne. 1997. Fine-grained
spatial and temporal population genetic structure in the marine bivalve
Spisulit ovalis. Evolution 51:1318-1322.

Hare. M. P., S. A. Karl & J. C. Avise. 1996. Anonymous nuclear DNA
markers in the American oyster and their implications for the hetero-
zygote deficiency problem in marine bivalves. Mol. Biol. Evol. 1.^:334-
345.

Heath. D. D.. P. D. Rawson &. T. J. Hilbish. 1995. PCR-based nuclear
markers identify alien blue mussel {Mytilus spp.) genotypes on the west
coast of Canada. Can. J. Fi.sli. Aqiial. Sci. 52:2621-2627.

Heath. D. D.. D. R. Hatcher & T. J. Hilbish. 1996. Ecological interaction
between sympatric Mytilus species on the west coast of Canada inves-
tigated using PCR markers. Mol. Ecot. 5:443^147.

Herbinger. C. M.. B. M. Vercaemer. B. Gjetvaj & R. K. O'Dor. 1998.
Absence of genetic differentiation among geographically close sea
scallop {Placofyeclen magelkinicus G.) beds with cDNA and microsat-
ellite markers. / Slwilfi.sli Re.s. 17:117-122.

Komaru, A., Y. Uchimura. H. leyama & K.T. Wada. 1988. Detection of
induced triploid scallop. Chtamys nobilis. by DNA microtlourimetry
with DAPI staining. Aquaeulture 69:201-209.

Manuel. J. L.. S. Burbridge. E. L. Kenchington. M. Ball & R. K. O'Dor.
1996. Veligers from two populations of scallop Ptacopcclen mai^el-
lunicus exhibit different vertical distributions in the same mesocosni. J.
SheUfish /ffv. 1 5:25 1 -257.

Marsh, A.G.. J.D. Gauthier & G.R. Vasta. 1995. A semiquantitative PCR

assay for assessing Perkinsus marinus infections in the eastern oyster,
Crassostrea virginica. J. Parisitotogy 81:577-583

Mitton. J. B. 1994. Molecular approaches to population biology. Annu.
Rev. Ecol. Sy.tt. 25:45-69.

Moore. J.D.. R.A. Elston, A.S. Drum & M.T. Wilkinson. 1991. Alternate
pathogenesis of systemic neoplasia in the bivalve mollusc Mytilus. J.
Invert. Pathol. 58:231-243.

Morse, M. P. & J. D. Zardus. 1997. Bivalvia. pp. 7-1 18. In: F.W. Harrison
(ed.). Microscopic Anatomy of Invertebrates, Vol. 6A. Mollusca II.
Wiley-Liss. New York.

Oliver. L.M. & W.S. Fisher. 1995. Comparative form and function of
oyster Crassostrea virginica hemocytes from Chesapeake Bay (Vir-
ginia) and Apalachicola Bay (Florida). Dis. Aquat. Org. 22:217-225.

Rawson, P. D.. K. L. Joyner. K. Meetze & T. J. Hilbish. 1996. Evidence for
intragenic recombination within a novel genetic marker that distin-
guishes mussels in the Mytilus edulis species complex. Heredity 11:
599-607.

Sarver, S. K. & D. W. Foltz. 1993. Genetic population structure of a
species' complex of blue mussels (Mytilus spp.). Mar. Biol. 117:105-
112.

Suchanek. T. H.. J. B. Geller. B. R. Kreiser & J. B. Minon. 1997. Zoo-
geographic distributions of the sibling species Mytilus galloprovincialis
and M. trossulus (Bivalvia: Mytilidae) and their hybrids in the North
Pacific. Biol. Bull. 193:187-194.

Taherlet. P.. L. P. Waits & G. Luikart. 1999. Noninvasive genetic sam-
pling: look before you leap. Tree 14:323-327.

Toro, J.E. 1998. Molecular identification of four species of mussels from
Southern Chile by PCR-based nuclear markers: the potential use in
studies involving planktonic surveys. J. Shellfish Res. 17:1203-1205.

JiHiriHil of Shellfish Research. Viil. 14, No. 2. W.'i-IOOl, :()(1().

EFFECT OF ARTIFICIAL DIETS ON GROWTH, LIPID UTILIZATION, AND GONAD
BIOCHEMISTRY IN THE ADULT SEA URCHIN PSAMMECHINUS MILIARIS

PANOS A. PANTAZIS,'* MAEVE S. KELLY,'
JOHN G. CONNOLLY,- AND KENNETH D. BLACK'

Scottish Association for Marine Science
Ohan. Argyll. PA34 4AD Scotland. UK
-Red Mills Ltd. Goresbridge. Kilkenny. Ireland
' Dunstajfnage Marine Lciboratorv
Ohan. Argyll. PA 34 4AD Scotland. UK

ABSTRACT Three artiticial extruded diets of high protein and varying Mpid content were manufactured from dried kelp fronds and
commercially available raw materials. The diets were fed to replicate groups of Psammechhws miliuris (Echinodermata- Echinoidea)
over a 14-wk period. Survivorship, weight gain, gonad index, and gonad biochemistry were monitored over the duration of the trial
The diets proved palatable to the urchins and were sufficiently .stable in seawater for the urchins to consume in tank-based trials All
of the artificial diets enhanced gonad growth as compared to a reference group fed Lammaria saccharine fronds. The satisfactory
performance of urchms fed a high-protein diet with a vegetable oil lipid .source, composed mainly of oleic and linoleic fatty acids
suggests that P. milums is able to utilize effectively diets low in polyunsaturated fatty acids (PUFAs). This has positive implications
for the manufacture of cost-effective urchin diet by excluding expensive raw materials (i.e., flshmeal, fish oil).

KEY WORDS: Sea urchin, Psammechiims iniliaris. gonad biochemistry, lipid utilization, artificial diets

INTRODUCTION

The need for a more systematic approach to .sea urchin culture
has been realized since the early 1980s, when the demand from the
French and Japanese markets created a collapse of both their local
fisheries and other supplying fisheries; for example, those of the
United States. Ireland, Spain, Chile, and the Philippines
(McLaughlin et al. 1996; Trinidad Roa 1989, Walker and Lesser
1996).

A concomitant increase in the price of the final product of up
to £1 1. kg"' (raw) in the French market or even up to £83.5.kg~'
(processed) in the Japanese market (Grosjean et al. 1998) has led
researchers worldwide to a consensus of specific priorities. Among
these is to ■• . . . determine the nutritional requirements needed to
optimize sea urchin gonad growth and whole animal growth. , . ."
(Parsons 1997).

Of the three most commonly found edible sea urchin species in
the British Isles, Paracentrotiis lividus (Lainarck), Echinus escii-
lentits Linnaeus, and Psammechimis iniliaris (Gmelin), the latter
has been identified as a potential aquaculture species (Kelly et
al. 1 998a). It has an advantage over the other species in terms of roe
quality and seems robust in culture (Cook et al.l998. Kelly et
al. 1998b). Gonad growth can be dramatically enhanced in this
species by additional feeding (Cook et al. 1998. Kelly et al.l998b):
however, further research is required to produce a refined urchin
diet that is cost effective and enhances roe quality. Such a diet
should enhance the marketability of the roe in terms of its texture,
color, and taste and, therefore, help create a uniforin product.

Natural populations of P. iniliaris are frequently found where
the macroalgae Laminaria saccharina (L) Lamour are abundant,
and they will graze freely on this species of algae. As a food source
in a inarine environment, fresh kelp fronds are extremely stable.
The pronounced seasonal variation in the biochemical composition

*Corresponding address: Institute of Aquaculture, Stirling University. FK9
4LA UK. E-mail: ppl@stir.ac.uk

of kelp (Black 1950). however, would create a serious drawback
tor using fresh fronds as a year-round food source for .sea urchins.
In addition, important palatability factors (substances on the algal
surface) are likely to be altered or destroyed during preservation
and storage (Renaud et al.l990). However, kelp contains large
amounts of such structural polysaccharides as alginates, galactans,
and galactans mixed with agar or various gums. All these sub-
stances act not only as binders but also as gelling agents, and it
would, therefore, be potentially advantageous to include dried kelp
with commercially available raw materials in the manufacture of a
stable, ready-to-use diet for such slow grazing species as P. ini-
liaris.

Artificial diets have been used to enhance gonad growth in
several echinoid species; however, the amount of biochemical in-
formation on the diets provided by the authors varies. Klinger et al.
(1986), Lawrence et al. (1989). Lawrence et al. (1992). Lawrence
et al. (1997), effectively used artificial diets (consisting of shrimp
meal, fishmeal. seaweed, wheat gluten, corn oil. fish oil. com
starch) to evaluate the effect of diet composition on physiological
indices (feeding rates, absorption-assimilation, gonadal growth,
somatic growth) in Lytechimis variegatus (Lamarck). Ui.xechimis
albus. and Paracentrotiis lividus. They provided information on
diet composition (percentage of raw materials used) but not on the
quality of these raw materials. Nagai and Kaneco (1975) provided
a more precise biochemical analysis of the artificial diet they used;
however. 37.9% of the proximate composition was not determined.
deJong-Westman et al. (1995) did not give the full proximate
analyses of the diets used but provided limited biochemical infor-
mation on the quality of raw materials. The.se authors also com-
ment that further research is required to refine their diets to the
optimum levels of nutrients needed.

Fernandez (1996) compared the performance of the sea urchin
P. lividus fed three diets made from commercial raw materials
(corn fiower. wheat, fishmeal, fish oil. sunflower oil) embedded in
12% gelatin solution. Although biochemical analyses for each diet
were given, the nutrient levels (energy, protein, lipid. Nitrogen

995

996

Pantazis et al.

Free Extracts [NFE], and oil source) varied in all three of the diets.
Therefore, it is difficult to identify which nutrients were respon-
sible for the enhanced growth rates.

Rapid gonad growth has been observed in P. miliaris sus-
pended in salmon cages, where salmon feed pellets contributed to
their diet, and in tank-based experiments where urchins were fed
exclusively on salmon pellets (Cook et al. 1998, Kelly et al.
1998b). However, the high cost of salmon feed dictates that it will
never be an economically viable urchin diet, unless the urchins are
obtaining it as a by-product of another system where it would
otherwise have been lost; for example, in polyculture with salmon.
Therefore, the need remains for a cost-effective alternative diet for
urchins produced in monoculture and as a preharvest diet for ur-
chins of variable roe quality.

The aim of this study was to design and manufacture a diet for
the sea urchin P. miliaris in which the lipid content was not based
on expensive animal origin oils and to evaluate the performance of
urchins fed these diets. Diets were designed with varying lipid
level, and a detailed biochemical profile of the diets and the gonads
of the urchins was then made with the aim of providing a better
understanding of the nutritional requirements of this species.

MATERIALS AND METHODS

Three experimental diets of equal protein but varied lipid con-
tent were formulated (Table 1) using prairie meal (gluten), yeast,
dried skimmed milk, corn, molasses, dried kelp fronds (Lamiiuiria
In'perborea. Fosl.). full fat soya, wheat, corn oil, and soya oil. The
diets were manufactured using extrusion technology to ensure bet-
ter adsorption efficiency of the incorporated lipids. The biochemi-
cal composition and energy content of the diets was confirmed by
the methods described below.

Four hundred P. miliaris were collected from local wild popu-
lations by SCUBA divers. Urchins (mean horizontal test (shell)
diameter 25.76 mm (SD 0.93) and mean initial weight 6.062 g (SD
0.523)1 were placed in "NorthWest" perforated plastic trays (0.5 x
0.5 X 0.1 m), which were then placed in rectangular seawater
aquaria (3 x 0.65 x 0.65 m) with a How rate 15 L h'. One of three
artificial diet treatments or a reference diet (L saccharina) was
assigned to each tray following the randomized block experimental
design (Woolf 1968). There were three (3) replicate groups of
urchins for each diet type. The average stocking density per tray
was 0.64 kg. M"- (SD 0.016, n = 21-23). Mean salinity recorded
over the experimental period was 34.4 %,> (SD 0.48) and the mean
oxygen concentration was 9.27 mg L"' (SD 0.29). Photoperiod
was kept constant at lOh: 14 h L: day.

The experiment was conducted from August to December

1997. The urchins were fed once daily, ensuring a pellet of food
was available for each individual. The reference treatment was fed
L. saccharina fronds ad libitum. Before each feed, any uncon-
sumed food on the tray and feces underneath each tray were col-
lected by siphoning and were dried until a constant dry weight was
achieved. Food consumption was estimated over the first 39 days
of the experiment by calculating the difference between the dry
weight of food consumed and the dry weight of uneaten food. This
was expressed as a percentage of dry food consumed in g live
weight"' day"', where live weight was the average between Day I
and Day 39 for each replicate treatment.

The urchins were sampled four times throughout the experi-
mental period. Before sampling, the urchins were starved for 48 h
and then blotted, and the weight and diameter of each was re-
corded. At Day 1 . Day 39, and Day 70. 10% of the urchins in each
treatment were dissected, and the wet weight of their gonads and
eviscerated test were recorded. At Day 97. all of the remaining
urchins were euthanized, and gonad weight and eviscerated test
weight were recorded.

The performance of the urchins was based on the following
parameters:

(1) survival rate: 100 x (number of individuals at the end of the
experimentA number of individuals at the beginning of the
experiment — total number of individuals euthani/ed)|; and

(2) gonad index (GI): 100 x [gonad wet weight/total wet
weight] (Lawrence et al. 1965).

At the end of the experimental period, the biochemical com-
position of the gonads of the urchins from the different artificial
diets treatments were compared by evaluating the levels of crude
protein, crude lipid, glycogen, ash. gross energy, and fatty acid
profile. On dissection, the color of the gonads was assessed im-
mediately by matching it to the closest representative color in the
Pantone® collection of color standards. The gonad color was al-
ways assessed by the same observer, in natural daylight. In addi-
tion, on dissection, a portion of gonad from a sample of urchins
was preserved in Bouins fluid and embedded in wax. The samples
were then sectioned and stained using hematoxylin and eosin for
microscopical confirmation of the reproductive stage (Byrne
1990).

Nested analysis of variance (ANOVA). following tests for nor-
mality and homogeneity of variance, was employed to examine the
significance of differences in the various indices recorded. Per-
centage data were arcsine transformed (Zar 1996). Where data did
not conform to the assumptions of ANOVA. the multiple range
Duncan test and the Student-Newman-Keuls test were employed
and tiave similar rcsuhs. Statistical evaluation of the results was

Crude prolcin

Crude lipid

Crude tibcr

Ash

NFH*

Energy in kj/g"

TABLE \.
Proximate analy.si$ of the formulated diets and Laminaria saccharina ( ''/€ on a dry matter basis).

Diet 1

Diet 2

Diet 3

.^h.%'(l.02)
5.10" (0.4)
2. 1 5-' (0.2)
7.93-' (0.5)

47.84'^^ (0.45)
9.67" (2.05)

.^7.66- (1.05)
6.83'M0.3)
2.01' (0.1)
7.02" (0.65)
46.44" (0.7)
11,37" (2.96)

36.05" (0.89)
9.18" (0.2)
2.46" (0.15)
7.95" (0.47)
44.34" (0.6)
1().9.V(2.72)

* Nitrogen-free extracts.

Slanilaiil ilcvia(i(in in paivndK'sis Oi

L. saccharina

8.69" (0.82)
2.57''(0..\5)
7.23" (0.85)
28. II" (1.05)
53.4'' (I. .^5)
12.97"(()..56)

3). Numbers in llic same rinv anil with the same superseripl are nol signilieandy dilTeienl (/' < 0.05).

Effect of Artimcial Diets on P. miliakis

997

performed using the statistical package SPSS for Windows (Re-
lease 6.1.3).

Analytical Methods

Diets

Before analyses, diet samples were ground with a mortar and
pestle until they would pass through a 1-mm sieve. Moisture con-
tent was determined by oven drying at 135 °C for 2 h. Analyses for
total nitrogen, crude fiber, crude lipid, ash. and gross energy were
performed on a dry matter basis and in triplicate. The total nitrogen
content of the raw materials was estimated using the Kjeldahl
method (Tecator-Kjeltec Auto Analyzer 1030). To convert the
determined nitrogen into crude protein, the nitrogen value was
multiplied by 6.2.'i. assuming that protein is composed of 16%
nitrogen. The percentage of crude fiber was determined (Tecator
Fibertec System M / 1020 Hot Extractor) as the difference between
the dried insoluble material (remaining after the acid and alkaline
hydrolysis) and its respective ashed inorganic fraction. The crude
lipid of diets was determined after extraction with petroleum spirit
(bp 40-60 °C). Ash was determined by incineration in a muffle
furnace at 600 °C for 2 h. and carbohydrates were estimated by
subtraction as nitrogen-free extracts (NFE). Energy was deter-
mined by chemical oxidation as originally described by O'Shea
and Maguire (1962) using potassium dichromate as the oxidizing
agent.

Urchin Tissues

Gonad analyses were done in triplicate from a pooled sample of
all the urchins within the same replicate. Total nitrogen, ash. and
gross energy were estimated as described above. The total lipid
content was determined by inserting a known quantity of external
standard (tricosanoic acid) during the preparation of fatty acid
methyl esters (FAMEs) and quantifying the total quantity of
FAMES based on the results of gas-liquid chromatography. Gly-
cogen was determined by the method of the anthrone reagent after
Good et al. (1933) as modified by Seifter et al. (1930) and Hassid
and Abraham (19571. The final quantity of determined glucose was
divided by the factor 1.11 to convert to glycogen (Morris 1948).
Test (shell) ash was determined as previously described for diets.

Fatty Acid Methyl Esters

FAMEs were prepared by the acid esterification method
(Christie 1982) after extraction by the method of Folch et al.
(1957). The resulting methyl esters were purified using 20 x 20 cm
thin layer chromatography (TLC) silica gel G plates with hexane:
diethyl ether: acetic acid (90:10:1 v/v) as developing solvent and
identified by comparison with known standards on a Carlo Erba
gas-liquid chromatograph equipped with a CP-WAX 58 CB (0.2
m) capillary column (25 x 0.25 mm). A thermal gradient of 4 °C/
min"' between 160-240 X was used. FAME identity was con-
firmed where possible by gas chromatography-mass spectrometry
(GCMS) (Thermo Quest / Finnigan. Trace 2000 series).

RESULTS

The artificial diets were palatable to the urchins, and after a
period of acclimatization to the new diet format, they fed freely.
Once in contact with a pellet, the urchins covered the pellet keep-
ing it in contact with their oral surface and grazed slowly from it.
Although stability tests were not performed (Caltagirone et al.
1991). the formulated pellets were stable enough that the urchins

Day 1 Day 39

CD Diet 1 CD Diel 2 1=1 Diet 3 I

Day 70

Day 97

I Reference -<^ Water temperature

Figure. 1. Live weight of sea urchins (g) fed artificial or reference diets
(error bars represent 95% confidence limits).

in trays could continue feeding on one pellet for 24-48 h. Feed
consumption (arcsine transformed values) was not statistically dif-
ferent (P = 0.44. F = 0.98. df = 3.10) among treatments. Ob-
served, nontransformed consumption rates were Diet 1 = 1.20% ±
0.77 SD. Diet 2 = 1 .74% ± 0.54 SD. Diet 3 = 1.36% ± 0.46 SD.
Reference group = 1.08% ±0.15 SD.

Survival rates were high and not statistically different (P <
0.05) among the groups fed the artificial diets: 93.5% ± 0.5 SD,
94.38% ± 0.58 SD, and 97.83% ± 0.6 SD for the sea urchins fed
Diets 1. 2, and 3. respectively, but were significantly higher than
that of the reference group (77.95% ± 0.54 SD). This level of
survivorship is within the range quoted by Cook et al. (1998) for
adult P. miliaris (21.3-21.7 mm test diameter) fed L siucharina
(80.3%) and salmon pellets (60.3%). respectively.

There was a statistically significant increase in total live weight
of all urchins fed the artificial diets overtime (Fig. 1 ). but no such
change was recorded for the live weight of the reference group.
The mean test diameter recorded at the beginning of the experi-
ment 25.58 ± 0.55 SD Ui = 67-92) was not statistically different
from the final mean diameter of urchins from all treatments 24.79
± 0.81 SD (/! = 33-75). All three artificial diets significantly and
rapidly enhanced gonad growth (Fig. 2). The Gl of all urchins on
experimental diets increased from an initial mean of 2.47 ± 1.38
SD to a final mean of 17.33 ± 5.72 SD. On day 70. urchins fed Diet
3 had a significantly higher GI than those fed Diet 1 (P = 0.03. F
= 4.27. df 2. 18). However, by the end of the experiment, there

Day 1 Day 39 Day 70

Sampling Days

I — I Diet1 CD Diet2 ^ Diet3 ^ Reference

Day 97

- Water temperature

Figure. 2. Gonad indices (GI) of sea urchins (error bars represent
95% confidence limits). Single bar (Day 1 ) represents mean GI of 10%
of the initial population.

998

Pantazis et al.

were no significant differences in GI of the urchins fed the artifi-
cial diets. The recorded increase in Gl was equivalent to a mean
increase of 1.52 g in gonad weight, and this accounted for most of
the recorded wet weight gain (Table 2). There was no statistically
significant increase in the 01 of the reference group over the ex-
perimental period.

Test ash increased from an initial value of 51.98% ± 5.03 SD
in = 8) on day 1 to a mean value of 86.7% ± 0.84 SD {» = 4) on
day 39, there were no significant differences between treatments or
between the treatments and reference group, although the reference
diet, L. saccharina. had a higher ash content. Test ash values (of all
the treatments) remained high until the end of the experiment
(88.75% ± 0.32 SD In = 10 of dried test).

The artificial diets also had a beneficial effect on gonad color.
On day 1 , the gonad color of the dissected urchins was gray or
brown (Pantone colors 139U, 146U, and 1535U) and classified as
unmarketable colors. Feeding a uniform substrate, such as the
artificial diets, seemed to limit the range of gonad colors produced.
Based on the Pantone collection of color standards, marketable
colors were classified as 155U, 156U. 157U, 714U, 1555U, peach
and pale yellow. At the end of the trial, percentages of marketable
colors were 67.4% for Diet 1. 65.12% for Diet 2, and 59.09% for
Diet 3, respectively.

Histological examination of the sectioned gonads at the end of
the experiment revealed the higher GIs observed in the urchins fed
the artificial diets was attributable to an increase in the nutritive
phagocytes, the storage cells in the gonad and not attributable to
the presence of developing gametes. The stage of gamete devel-
opment in the gonad of both the males and females appeared
typical of the postspawning recovery phase (Byrne 1990). The
urchins seemed to be following the typical pattern of the annual
reproductive cycle in P. mil'ums (Kelly et al. 1998b, Kelly 2000).

Gonad Biochemistry

The biochemical analyses (Table 3) of the gonads of the ur-
chins at the end of the experiment (Day 97) revealed that the
reference group had significantly higher proportion of total lipid in
the gonad than the urchins fed the artificial diets. However, the
total quantity of gonadal lipid accumulated in the reference group
was not higher than the rest of treatments as, at that time, the GI
(indicating relative gonad size) of the reference group (Fig. 2) was
significantly lower than that of the urchins fed artificial diets.

There is no obvious relationship between the amount of lipid in
each diet (as the mean of three determinations) and the lipid con-
tent of gonads from each treatment (Tables 1 and 3). In addition,
there was a surprising variation in the gonad lipid content between
replicate samples of urchins fed the same diet. For example. Diet

1 gave gonadal lipid contents ranging from 4 to 17% (on a live
weight basis). There is no satisfactory rationalization of this result.

There was no significant difference in gonad protein levels
among urchins fed artit~icial diets or between those fed artificial
diets and the reference group. Urchins also accumulated equal
levels of energy (per gonad unit weight) when fed the artificial
diets, but those fed diet 3 accumulated significantly more than the
reference group (Table 3).

Although differing in lipid content, all three artificial diets
shared similar lipid sources and, therefore, had similar fatty acid
profiles. In turn, this led to similar fatty acid profiles in the gonads.
Table 4 shows the mean level (%) and standard deviation for each
fatty acid for all the artificial diets (mean of duplicate determina-
tions from each diet) and for the gonads (mean of triplicate deter-
minations from each treatment).

The dietary lipids had a simple vegetable source dominated by
16:0 (palmitic). 18:0 (stearic), l8:!n-9 (oleic), 18:2n-6 (linoleic).
and 18:3n-3 (linolenic) acids. The gonads showed reduced
amounts of each of these but significantly increa.sed amounts of
their elongation-desaturation products 20:1*, (double bond posi-
tion not determined), 20:2* (possibly one or more non-methylene-
interrupted dienes (Cook et al. in press), 22:ln-9, 20:20n-6, 20:
3n-6 and 20:4n-6 (arachidonic).

DISCUSSION

All the manufactured diets were successful in prt)moting rapid
gonad growth and created a more uniform gonad in terms of color
and texture. The diets were palatable to the urchins, which adapted
well to feeding on the pellets. Because the artificial diets did not
differ greatly in composition, it is unsurprising their consumption
rates were the same. However, it was anticipated that their con-
sumption rate would have differed from that of the reference group
fed L. saccharina. which had a higher fiber content. Bedford and
Moore (1985) showed that adult P. miliaris reduced their gut re-
tention times to compensate for less digestible materials. In the
current experiment, the consumption rate data were collected from
replicate groups of urchins; ingestion and assimilation could be
re-examined for individual urchins fed artificial and reference di-
ets.

Various artificial diets were able to promote somatic growth in
other adult sea urchin species (Nestler and Harris 1994, Fernandez
and Bourdouresque 1998). Protein is a constituent of the organic
material (intrastereomic matrix) of the echinoderm body wall
(Dubois and Chen 1989), and its incorporation in the diet has been
proved beneficial for the somatic growth of other sea urchin spe-
cies (Fernandez 1997, McBride et al. 1998). Adult P. miliaris (test
diameter 15 mm) have also experienced increased somatic growth
after the adniinistralion of a commercial salmon feed and for a

TABLK 2.
(ionad live weight (g) of sea urchins fed the artificial diets and Laminaria saccharina (»er the experimental period.

Did 1
Diet 2
Diet 3

Reference

Day 1

(n = 8)

Day 39

(;i = 6-8)

Day 70

(H = 6-10)

Day 97

(n = 25-70)

0.26(0.1.54)
().26(().l.'i4)
0.2(1(0.1.^41
0.26(0.1.54)

0..^4"(0.12)
0.4.5" (0. II )
0..W(0.I2)
0.06" (0.026)

l.09-'(0.2y)
1.67" (0.24)
1.8.3" (0..57)
0.26' (0.13)

1.67" (0.71)
1.9.5" (0.6)
1.71" (0.56)
0.22" (0.09)

Slanilaiii ncvinliiin ill |i:ia-iilliosis. Nuiiihc-rs in iIk- same column ami uilh llic saiiK- MipL-iscri|il are iicit sijinil'icanlly dilTL-rcnl (/*< O.O.'il.

Effect of Artificial Diets on P. miliaris

999

TABLE 3.
Comparative gonad biochemistry of urchins fed experimental Diets 1-3, Day 97.

Diet 1

Diet 2

Diet 3

Reference

Dry matter

Crude protein

Crude lipid

Glycogen

Ash

Energy kJ/g"'

27.30-'

'(1.54)

42.46-'

(0.77)

25.20'-

(1.11)

27.56-'

(3.23)

4.02"

(0.08)

12.7r'-

"(1.43)

26.07"

(1.74)

42.40-'

(1.15)

15.05^

(5.84)

22.62"

(6.58)

5.01"

(1.85)

14.71^'

"(3.28)

26.736"

"(0.72)

41.51"

(1.98)

23.18"

(4.68)

26.53"

(13.'-)7)

5.16"

(0.65)

17.41 =

(7.29)

28.59" (1.22)
33.11" (0.32)
42.6.5" (1.56)
15.15" (3.42)
8.33" (1.55)
ll.99"(2..'!5)

Values are expressed as percentage of the sample on a dry matter basis. Standard deviation in parenthesis (» = 3). Numbers in the same row and with
the same superscript are not significantly different (f < 0.05). Statistics generated with arcsine transformed data.

peinod of 6 months (Cook et al. 1998). The artificial diets in this
experiment did not promote measurable somatic growth; however,
the trial was of relatively short duration for recording lest growth.
Calcium carbonate represents, roughly, 487^ of the total shell vol-
ume of echinodeims (Weber 1969, Kaneko et al. 1982) and even-
tually accounts for a considerable portion of the inorganic carbon
of the shell. Although the artificial diets used in this experiment
had much lower ash levels (7-8'7r approximately. Table I ) than L.
saccharina (28% of its dry weight; Black 1950), the urchins fed
artificial diets maintained test ash values as high as those of the
reference group, indicating that a high ash content in an artificial
diet may not be essential for maintaining inorganic test content.

TABLE 4.
Fatty acid profiles of gonads and diets

Gonads

Diets

Mean

SD

Mean

SD

14:0

3.3

0.9

0.3

0.3

15:0

0.2

0.2

0.0

0.0

16:0

11.3

1.3

11.3

0.7

16:ln-7

1.7

0.6

2.0

0.5

16:1*

0.4

0.6

0.0

0.0

18:0

2.5

0.4

4.0

0.5

18:ln9

5.6

0.9

22.4

0.8

18:ln-7

1.3

0.4

1.0

0.2

l8:2n-6

24.2

2.7

52.0

2.3

18:3n-3

0.0

0.0

5.7

0.5

18:4n-3

0.0

0.0

0.0

0.1

20:1*

5.3

0.5

0.4

0.5

20:0

2.6

0.4

0.2

0.0

20:2*

8.2

0.6

0.0

0.0

20:2n-6

7.2

1.5

0.1

0.1

20:3n-6

2.9

0.6

0.0

0.0

20:4n-6

12.2

1.5

0.0

0.0

20:3n-3

0.2

0.3

0.0

0.0

20:4n-3

0.1

0.1

0.0

0.0

20:5n-3

1.7

0.7

0.4

0.2

22:ln-9

3.3

0.4

0.0

0.0

22:5n-3

0.0

0.0

0.0

0.0

22:6n-3

0.4

0.4

0.0

0.0

U/K

5.4

2.5

0.2

0.5

Totals

100.0

100.0

Methyl esters as % of total methyl esters on a wet basis

U/K = unknown peaks.

* = Double bond position not determined.

The higher mortality recorded for the reference group could
possibly be attributed to the seasonal variation of the biochemical
coniposition of the kelp (Black 1950) resulting in a deficient diet
incapable of meeting the nutritional requirements of the urchin at
this life stage. Similarly, Fong and Mann (1980) suggested that the
amino acid profile of the L. lonfiicnms was of low nutritional
value ioT Stioniiyloceiilidtiis dwehachieusis (nutritional value as-
sessed as percentage of each essential amino acid found in L.
longicniris compared to the respective one found in egg albumin
(Cowey and Sargent 1972).

The rapid gonad growth seen in the urchins fed the artificial
diets is a positive attribute for a potential aquaculture species. The
increa.se in 01 from a mean of 2 to 17% over approximately 14
weeks compares favorably with the increase in GI found in urchins
maintained in polyculture with Atlantic salmon, and accessing
salmon feed, over a 16-wk period |G1 increased from 2.56%- ( 1.38
SD, n = 20) to 18.95 (2.39 SD, n = 20); data converted from that
of Kelly et al. 1998b]. It is not known, however, if the GI would
have continued to increase over time, as found for P. inUiaris fed
exclusively on salmon feed (Cook et al.l998). Because the manu-
factured sea urchin diets were composed of 36-37% crude protein
and 5-9% crude lipid, they seem to be equally or even more cost
effective, in ternis of promoting gonad growth, to the salmon feed
containing 40-45% crude protein and 20-30% crude lipid.

Although very different to natural dietary lipid sources, the
mainly vegetable oil lipids in the diets seemed to allow good gonad
growth in this species. There was no relationship between dietary
lipid content and gonadal lipid storage, the level of which, there-
fore, must have been controlled by some other dietary or rnetabolic
factor. The dietary lipid was provided by vegetable-derived oils
rich in 18:1 and 18:2, comprising about 75% of the total dietary
lipid. Using such starting materials with a low level of polyun-
saluration. the urchins were capable of accumulating a range of 20
carbon fatty acids (20:2*, 20:2n-6, 20:3n-6 and 20:4-6) as well as
some 22:ln-9, which must, therefore, be regarded as nonessential
fatty acids for this species. The urchins also had small amounts
(1.7%) of 20:5n-3 in their gonads, but this may have been accu-
mulated directly from the diet, which also contained small amounts
(0.4%). In addition, small amounts (0.4%) of 22:6n-3 were also
present in the gonads despite its absence in the diets. This may
have originated from dietary sources before the start of the experi-
ment, because this species lacks the enzyme systems for desatu-
ration/elongation necessary for synthesis of this fatty acid (Bell et
al. in press). Because gonad growth was dramatic over the course
of the experiment, the lipid profile of the gonads presumably
largely results from the diets they were fed during the trial. Cook

1000

Pantazis et al.

et al. (in press) found that urchins held in aquaria and fed primarily
on salmon feed had high levels of 22;6n-3. probably accumulated
directly from the high levels in their diet. Further experimentation
using labeled fatty acids will better clarify the biosynthetic ability
of P. miliaris at this high level of polyunsaturation.

High levels of good quality fishmeal and fish oil rich in PUFAs
constitute the basis for salmon diets, characterized by approxi-
mately 40-45'7f crude protein. 20-30% crude lipid and a high
manufacturing cost. The satisfactory gonad growth of sea urchins
fed diets composed of inexpensive vegetable oils, cereal grains,
and agricultural by-products, advocates for the use of such diets in
sea urchin culture. More experimental work to fully meet the sea
urchins' nutritional requirements using cost-effective, low PUFA
diets is needed.

Although the gonad color produced by the artificial diets was

not the color the marketplace prefers (often described as pumpkin),
the colors were bright and clear tones, and, therefore, not unat-
tractive (except for those of the reference that were dark and
brown). The noted improvement of the roe color in most of the
experimental urchins calls for further research into the incorpora-
tion and expression of dietary pigments.

ACKNOWLEDGMENTS

This research was funded by Highlands & Islands Enterprise
and the Highland Council. We thank the Director of SAMS for the
use of the facilities. We also thank Dr. Nick Lake and Dr. Debbie
Cashmore of Seafish. Ardtoe; Atlantic Resources Development
Ltd. for providing raw materials and Dr. Michael Bell, Institute of
Aquaculture Stirling University, for his valuable comments and
help in fatty acid analyses and identification.

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Jcmrmil of Shellfish Research. Vol. 19. No. 2. 1()0.V10()6. :()()().

SPATIO-TEMPORAL DISTRIBUTION OF PROROCENTRDM LIMA IN COASTAL WATERS OF

THE GULF OF MAINE: A TWO-YEAR SURVEY

LUCIE MARANDA.' MAUREEN D. KELLER,^

JOHN W. HURST, JR./ LAURIE L. BEAN/ JAY D. MCGOWAN/

AND PAUL E. HARGRAVES'

^Graduate School of Oceanography.

University of Rhode Island. Narragansett. Rhode Island 02882
'Bigelow Laboratory for Ocean Sciences.

West Boothbay Harbor. Maine 04575

Maine Department of Marine Resources,

West Boothbay Harbor, Maine 04575
^ Maine Department of Marine Resources.

Lamoine. Maine 04605

ABSTRACT The dinoflagellate Proroeentnim lima (Ehrenberg) Dodge was found at several sites along the coast of Maine in 1998
and 1999, some in areas where shellfish are harvested coininercially. Identity was confinned by scanning electron microscopy (SEM).
Although this dinoflagellate is known to produce toxins (okadaic acid and derivative compounds), incidence of diarrhetic shellfish
poisoning in coastal Maine is not well understood, despite confirmed toxicity events in the early 1990s in Nova Scotia. Canada in
adjoining waters to the north. Some samples containing the dinoflagellate came from wild mussel populations collected at low tide,
while others originated from aquaculture sites. Many of the cells were isolated from water samples and net tows and on a few occasions
were associated with filamentous macroalgae. Proroeentnim lima appears to be relatively rare in Maine coastal waters, but its
widespread distribution over several months warrants increased monitoring to allay public health concerns.

KEY WORDS: Proroeentnim lima, diarrhetic shellfish poisoning. DSP. Gulf of Maine

INTRODUCTION

Diarrhetic shellfish poisoning (DSP) is a severe gastrointestinal
illness in humans resulting from the ingestion of shellfish contain-
ing DSP toxins (okadaic acid and derivatives). These toxins accu-
mulate in shellfish that have been feeding on dinofiagellates of the
genus Prorocentriim Ehrenberg or Diiiophysis Ehrenberg. In
coastal northeast USA, knowledge of DSP incidence is fragmen-
tary and until recently, no DSP monitoring has been judged nec-
essary. However, three events of the past 10 years challenged this
complacent perspective: ( 1 ) in the late 1980s, several shipments of
the oyster Ostrea ediilis Linnaeus, originating from Maine and sent
oversea, tested positive for DSP and were refused at a great eco-
nomic loss to shellfish farmers (Shuniway 1990). The source of
DSP toxins was not determined and remains controversial. (2) In
1990, the first confirmed DSP event in North America involved
cultured mussels on the Atlantic coast of Nova Scotia, Canada, in
waters contiguous with the Gulf of Maine (Quilliam et al. 1993).
The DSP toxicity was linked to populations of P. lima (Jackson et
al. 1993, Lawrence et al. 1998). (3) Proroceiitriun lima, a known
toxin producer, was first observed in the Gulf of Maine in 1994 in
an offshore plankton net sample collected in the Great South Chan-
nel, west of Georges Bank (Maranda et al. 1999). but, until then,
had not been reported from coastal locations.

These events prompted us to investigate the extent, in space,
time and abundance, of the presence of P. lima in the coastal
waters of the Gulf of Maine. Recently. Morton et al. (1999) de-
tected low levels of okadaic acid-like activity in blue mussels
Mytilus edulis Linnaeus and reported on the presence of P. lima in
one area along the coast of Maine.

MATERIAL AND METHODS

Wild blue mussels M. edidis and associated sediments were
collected at low tide once a month in March. April, May, and

October, and twice a month in June, July, and August, at ten
monitoring stations along the coast of Maine in 1998 and 1999
(Fig. 1 ). This sampling strategy was driven by the economic im-
pact P. lima can potentially have on the shellfish industry. Epibiota
and associated sediments were rinsed off mussels with 0.45-(jLm
filtered seawater and the 10- to 90-|xm size fraction was examined
in a Sedgwick-Rafter chamber by bright field microscopy using a
lOX or 20X objective. Cells resembling P. liimi were isolated with
a stretched pipet for observation at higher magnification or for
preparation for scanning electron microscopy (SEM). Whenever
possible, samples were observed fresh, otherwise they were pre-

figure 1. Coast of Maine. Location of ten stations (•) sampled for
epibionts on mussels, three subtidal (O) and three inlertidal (A) sta-
tions sampled for epibionts on macroalgae, two commercial shellfish
sites (■) and Clen Cove, also known locally as Clam Cove, a mid-coast
station (A).

1003

1004

Maranda et al.

served in formaldehyde:acetic acid (1% final concentration) and
examined within 6 mo of collection. Sample size (mussel number
and size) varied with stations: total weight of material ranged
between 0.2 and 1 .7 kg. with an average of 45% in sediments and
epibionts.

Whole water samples and/or plankton samples (>10 ixm) were
collected at additional stations on an irregular basis: a shellfish
nursery site (fall 1998 and summer 1999). a shellfish depuration
site (summer 1999), and Glen Cove, a shallow mid-coast site with
abundant wild mussels (summer and fall 1999) (Fig. 1). For 3 mo
during the summer of 1998, whole specimens of filamentous mac-
roalgae and associated epibiota were collected underwater twice a
month at three subtidal stations by a scuba diver (Fig. 1); phae-
phophytes and rhodophytes were harvested down to 10-m depth. In
the fall of 1999, macroalgae and associated epibiota were collected
at three intertidal sites in the northeast section of coastal Maine
(Fig. 1 ). The epibiota was later shaken off seaweed specimens; the
samples were settled and concentrated 20 to 50 times prior to
observation in a Sedgwick-Rafter chamber.

Criteria used for the identification of P. lima were those defined
by Faust (1991).

RESULTS

1998

From the epibionts collected on mussels (99 samples), cells of
P. lima were detected in a tide pool at Head Beach. 43°43'N
69°5rW, in October (Fig. 2 and Fig. 3 A and B). During that same
month, one of us (M. D. K.) collected P. lima at a shellfish nursery
site, 43°55'N 69°52'W (filled square #1, Fig. 2) and established a
mixed culture. P. lima was reisolated in clonal cultures several
months later (Fig. 4) and deposited at the Provasoli-Guillard Cen-
ter for Culture of Marine Phytoplankton (CCMP1966). Size and
pore pattern of the cells falls within published values for the spe-
cies (Table I ).

The epibiota of filamentous macroalgae from subtidal locations
did not harbor any P. lima cells.

1999

In August, September and October, cells of P. lima were iden-
tified by SEM in whole water and plankton samples, and with

4

'^^'^ijs

Figure 2. i.ocalion of stations where P. lima was found in 1998 and in
1999. Symhols as in Klgure 1, except for the sites (crosses) reported by
Morton el ul. (1999 and personal communication).

Figure 3. Prnrocenlruin lima Ironi a tide pool near Head Beach, ME,
October 1998. (A) Bright Held photomicrograph of one cell within
detritus as seen in a Sedgwick-Rafter counting chamber. The arrow
points to the central pyrenoid. Note the depression in the apical area
on the right thecal plate. (I? I SEM micrograph of the left thecal plate
of a second cell isolated from Head Beach. Note the row of marginal
pores (arrowhead) and the pattern of the valve pores (arrow I.

filamentous macroalgae troni Glen Co\e. 44"'08'N 69"()6'W and.
in August and September, were found at the same shellfish nursery
site sampled in 1998 (Fig. 2).

Cells of P. lima were observed by light microscopy in 5 of 105
samples of mussel epibionts analysed. They came from five dif-
ferent stations spanning the whole coast of Maine and were col-
lected between June and September. In September, cells were
found in water samples from a shellfish depuration site (filled
square #2) and within the epibiota of two niacroalgal collections
(Fig. 2|. Shape and si/c ol cells fall within the reported range for
P. lima.

Prorocf.ntrvm lima in Coastal Gulf of Maine

1005

Figure 4. Prorocentrum lima from cultured material originating from
the shellfisli nursery site #1. Collapsed right thecal plate, treated with
chloral hydrate. Oil immersion phase contrast.

DISCUSSION

Prorocentriiin lima is present in coastal waters of the Gulf of
Maine. The SEM-based identification at three sites is unequivocal
and included si/e. shape and micro-morphological characters of
cells associated with mussels and in water samples. The pattern
and number of marginal and valve pores, along with the apical
depression on the right theca. constitute the most compelling evi-
dence of the identity of the cells. Priirocenuiiin lima was also
detected at several other sites along the coast of Maine, although
micro-morphological characters could not be resolved in all cases
by light microscopy. We recommend that cells be identified with
attention to micromorphology. especially at low magnification, as
non-toxic Prorocenintm species such as cassiihiciim (Woloszyn-
ska) Dodge (Fig. 5) can easily be mistaken for lima especially in
samples loaded with sediments and detritus.

It is surprising that, despite many decades of phytoplankton
studies in the Gulf of Maine and surrounding coastal waters, no
reports off. lima can be found prior to 1994 (Maranda et al. 1999).
Similarly, P. lima was not recorded from eastern Canadian waters
prior to the 1990 DSP incident. Whether this predominantly
benthic/epiphytic toxin producer was a previously "hidden" or not
appropriately sampled native member of local populations or
whether a successful genotype has been recently introduced in the
northwest Atlantic cannot be resolved at this time. Nevertheless,

Figure 5. Prorocentrum cassubicum from Mosquito Harbor (44°23'N
68-04'W), October 1997. SEM micrographs reveal a high number of
marginal and valve pores (ca. 120 and 218, respectively). The size (51
X 30 pm, LAV' ratio: 1.70) and shape of the cell come close to that of P.
lima.

the presence of P. lima signals the potential for DSP incidence
given that, to date, all cultured clones identified as P. lima, re-
gardless of their origin, produce OA and/or some derivatives
(McLachlan et al. 1997, Morton et al. 1999).

In Canadian waters, P. lima grows in association with filamen-
tous macroalgae fouling mussels suspended in long-line cultures
(Lawrence et al. 1998). Shellfish may thus become toxic after
feeding on the epiphytic P. lima when it becomes available as a
food item following turbulence in the water column. In Maine,
mussel growers use predominantly on-bottom cultivation methods,
less susceptible to fouling macroalgae, while wild mussels still
constitute an important portion of annual landings. However re-
newed interest in and conversion to suspension methods are gain-
ing momentum; this could lead to increased exposure of mussels to
fouling macroalgae and associated flora, including the toxic P.
lima. This specific association between P. lima and filamentous
macroalgae is documented in tropical/subtropical habitats as well
(Tindall and Morton 1998) and implies that our spatio-temporal
survey of mussel epibionts may underestimate the dinoflagellate
population. Appropriate quantitative sampling of plankton and epi-
phytic populations would thus be needed to undersatnd the ecology
of P. lima in Maine coastal waters.

Although our survey suggests that P. lima is relatively rare at
most stations, the widespread distribution of the dinoflagellate, its
recurrence 2 y in a row and over several months, and presence
close to mussels and in the plankton warrant increased monitoring
to address public health concerns, especially in light of coming
shifts in cultivation methods.

TABLE 1.
Comparison of Prorocentrum lima from the coast of Maine with some published micro-morphological features (length and width in pm).

Reference/Sou rce

Length

Width

LAV Ratio

Marginal Pores

Valve Pores

Dodge 19X2

32-5U

2()-2S

—

—

—

Faust 1991

31-47

22^0

1.18-1.52

55-72

58-86

Marret al. 1992

46

32

1.44

ca. 55

ca. 70

Morton and Tindall 1995

38^1

32-36

1.18-1.34

—

—

Head Beach (two cells)

43,48

28,35

1.39, 1.54

73. 79

76.91

Glen Cove

46-50

32-33

1.44-1.51

56-60

ca. 60

Shelitlsh nursery site

41 -.50

26-36

1.49

56-60

ca. 70

1006

Maranda et al.

ACKNOWLEDGMENTS

This work was funded by a grant from the National Oceanic
and Atmospheric Administration (Sea Grant #NA66RG0303) to
LM and PEH. M. D. Keller joined this project in the summer of
1999. Her untimely passing a few months later sadly interrupted
this collaboration. This paper is dedicated to her memory. We

thank numerous mussel collectors from the Maine Department
of Marine Resources, J. E. Prentice of the University of Rhode
Island for processing mussels in 1998 and S. Zimsen of the
University of Maine for collection by scuba diving and sea-
weed identification. We are grateful to M. A. Faust, Smithsonian
Institution, for her identification of P. cassithiciim from SEM
micrographs.

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diarrhetic shellfish toxins by an isolate of Prorocentrum lima from
Nova Scotia. Canada, pp. 513-518. In: T. J. Smayda & Y. Shimizu
(eds.). Toxic Phytoplankton Bloom in the Sea. Elsevier Science Pub-
lishers, New York.

Lawrence, J. E., A. G. Bauder, M. A. Quilliam & A. D. Cembella. 1998.
Prorocentrum lima: a putative link to diarrhetic shellfish poisoning in
Nova Scotia, Canada, pp. 78-79. In: B. Reguera, J. Blanco, Ma. L.
Fernandez & T. Wyatt (eds.). Harmful Algae. Xunta de Galicia and
IOC of UNESCO, Vigo, Spain.

Maranda, L., C. Martin & C. Chan. 1999. Prorocentrum lima (Dino-
phyceae) in waters of the Great South Channel near Georges Bank. /
Phycol. 35; 1 158-1 161.

Marr, J. C, A. E. Jackson & J. L. McLachlan. 1992. Occurrence of Pro-
rocentrum lima, a DSP toxin-producing species from the Atlantic coast
of Canada. / Appl. Phycol. 4: 17-24.

McLachlan, J. L., G. T. Boalch & R. Jahn. 1997. Reinstatement of the

genus Exuviaella (Dinophyceae, Prorocentrophycidae) and an assess-
ment of Prorocentrum lima. Phycologia 36: 38^6.

Morton, S. & D. R. Tindall. 1995. Morphological and biochemical vari-
ability of the toxic dinotlagellale Prorocentrum lima isolated from
three locations at Heron Island. Australia. J. Phycol. 31: 914-921.

Morton, S. L., T. A. Leighfield, B. L. Haynes, D. L. Pelitpain, A. A.
Busman. P. D. R. Moeller, L. Bean, J. McGowan, J. W. Hurst Jr. & F.
M. Van Dolah. 1999. Evidence of diarrhetic shellfish poisoning along
the coast of Maine. J. .Shellfish Res. 18: 681-686.

Quilliam, M. A., M. W. Gilgan, S. Pleasance, A. S. W. Defreitas, D.
Douglas, L. Fritz, T. Hu, J. C. Marr, C. Smyth & J. L. C. Wright. 1993.
Confirmation of an incident of diarrhetic shellfish poisoning in eastern
Canada, pp. 547-552 In: T J. Smayda & Y. Shimizu (eds.l. Toxic Phy-
toplankton Bloom in the Sea Elsevier Science Publishers, New York.

Shuniway. S. E. 1990. A review of the effects of algal blooms on shellfish
and aquaculture. J. World Aquacult. Soc. 21: 65-104.

Tindall, D. R & S. L. Morion. 1998. Community dynamics and physiology
of epiphytic/benthic dinoflagellates associated with ciguatera, pp. 293-
313. In: D. M. Anderson, A. D. Cembella & G. M. Hallegraeff (eds.).
Physiological Ecology of Harmful Algal Blooms. Springer-Verlag,
New York.

Jitnrmil of Slwllfish Rfsccinh. Vol. 14. No. 2, 1()07-I01.'i, 2001).

ENVIRONMENTAL CONDITIONS ASSOCIATED WITH DOMOIC ACID IN RAZOR CLAMS ON

THE WASHINGTON COAST

NICOLAUS G. ADAMS,*' MITCH LESOING," AND
VERA L. TRAINER'

'National Marine Fisheries Sen ice

Northwest Fisheries Science Center

Environmental Conservation Division

2725 Montlake Blvd. E.

Seattle. Washington 98112
'Qiiileute Natural Resources

Quileute Indian Tribe

234 Front St.

La Push. Washington 98350

ABSTRACT In October 1998, record levels of the neurotoxin domoic acid (DA) were detected in razor clams {Siliiiua putiila. Dixon)
resulting in the closure of shellfish harvesting areas along the Washington coast. This toxin was detected in seawater samples collected
at Kalaloch Beach and Second Beach on the central Washington coast using a receptor binding assay and liquid chromatography-
tandem mass spectroscopy. Domoic acid levels ranging from 0-2700 ng/L were measured in seawater samples containing from
70-100% PseuJo-iiitzscliici psemhdelkatissimu (Hasle) Hasle at concentrations of 1.0-15 x 10'' cells/L. resulting in maximum levels
of cellular toxin of approximately 500 fg/cell. A cultured isolate of this species collected from Kalaloch Beach also produced DA, as
determined by the receptor binding assay, durmg late exponential and stationary stages of growth. The toxic P. pseudodelicatissima
bloom in the late summer and autumn of 1998 occurred 2-3 weeks after strong coastal upwelling during a period of anomalously low
rainfall, typical in post-El Nino years. Higher toxin levels m seawater at Kalaloch Beach compared to Second Beach were attributed
to the periodic nature of upwelling at Kalaloch Beach, demonstrated by a 175-fold increase in nitrate in seawater coincident with a 5
°C decrease in sea surface temperature on September 1. The upwelling event in September was followed by wind relaxation and
reversal at the end of that month, resulting in the transport of toxic cells toward the coast where nutrients were already present to fuel
the algal bloom. A pulse of nutrients, either from rainfall or upwelling, to coastal regions that have experienced several weeks of low
nutrients, followed by wind relaxation or reversal events that transport cells to inshore regions, are suggested to be important factors
in the initiation of the most toxic Pseiido-nitzscliia species blooms on the Washington coast.

KEY WORDS: Pseudo-iut:..schia. domoic acid, razor clams, upwelling

INTRODUCTION

The first domoic acid (DA) poisoning of humans was reported
in 1987 in eastern Canada (Todd 1993). A series of collaborative
studies demonstrated that the toxin was concentrated in mussels
that had fed on a bloom of Pseiido-nitzschia imilliseries, the first
pennate diatom from which DA was isolated (Bates at al. 1989). In
1991, DA was implicated in the illness and death of brown peli-
cans (Pelecamts occidentalis) and Brandt's cormorants (Phalac-
rocorax penicillatus) in Monterey Bay, California (Work et al.
1993). Using laboratory isolates. Garrison et al. (1992) determined
that P. australis was the DA-producing diatom responsible for the
mortalities. Unlike the outbreak in eastern Canada, where mussels
were the toxin vector (Wright et al. 1989). anchovies became toxic
to seabirds after feeding on the diatom, P. australis. in Monterey
Bay (Buck et al. 1992. Fritz et al. 1992), illustrating that DA has
at least two means of entering the higher food web, through filter-
feeding moUuscan shellfish and suspension-feeding finfish.

The poisoning event in Monterey Bay resulted in the establish-
ment of a DA monitoring program in the states of Washington and
Oregon. In October 1991, about 1 month following the toxic
bloom event in California, levels of DA above the regulatory limit
of 20 fjLg/g shellfish tissue were found in the edible parts of razor
clams (Siliqua patula) and Dungeness crabs {Cancer magister)

♦Corresponding author. E-mail: nicolaus.adams@noaa.gov

collected on the Washington coast (Wekell et al. 1994). Conse-
quently, beaches were temporarily closed to recreational and com-
mercial shellfish harvesting (Horner and Postel 1993). resulting in
a substantial loss of revenue ($15-20 million) to local fishing
communities (Anderson 1995). The P.seudo-nitzschia species re-
sponsible for the DA poisoning event was not determined, because
there were no phytoplankton samples collected on the Washington
coast immediately before the measurement of toxin in razor clams
(Homer et al. 1993). Several species that had been determined to
be toxic in some, but not all, geographical regions (i.e., P. multi-
series, P. australis. P. pungens. and P. pseudodelicatissima) were
found in Washington coastal waters (Horner et al. 1997). Among
those species. P. australis was thought to be responsible for the
presence of DA. because it was identified in samples collected off
Grays Harbor. Washington, several months after the initial event
(Horner and Postel 1993). Taylor and Homer (1994) suggested that
the 1991 toxic bloom event off the Washington coast might have
been part of a widespread bloom of P. australis. It was estimated
that a bloom starting in California in September 1991 and carried
up the coast by currents at speeds of nearly 20—40 cm/sec could
have reached the Washington coast by late October to November
(Horner et al. 1997).

More recently, in May and June of 1998. the first confirmed
deaths of a marine mammal species attributable to DA poisoning
were documented in sea lions along the central California coast. In
this mortality event, sea lions fed on anchovies and sardines that

1007

1008

Adams et al.

had ingested toxic P. aiistralis cells (Gulland et al. 1999. Lefebvre
et al. 1999, Scholin et al. 2000). Because razor clam toxification on
the Washington and Oregon coasts in 1991 was preceded by the
death of seabirds in Monterey Bay earlier that summer, we sus-
pected that the poisoning of sea lions by DA in 1998 was an early
warning of impending toxicity on the Washington and Oregon
coasts.

Because concentrations of Pseiido-nitzschia spp. increase and
subside rapidly in Washington coastal waters (R. Horner, pers.
comm.), a more rigorous and frequent monitoring effort was
implemented by sampling at two beaches in order to investigate
the relationships among Pseudo-nitzschia spp. cells, DA levels in
seawater and shellfish, nutrients, and a variety of environmental
variables. This paper describes weekly sampling of the surf zone,
which enables us to describe the chemical, biological, and physical
processes preceding and during the razor clam toxification event
that occurred in the late summer and early autumn of 1998.

METHODS

Sample Collection

Seawater was sampled on a weekly basis from the surf zone of
two accessible beaches on the Pacific coast of Washington State:
Second Beach, near La Push, and Kalaloch Beach, approximately
65 km south of La Push (Fig. 1). Seawater samples were collected
using a bucket and preserved for phytoplankton species identifi-
cation and enumeration, as well as DA, chlorophyll a, and nutrient
analyses as described below.

Nutrient and Chlorophyll a Analyses

Seawater was filtered through a 25-iiim Whatman #1 filter and
collected in a 60-mL polyethylene bottle, frozen, and later ana-
lyzed for nutrient concentrations using standard autoanalyzer
methods (Whitledge et al. 1981). Aliquots (50 mL) of seawater
samples were filtered through Whatman GF/F filters for chloro-
phyll a detection. Filters were stored at -20 °C until analysis by
extraction with 10 mL 90% acetone overnight at 4 °C in the dark.
Extracts were analyzed using the standard fiuorometric method
(Welschmeyer 1994) using a Turner Designs (TD-700) fluorom-
eter with narrow bandpass filters.

DA Analysis in Phytoplankton

Cellular DA was measured by filtering 2 liters of seawater
through a 47-mm 0.45-p.m (Millipore HA) filter. Depending on the
density of the material in the sample, up to three filters were used.
Filters were folded in half with forceps, wrapped in aluminum foil,
and frozen until analysis using the method described by Van Dolah
et al. { 1997). A glutamate decarboxylase digestion step was used
before analysis to remove endogenous glutamate in all samples.
Binding experiments were initiated by incubation of 100 jjlL of
cloned GluR6 membrane preparation (Taverna and Hampson
1994) with 50 |jiL of a 5 nM solution of ['H] kainic acid and 50 jjlL
of standard or sample in a 13 x 10 mm glass test tube. Samples
were vortexed briefly, incubated at 4 °C for 1 h, poured over
Whatman GF/C filters (25-mm diameter), and rinsed twice with 5
mL of 50 mM Tris-citrate buffer (pH 7.4). Filters were placed in
scintillation vials, soaked overnight in 10-mL .scintillation fluid.
and radioactivity was measured using scintillation spectroscopy.
For quantification of DA in selected seawater samples, liquid chro-
matography-tandem mass spectroscopy (LC-MS/MS) was per-
formed according to standard protocols (Scholin et al. 2000).

Cell Counts

Phytoplankton cells were counted by first pouring 100 mL of
seawater into a graduated cylinder, then fixing with formaldehyde
to a final concentration of approximately I 9t and settling for
24-72 hours. Ninety mL of water were carefully drawn off with a
pipette and the settled material was resuspended. A 0.1 mL sub-
sample was loaded into a Palmer-Maloney slide, and a minimum
of 100 individual algal cells were counted at lOOX magnification
using light microscopy. Phytoplankton cells were identified to the
lowest possible taxon and to species when possible. The percent-
age contribution of the Pseudo-nitzschia species for the whole
phytoplankton assemblage, viewed at lOOX, was calculated.

Scanning Electron Microscopy

In samples where Pseudo-nitzschia spp. were numerous, ali-
quots were prepared using a modified KMnOj/HCI oxidation
method (Miller and Scholin 1998). Filter membranes with pro-
cessed samples were bonded to aluminum stubs, air-dried, coated
with gold-palladium, and examined with an AMRAY 1000 SEM.

47 N

Second Bcacb

Seattle

Kalaloch B«ach •

Destruction Island
Quilluyuttr Airport
Cape Elizabeth Buoy

Washington

•^S-"^!^ Columbia River

Hgure. I, .Saniplinj; silts (>n the central Washinjjion coast and loca-
tions of coa.slal environincntai monitoring stations.

Growth Study

Pseudo-nitzscliia psciidiidcliciilissinui. clone NWFSC-()47, iso-
lated from a plankton sample collected at Kalaloch Beach, Wash-
ington on July 31, 1999, was grown in batch culture. The isolate
used in the growth study was not axenic, but sterile technique was
used throughout the experiment. Cells were maintained in f/2 me-
iliurn (Ciulllard and Rylher 1962) made with filtered natural sea-
water. An inoculum of exponentially growing cells was used. Du-
plicate cultures (200 mL each) were grown in l-L borosilicate
culture flasks al 13 °C. Irradiance was provided by a bank of
Cool-White fluorescent lamps (15 W). Cells were exposed to a
12: 12 h light:dark cycle al a light intensity of 80 (xE nr" s~'. Every
3—4 days during the study, 2()-mL aliquots from each flask were
filtered onto Millipore HA, 0.45-p.m, 25-mm diameter filters using
\acuum (< 15 psi). Filters were stored and analyzed for DA by
receptor binding assay as described above, and a 0. l-niL aliquot of
culture was collected for cell enumeration.

DoMoic Acid in Razor Clams

1009

Environmental Data

Wind speed and direction were obtained from the National
Data Buoy Center's weather station on Destruction Island. Wash-
ington (NDBC #DESW1. 47.68 '^N. 124.49 ^W). which is about 7
km nonhwest of Kalaloch Beach. Rainfall data were obtained from
the Quillayute airport, near La Push, Washington and from the
Kalaloch Ranger Station in Kalaloch. Washington. Water tempera-
ture at beach sampling sites was measured with a thermometer. Sea
surface temperatures were obtained from the Cape Elizabeth buoy
(NDBC #46041) at 47.4 °N. 124.5 °W. The locations of coastal
monitoring stations are shown in Figure 1.

Domoic Acid Analysis in Shellfish

Razor clams were collected at Kalaloch Beach for DA analysis.
Concentrations of DA were determined utilizing a methanol/water
extraction and analysis by high-performance liquid chromatogra-
phy (HPLC) (Hatfield et al. 1994). Additional razor clam to,\in
concentration data were obtained from samples collected by
Quileute Natural Resources staff and analyzed by the Washington
Department of Health (WDOH). Only the edible parts of the clams
were analyzed for DA (i.e., viscera were not analyzed).

RESULTS

Pseudo-nitzschia Cell Numbers and Domoic Acid Levels

As Pseudo-nilzsthia spp. cell counts increased, there was a
corresponding increase in the level of DA activity detected in
seawater at Kalaloch Beach (Fig. 2a). Both cell counts and DA
activity in seawater reached a maximum on September 22 at 17.1
X 10^ total Pseudo-nitzschia spp. cells/L and 2.700 ng DA/L sea-
water. A smaller peak of DA activity on August 25 also corre-
sponded with elevated Pseiido-nitzschiu spp. cell counts. The high-

est recorded level of DA detected in razor clams (295 |JLg/g) oc-
curred within 18 days after the maximum Psciido-niizMhiu spp.
cell number and level of seawater toxicity were measured.

At Second Beach (Fig. 2b). the highest number of Pseudo-
nitzschia spp. cells was about three times lower than that at Kala-
loch Beach, reaching a maximum of 5.9 x 10'' cells/L on Septem-
ber 2.^; whereas, the level of DA activity detected in seawater
reached a maximum of 350 ng/L on October 6. There were also
smaller peaks in Pseudo-nitzschia spp. cell counts on August 12
and 26 associated with increased DA activity in seawater. An
increase in Pseudo-nitzschia spp. cells on September 9 did not
correspond to elevated DA in seawater; however, toxin levels in-
creased by September 13, corresponding to levels of 2.2 x lO'' total
Pseudo-nitzschia cells/L. Razor clams were not collected from
Second Beach for measurement of DA.

Phytoplankton Assemblage Observations

Pseudo-nitzschia pseudodelicatissima (Fig. 3) was the only
known DA-producer present in significant numbers within the
phytoplankton assemblage (Table 1). Most of the other Pseudo-
nitzschia were P. cf. heiniii (up to 907f of the total Pseudo-
nitzschia on August 25 at Kalaloch Beach) plus relatively small
amounts of P. pungens (less than 3% on dates when SEM was
done) and P. delicatissima (8% on September 1 at Kalaloch Beach,
the only sample in which this species was observed).

Table 1 shows Pseudo-nitzschia spp. as a percentage of the
whole phytoplankton assemblage and P. pseudodelicatissima as a
percentage of total Pseudo-nitzschia spp. at Kalaloch and Second
Beaches. At both sampling sites, Pseudo-nitzschia spp.. as a per-
cent of total phytoplankton species, increased until the end of

Kalaloch Beach

Pseuda-nitzichia spp- cell counts
Domoic acid in razor clams (Mg/gl
Domoic acid in seawaicr 1 10 ng/Li

Gi. 10

I 6

I "^

I 2
a.
0

18 1
§16-

B Second Beach

— * — Pietido-mtzjchia spp.ccll counis

1"

S 14-

A

+ 30
- 25

5 12

a-s™-«i

/\

1:10-

\

/ \

I 8^

H

-.-*-i\-f-

Jul Aug Sep Otl Nin

Figure, 2, Weekly Pseudo-nitzschia spp, cell counts and domoic acid in
seawater from July through October 1998. at Kalaloch Beach (A) and
Second Beach (B). Domoic acid levels in razor clams are shown for
Kalaloch Beach.

^A.. ■-

Figure, 3, Scanning electron micrographs of a field isolate of P.
pseudodelicatissima from Kalaloch Beach, (top) a whole valve, (bot-
tom); higher magnification showing 1 row of square poroids between
interstriae. Scale bars indicate size.

1010

Adams et al.

TABLE 1.
Pseudo-nitzschia spp. abundance and domoic acid levels on the central Washington coast in 1998.

Kalaloch

Pseudo-nitzcliia spp.

Pseudo-nitzchia spp.

Domoic Acid

Domoic Acid

Domoic Acid

P. pseudodelicatissima

Date

(lO'-cells/L)

abundance'

(ng/L)

(fg/cell)^

by LC-MS/MS

abundance'^

7 July

1.4

37

0

13 July

0.1

30

0

26 July

0.5

34

0

-

4 Aug

1.7

48

0

11 Aug

3.2

30

0

19 Aug

4.1

59

0

40

25 Aug

3.0

64

410

1 Sept

0.6

41

0

70

8 Sept

3.8

83

10

90

14 Sept

2.0

93

330

18 Sept

10.6

97

1700

170

+

90

22 Sept

17.1

99

2700

180

+

90

29 Sept

7.8

99

1600

210

+

100

5 Oct

1.8

92

640

500

+

70

9 Oct

0.1

41

10

20

15 Oct

0.0

15

0

21 Oct

0.0

16

0

29 Oct

0.0

4

no data

3 Nov

0.0

3

0

Second Beach

Pseudo-nitzchia spp.

Pseudo-nitzchia spp.

Domoic Acid

Domoic Acid

Domoic Acid

P. pseudodelicatissima

Date

(10''cells/L)

abundance'

(ng/I.l

(fg/cell)-

by LC-MS/MS

abundance'^

7 July

0.0

18

0

13 July

0.1

38

0

27 July

0.3

54

0

5 Aug

0.5

56

0

60

12 Aug

1.4

78

10

60

20 Aug

0.4

71

0

26 Aug

1.2

88

170

160

90

31 Aug

0.3

84

30

60

9 Sept

3.9

95

0

13 Sept

2.2

97

140

109

90

17 Sept

1.7

99

30

23 Sept

5.9

99

160

40

70

30 Sept

3.9

100

250

6 Oct

1.6

98

360

230

+

100

15 Oct

0.0

50

0

22 Oct

0.0

3

no data

26 Oct

0.0

26

no data

4 Nov

0.0

6

no data

' as a percentage of the phyloplankton assemblage viewable at lOOX; 'domoic acid per cell was only calculated where DA was > 100 ng/L. and P.
pseuJodeliculissima abundance was >70'7r; 'estimated values as a percentage of total Pseudo-nitzschia spp.

September and early October, at which time the percentage de-
clined. The increase was due to a bloom of P. pseudodelicatissima.
which comprised the majority of all Pseudo-nitzschia obsei'^'ed.
Pseudo-nitzschia pseudodelicatissima reached maximum num-
bers on September 22-23. corresponding to measurable levels
of DA at both sites and the highest measured DA levels al Kala-
loch Beach. After October 6, numbers of Pseudo-nitzschia
spp. declined dramatically, as did toxin levels at both sites. The
LC-MS/MS results confirm that DA was present on September 18.
22. 29. and October .S at Kalaloch Beach and on October 6 at
Second Beach.

Nutrients and Chlorophyll a

Figure 4 shows the concentrations of phosphate, silicate, and
nitrate relative to Pseudo-nitzschia spp. cell counts at Kalaloch
Beach and Second Beach {Pseudo-nitzschia spp. cell counts are
duplicated froin Fig. 2). On July 26. there was an increase in the
levels of all three nutrients at Kalaloch Beach (Fig. 4a). Phosphate
and silicate increased by a factor of approximately 4 compared to
the previous sampling date, and nitrate increased by a factor of
about 40. Approximately 3 weeks after the influx of nutrients,
there was a subsequent rise in the number of Pseudo-nitzschia spp.

DoMoic Acid in Razor Clams

1011

Kalaloch Beach

B

Second Beach

90

— • — Phosphaic

.

80

— ■ — Silicaic

70 -

— • — Nitrate

60

— *— ■Pseudo-niizsvhiii spp cell counts

50

40

30

»^ , , /^ ~*~*-«~-_4^

ir-a

20
10

n

•-

, I -n-r , I ~r ■ >:< 1 ■ ■ , ■

^

^

-u

Aug

Sep

c

45

1

-^Kalaloch Beach

40

\

-•-Second Beach

.15

\

30

V-^

A /V

20

\ ,

/ v^ /\

15

\ X

A / v,,-*-..

10

5
n

^^o^

.^OLJC^

Jul Aug Sep Oct Nov

Figure. 4. Weekly concentrations of nitrate, silicate, phosphate from
July through October 1998, at Kalaloch Beach (A) and Second Beach
(B). Pseudo-nitzschia spp. cell counts are duplicated from Figure 2 for
reference. Weekly chlorophyll a concentrations at Kalaloch Beach and
Second Beach are shown (C).

cells that reached a maximum of 4 x 10'' cells/L in August. There
was another increase in nutrient levels on September 1; phosphate
and silicate increased by a factor of 5 from the previous sampling
date, and nitrate increased by a factor of 175. Until about Septem-
ber 14. nutrient levels remained relatively constant at Kalaloch
Beach, with phosphate ranging from 1-2 |a.M, silicate 37^6 |jlM.

and nitrate 22-24 (jiM. with the highest levels occurring on Sep-
tember 1. About 2-.'^ weeks following this second increase in
nutrients, Pseudo-nitzschia spp. cell numbers increased from 0.5 x
10" cells/L on September I to 2.0 x 10" cells/L on September 13.
Nutrient levels decreased substantially on September 18. corre-
sponding to an increase in the number of Pseudo-nilzschia spp. to
10.6 X 10" cells/L. Pseudo-iiii:sihia spp. cell numbers reached
their maximum of over 17 x 10" cells/L several days later on
September 22, while nutrients continued to decline. After reaching
this peak, Pseudo-nitzschia spp. cell counts rapidly decreased and
remained low throughout the remainder of 1998. Chlorophyll a
levels at Kalaloch Beach were routinely higher than those at Sec-
ond Beach, except for the August 5 sampling date, when they were
similar (Fig. 4c).

Second Beach showed more sustained high levels of nutrients
compared to Kalaloch Beach (Fig. 4b). At Second Beach from July
7 to September 30. phosphate ranged from 1-2 |jlM, silicate from
26-36 ^lM, and nitrate from 12-22 \xM. The concentrations of
these nutrients peaked on August 31. After nutrient concentrations
reached their maximum, there were two distinct peaks in Pseudo-
nitzschia spp. cell numbers (4 x 10" cells/L on 20 August and 6 x
10" cells/L on September 23). which coincided with slightly de-
creased nutrient concentrations. On September 30, the number of
Pseudo-nitzschia spp. cells began to decrease and on October 6 the
levels of nutrients also began to decline. Chlorophyll a levels at
Second Beach were less than 10 p-g/L on all sampling dates
(Fig. 4c).

Meteorological and Oceanographic Conditions

Monthly precipitation and sea surface temperatures (SST) are
shown in Table 2 (see Fig. 1 and Methods for the locations of these
monitoring sites). Average values for June through October for the
period of record are compared to data for those months in 1998.
Water temperatures for 1998 were similar to levels documented
during the period of record (1987-1993). However, levels of pre-
cipitation for the summer of 1998 were lower than the average for
the period of record (1948-1997). with September 1998 values
almost an order of magnitude less than the average. There were
also comparatively low amounts of rainfall at Kalaloch Beach
during August and September of 1998 (B. Rhode pers. comm.).

Wind vectors are shown in Figure 5a. Data from the end of July
through September showed periods of upwelling favorable winds
(vectors pointing toward the southeast), with the most sustained
period of strong upwelling in late August through early September.
Periods of wind relaxation and mild reversal were common

TABLE 2.
Monthly mean sea surface temperature and precipitation on the Washington coast.

Period of Record

June

July

August

September

October

Precipitation (cm) 1948-1998

Sea surface temperature (°C) 1987-1993

8.0
12.9

6.6

13.,S

.5.7
L3.8

12.2
13.2

26.4
12.3

1998

June

July

August

September

October

Precipitation (cm)

Sea surface temperature (°C)

3.2
12.6

5.3
13.8

0.4
14.0

1.3
124

20.8
12.6

1012

Adams et al.

•a
a

Jul 1

Aug 1

Sep 1

Octl

Nov I

10 (xm, 18-21 fibulae in 10 jjim. and 5-6 poroids per 1 ixm. Except
for the apical axes, which were approximately half of the reported
values, these measurements are similar to those describing P.
pseudodelicatissima in Hasle et al. (1996). Figure 6 shows average
growth rate and DA production for duplicate cultures of P.
pseudodelicatissima. DA activity increased steadily through expo-
nential growth pha.se. and as the cells reached the late stationary
growth phase, DA production dramatically increased. This experi-
ment illustrates that an isolate of P. pseudodelicatissima from
Washington coastal waters does produce DA as measured by a
receptor binding assay, and toxin production increases as the cells
reach stationary phase.

DISCUSSION

Domoic Acid Observations on the U.S. West Coast in 1998

B

a
E

18 — I

16 —

14 —

12

10 —

- Kalaloch Beach

- Second Beach

Jul 1

Aug 1

Sep 1

Oct 1

Nov 1

Figure. 5. A. Vector time series of daily average winds from June
through October 1998. Length of lines refer to speed, angle of vector
refers to direction. B. Weelily sea surface temperature at Kalaloch
Beach and Second Beach from July through October 1998. Solid ar-
rows indicate the date when the maximum numbers of Pseudo-
nitzschia spp. cells were seen al both beaches (September 22-23). Wind
reversal immediately before arrow is on September 16-19.

through the summer months. Upwelling favorable winds were sus-
tained from late August through early September, and then began
to weaken in strength toward the end of September. In the begin-
ning of October, a strong transition to northward (non-upwelling)
winds was observed.

A decrease in water temperature was recorded at both beaches
in mid-August, followed 1 week later by a warming event, which
corresponded to the first observation of toxic Pseudo-nitzschia
spp. (Fig. 3b). Water temperature decreased by approximately
6.0 "C at Kalaloch Beach and 4. .3 "C at Second Beach from late
August to mid-September, coincident with strong northwesterly
winds. Water temperature increased at both sites during the last 2
weeks in September and into early to mid-October. At both
beaches, the maximum numbers of Pscudo-nitzschiu spp. cells
were seen during this temperature increase (solid arrow).

Pseudii-nitzMhia pseudodelicatissima (irnwih Study

Pseudo-nitzschia pseudodelicatissima in culture had apical
axes of 38-60 (xm, transapical axes of 2-2.5 |j.m. .V^-40 striae in

The same species of Pseudo-nitzschia was not responsible for
all toxic events along the U.S. west coast during 1998. DA was
first detected in sardines and anchovies in California coastal waters
in May 1998. then subsequently measured in razor clams on the
Oregon coast beginning in late July and in Washington State razor
clams later that summer. Pseudo-nitzschia austialis was respon-
sible for the death of over 50 sea lions in May and June 1998 along
the central California coast (Gulland et al. 1999, Lefebvre et al.
1999. Scholin et al. 2000). Levels of DA in razor clams in Oregon,
above the 20 (Jtg/g regulatory limit, coincided with the presence of
P. australis in coastal waters beginning in late July (D. Cannon
pers. comm.. Trainer et al. in press.). In contrast, we report here
that the toxic event on the Washington coast that resulted in record
levels of DA in razor clams in October 1998 was attributable to a
bloom of P. pseudodelicatissima.

The association of different Pseudo-nitzschia species with dis-
tinct toxification events indicates that the hydrographic factors
influencing toxic bloom occurrence and toxic cell transport may be
unique to a given U.S. West Coast region. Given the southward
surface flow of the California current system and maximum Co-
lumbia River discharge as a low salinity, low density, offshore
plume lying to the southwest during summer months, it is unlikely
that a northward transport of phytoplankton cells is a primary
means by which toxic cells are spread northward along the coast
from California to Washington. We hypothesize that onshore ad-
vection of localized offshore populations of Pseudo-nitzschia spp.
cells that are present in U.S. West Coast waters during summer
months (Horner et al. in press) is the likely means of toxic bloom
initiation in Washington State waters.

Time Id)

Figure. 6. (ell counts and domoic acid levels in a cultured i.solate of P.
pseudodelicatissima. llic error bars indicate the range of values from
duplicate measurements at each lime point.

DoMoic Acid in Razor Clams

1013

Environmental Conditions

What were the environmental conditions that allowed a toxic
bloom to occur? Observations of toxic blooms in eastern Canada,
central California, and Puget Sound, Washington, have indicated
that rainfall may pro\ ide a significant source of nutrients that are
important in bloom initiation. High precipitation following an un-
usually dry summer was suggested as a causative factor in 1987
DA incident in eastern Canada (Bird and Wright 1989. Smith et al.
1990). with runoff supplying the nitrogen source. Similar condi-
tions were documented in the 1991 DA episode off the Washing-
ton coast, which occurred after a hot. dry period followed by rain
(Horner and Postel 199.^). Lack of runoff in central California
during spring through autumn is the norm (Buck et al. 1992);
however, record levels of rainfall were experienced in central Cali-
fornia during the early months of 1998 before the outbreak of P.
australis (Trainer et al. 2000, Scholin et al. 2000). Rainfall and
corresponding river runoff, followed by calm weather, were im-
portant factors in the development of a Pseudo-nitzschia spp.
bloom in Penn Cove. Puget Sound in the summer of 1997 (Trainer
et al. 1998).

Rainfall, however, is not the only source of nutrients to coastal
environments. The small upwelling events that punctuate the oce-
anic autumn/winter transition in the northeastern Pacific (Bolin
and Abbott 1963) may substitute for freshwater runoff as a nitro-
gen source. The toxic P. australis blooms observed off the Cali-
fornia coast in 1998 were positioned in coastal upwelling zones
(Trainer et al. 2000). The present study shows that nutrient inputs
from upwelling in the absence of significant rainfall fueled the
Pseudo-nitzschia spp. bloom off the central Washington coast in
late September 1998. These case histories make it clear that a pulse
of nutrients, whether from rainfall and subsequent river runoff or
from coastal upwelling events, is required to fuel toxic Pseudo-
nitzschia spp. blooms.

The transition to wind relaxation and reversal events in mid- to
late September and a strong reversal in early October, which
marked the beginning of the autumn transition, resulted in the
Ekman transport of surface water toward the coast beginning in
mid-September. When upwelling winds reverse direction or relax,
surface waters can be transported rapidly into the nearshore region
where algal cells can mix vertically to the bottom (Donaghay and
Osborn 1997). The beginning of the autumn transition in 1998
corresponded with a dramatic increase in water temperature at
Kalaloch Beach ( -i- 4 °C) and at Second Beach ( -I- 2 °C; Fig. 5b).
The subsequent relaxation of upwelling winds (Fig. 5a) corre-
sponded to an increase in the number of cells observed at both
beach sites, reaching a maximum on September 22-23 (Fig. 4a,b).

Difference between Kalaloch Beach and Second Beach

Although species composition and cellular levels of DA were
similar at both beaches during the course of this study, the maxi-
mum number of Pseudo-nitzschia spp. cells, specifically P.
pseudodelicatissima, measured at Kalaloch Beach was at least
three times higher than the levels measured at Second Beach. Our
results suggest that a pulse of nutrients, especially nitrate, rather
than a sustained nutrient supply could account for the difference in
the intensity of the bloom and, thus, the total levels of DA in the
seawater. Pulses of nitrate, possibly from resuspended sediments
after wind events or from river runoff have been previously asso-
ciated with blooms of P. muhiseries in eastern Canada (Smith et al.

1990). The highest concentration of nitrate at Kalaloch Beach was
measured on September I. a significant increase (175-fold) over
levels recorded on August 25. Although nitrate levels increased
slightly on September I at Second Beach, the change in concen-
tration of this nutrient was not significant through September.
Chlorophyll a levels were also routinely higher at Kalaloch Beach
than at Second Beach (Fig. 4c). This clearly indicates that hydro-
graphic factors at Kalaloch Beach were more supportive of phy-
toplankton blooms in the summer and early autumn months. The
physical oceanography that explains the different productivity at
Kalaloch Beach compared with Second Beach will be detailed in
future studies.

Toxigenic Pseudo-nitzschia pseudodelicatissima

Our measurement of increasing DA levels in a cultured isolate
of P. pseudodelicatissima, and confirmation of DA by mass spec-
troscopy of field samples consisting of 90-100% P. pseudodeli-
catissima. show that this species is a DA producer in Washington
coastal waters. Significant numbers of other known toxigenic
Pseudo-nitzschia species were not found in field samples collected
before and during the razor clam toxification event in 1998. The
1998 coastal event was not the only time that P. pseudodelicatis-
sima has been linked to toxin production in Washington waters.
This organism was the primary Pseudo-nitzschia species seen in
offshore areas of DA production during cruises aboard the RA^
McArthur in the summers of 1997 and 1998 (Homer et al. in
press). However. P. pseudodelicatissima does not produce toxin in
all areas of the world. For example. DA has not been measured in
seawater at times when P. pseudodelicatissima formed dense
blooms in Monterey Bay (Walz et al. 1994, Scholin et al. 2000) but
it is known to produce toxin in the Bay of Fundy (Martin et al.
1990). in the Gulf of Mexico (M. Parsons pers. comm.). and in
certain clones isolated from Danish waters (Lundholm et al. 1997).
Genetic variability or differences in gene expression in various
strains of P. pseudodelicatissima may explain the variation in toxin
production by this organism in different regions.

Indeed. P. pseudodelicatissima did not produce measurable
amounts of toxin at all times in both cultured and field samples.
For example, it was observed at Kalaloch Beach on August 19
(Table I ) when no toxin was measured in seawater. Laboratory
cultures showed that this alga produces maximum amounts of
toxin in late exponential and stationary phases of growth. The
highest cellular levels of DA in field samples were measured in
early October as the bloom was declining. Therefore, to predict
accurately the levels of toxin that will be produced by a bloom,
some knowledge of the growth stage of the bloom population must
be obtained. In the field, maximum numbers of P. pseudodelicatis-
sima were observed at Kalaloch Beach about 2-3 weeks after a
pulse of nutrients was recorded. Similarly, the cultured isolate
reached stationary pha.se of growth in approximately 12 days. Per-
haps similar growth rates and dependence of toxin production on
growth stage can be expected in field samples compared to labo-
ratory cultures, a possibility that needs to be determined empiri-
cally in future studies. This information will assist in the complete
characterization of natural blooms of toxic Pseudo-nitzschia.

Pseudo-nitzschia spp. cells were present in nearshore waters
through most of the summer at both beaches, indicating that they
may be able to bloom during much of the year, given the appro-
priate environmental conditions. During most of the summer of
1998. nontoxic or weakly toxic species such as P. cf heimii and P.

1014

Adams et al.

pungens, respectively, were present at times when DA was not
detectable in the seawater. When a rich supply of nutrients became
available, toxic P. pseudodelicatissima cells increased in number
and reached bloom proportions (over 10'' cells/L) over a 2-3 week
period. It is not clear which specific environmental conditions
caused P. pseudodelicatissima to become dominant and not any of
the other species of Pseudo-nitzschia that were present in the as-
semblage during the summer months.

Prediction

The influx of nutrients in early September 1998 was a result of
one of the strongest coastal upwelling events on the Washington
coast that season as evidenced by persistent northwest winds (Fig
4a). Although toxic blooms in Monterey Bay occur in the spring
and autumn, the Washington coast has recorded DA events pri-
marily in autumn months. This observation makes toxic blooms in
Washington potentially more predictable and perhaps more
strongly linked to nutrient pulses during coastal upwelling events,
especially in years of low rainfall. If nutrient concentrations and
environmental conditions are to be used as predictive factors, more
in-depth studies of these parameters as they relate to the compo-
sition of the phytoplankton assemblage must be undertaken.

Close observation of the developing Pseudo-nitzsclua popula-
tion during late summer and early autumn months and daily moni-
toring of environmental factors such as upwelling indices could
provide an early warning of toxic blooms that affect coastal razor
clam populations. Molecular probes able to detect toxic Pseudo-
nitzscliia species found seasonally in Washington coastal regions,
possibly in the form of automated sensors placed on buoys, could
warn recreational and subsistence shellfishers of an impending
bloom event (Scholin et al. 1999). Although we know that razor
clams can become toxic within 18 days of the appearance of a
toxic bloom, the precise timing between the appearance of high
levels of DA in seawater, and the accumulation of toxin in clams

must still be determined in order to fine tune our predictive capa-
bilities.

CONCLUSIONS

The late summer/early autumn outbreak of toxic Pseudo-
nilzscliia was strongly linked to the relaxation of seasonal up-
welling in a year of unusually low rainfall. The bloom occurred at
a time when winds reversed direction, allowing cells to be brought
to the coast. This scenario presumes an established, offshore popu-
lation of P. pseudodelicatissima, of which recent research cruises
provide evidence (Homer et al. in press. Trainer et al. in press). We
conclude that the pennate diatom P. pseudodelicatissima was the
major source of DA on the central Washington coast in the late
summer and autumn of 1998. with record levels of toxin in razor
clams detected in October of that year. Our study illustrates that
toxic Pseudo-nitzschia can, indeed, contribute to the surf-zone
diatom community on which razor clams feed. Natural depuration
of razor clams was still not complete as of October 1 999. This slow
depuration of toxin from razor clams has made a further impact on
the already depressed coastal economies of Washington State by
necessitating a second season of beach closures likely resulting
from one toxic bloom.

ACKNOWLEDGMENTS

We thank Carla Stehr for performing SEM, Keri Baugh for
receptor binding assays, Mark Busman and Peter Moeller for LC-
MS/MS, and Mick Spillane for assistance with the wind vector
plot. We acknowledge Brian Bill and Ryan Colyer for cell counts
and isolation and culturing of P. pseudodelicatissima. Fran Van
Dolah and David Hampson kindly provided the cloned glutamate
receptor. We gratefully acknowledge June Schumack for sample
collection and processing and Bich Thuy Le-Eberhart for chloro-
phyll a analyses, data processing, and organization. Our thanks go
to John Wekell for helpful discussions.

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Journal <•/ Slwllllsli Rcsccinh. Vol. 19. No. 2. 1017-1020, 2000.

A CASE OF CONSISTENT SPATIAL DIFFERENCES IN CONTENT OF DIARRHETIC
SHELLFISH TOXINS (DST) AMONG THREE BIVALVE SPECIES: MYTILUS EDULIS, OSTREA

EDULIS, AND CERASTODERMA EDULE

SUSANNE SVENSSON,'* CARL ANDRE,^'^

ANN-SOFI REHNSTAM-HOLM/ AND JONAS HANSSON'

Department of Zoophysiology

Goteborg University

Box 463

S-405 30 Goteborg. Sweden
'Department of Zoology

Stockholm Universitv

S-J06 91 Stockholm. Sweden
' Tjdrno Marine Biological Laboratory

S-452 96 Stromstad, Sweden
''clinical Bacteriology

Goteborg University

Giddhedsgatan 10

S-413 46 Goteborg. Sweden

ABSTRACT Content of diarrhetic .shellfi.sh toxins (DST) was compared among mussels (Mytilus edulis), oysters (.Ostrea edulis). and
cockles {Cercuioderma edule) at two spatial scales: regions (100 km apart) and locations within regions (5 km apart). Samples were
analysed for DST using protein phosphatase inhibiton assay in individual digestive glands. Concentrations of DST in all oysters and
cockles were below the detection limit in the assay, whereas mussels from both regions and all locations contained mean levels of DST
above the regulation limit for harvest and marketing. Thus interspecific differences in content of DST were found along the Swedish
west coast. Some behavioral and physiological phenomena are proposed to explain the differences among species. These include
differential uptake and processing of toxic algae, biotransformation of toxins, and reduced filtration at low temperatures. These findings
may have some implications for harvest and cultivation of bivalves and suggest a possibility that cockles and oysters could be marketed
for human consumption during periods of elevated levels of DST in mussels.

KEY WORDS: CerasUnierma edule. cockles, diarrhetic shellfish toxins. DST, interspecific differences
okadaic acid. OA, Ostrea edulis. oysters, protein phosphatase inhibition assay

Mylilus edulis. mussels.

INTRODUCTION

Diarrhetic shellfish poisoning (DSP) is one of several illnesses
caused by the consumption of shellfish containing toxic substances
produced by marine microalgae. The most common toxins asso-
ciated with DSP belong to the okadaic acid (OA) group (Yasumoto
et al. IQS.'i). DSP was first described in Japan during the late
1970s, but has since been reported from temperate waters around
all continents where it causes considerable problems for harvesters
and consumers of shellfish.

In Sweden, monitoring for DSP toxins (DST) in blue mussels,
Mytilus edulis. has been ongoing since 1988. This has revealed
large seasonal, geographical, and individual vaiiations in content
of DST in mussels along the West Coast (Lindegarth 1997). For
example, mussels generally contain DST above the quarantine
levels ( 160 (xg OA kg"' mussel meat, EC regulations) for harvest
during the autuinn and winter period, sometimes for up to 6 mo
each year.

DST is the most serious threat to a sustainable industry based
on cultured blue mussels in Sweden and there is a need for mana-
gerial actions to reduce its impact. Aquacullure and fisheries for a
more diverse range of bivalve species, which may vary in content
of toxins, could be an alternative during periods of high levels of

*Corresponding author. E-mail: susanne.svensson@zool.gu.se

DST in mus.sels. As an example, the knowledge about interspecific
differences in levels of paralytic shellfish toxins (PST) are used by
authorities in Maine, who have been practising species-specific
harvest and closure of bivalve fisheries for many years (Dr. S.
Shumway pers. comm.). In Sweden, small-scale fisheries for the
European oyster, Ostrea edulis. and cockles, Cerastoderma edule
and Ceratoderma lamarcki, are in operation today. These species
are currently not included in DST monitoring and information
about the presence of DST in oysters and cockles is sparse.

As mentioned earlier, differential patterns of accumulation and
depuration of PST among species of bivalves have been observed
(Bricelj and Shumway 1998). In general, Mytilus sp. rapidly ac-
cumulate and detoxify PST compared to most other species. Some
observations on differences in content of DST among species of
bivalves collected in the field have also been made (MacKenzie et
al. 1998, Poletti et al. 1998). Mytilus spp. were reported to contain
higher levels of DST compared to the other species included in
these studies. However, these studies were not specifically per-
formed to investigate differences among species concerning their
ability to accumulate DST. For that purpose shellfish should be
collected at the same time and habitat to reduce effects of differ-
ences in exposure to toxic algae prior to sampling.

During the summer of 1998, high levels of OA (>1 mg OA kg"'
mussel meat) were detected in blue mussels from a farm located in
north Bohusliin on the Swedish west coast. At the same time, we

1017

1018

SVENSSON ET AL.

observed that oysters and cockles, collected from the vicinity of
the farm, contained non-detectable levels of DST measured by
HPLC. To test whether the observed differences in DST among
these species were real and consistent in space, we sampled at two
different spatial scales. This paper reports the results from this
study.

MATERIALS AND METHODS

Naturally occurring cockles (Cerastodenna edule), oysters (Os-
trea edulis). and mussels (Mytiliis ediilis) were collected during 1
wk in late November to early December 1998 from two regions
separated by approximately 100 km along the coast of Bohuslan
County, Sweden (A; Ljungskile and B: Tjarno, Fig. 1). In these
regions mussels are commercially farmed and fishing for both
cockles and oysters occurs. Sampling was performed when levels
of OA in farmed mussels had been reported to be high for more
than 2 mo in both areas (OA data from the weekly monitoring
program). Within each region sampling was done at each of two
locations, separated by approximately 5 km (Buvenas and Sparre-
viken in region A. Tenskiir and Kockholmen in region B. Fig. I).
These locations were selected because all three species were found
to co-exist on a small scale. Six individuals of each species were
taken from each location at depths between 0.5 and 3 m within a

0 lOkm

Tenskar '^ ^V B
Kockholmen ' .'/

Buvenas -j"/ _^
SparTeviken^7'~

Figure 1. Map of the coastal area or Bohuslan County showing the
sampling locations ol'Af edulis, O. edulis, and C. edule. (Al Ljungskile
region. (U) Tjarno region.

radius of 20 m. Sampling depths and size characteristics of the
samples from each location are shown in Table 1 . Water tempera-
tures were between 3 °C and 5 °C at the time of collection. The
specimens were frozen and stored at -20 °C until toxin analysis
was performed.

Preparations of shellfish extracts were done on individual di-
gestive glands using the sample clean-up protocol according to Lee
et al. (1987). Concentrations of DST in the resulting chloroform
extracts were then analyzed using the fluorescent microplate phos-
phatase inhibition assay (PIA) according to Vieytes et al. (1997)
with some modifications which will be published elsewhere
(Rehnstam-Holm et al. in prep.). Toxin content was expressed as
micrograms of OA equivalents g'' of digestive gland and micro-
grams of OA equivalents kg~' mussel meat. The detection limit for
shellfish extracts was 5 ng OA equivalents g"' digestive gland
when dilution factors were considered.

To confirm the accuracy of the PIA method, 4 individuals of
each species were chosen at random and analyzed for OA and
DTX-I by HPLC according to Lee et al. (1987) using 1-pyrenyl-
diazomethan (PDAM) instead of ADAM.

RESULTS

Results from the PIA analyses are shown in Table 1. All the
cockle and oyster samples contained levels of DST below the
detection limit for the PIA, whereas toxins were detected in all
individual mussels. Mean concentration in mussels varied between
1 ..5 to 2.6 |j,g OA equivalent g"' digestive gland or 209 to 241 |jLg
OA equivalent kg~' mussel meat which is above the regulation
limit for marketing of mussels.

When analysed by HPLC, neither OA nor DTX-1 were de-
tected in the oyster and cockle extracts, whereas OA was detected
in all mussel samples. Low amounts of DTX-1 compared to OA
010%) were found in two of the mussel samples. OA equivalent
measured by the PIA method correlated well to concentrations of
OA in the HPLC method (data not shown). Thus HPLC confirmed
the results from the PIA that OA and DTX-1 were absent (non-
detectable) in the oyster and cockle samples.

This study was designed to test hypotheses about interspecific
and spatial variability in DST using multifactorial ANOVA. Since
no toxins were detected in C. cdiilc and O. edulis. tests with normal
parametric procedures could not be justified in order to test hy-
potheses about interspecific differences in mean content of DST.
Nevertheless, confidence intervals did not include the value for
detection limit for the PIA which indicated that the sample si/e (;;
= 6) was sufficiently large to allow unambiguous conclusions
about interspecific differences.

DISCUSSION

The purpose of this study was to investigate interspecific and
spatial variability of DST in 3 bivalve species that co-exist in
Swedish waters. To our knowledge this is the first study where
levels of DST have been compared among bivalve species that
were sampled from the same locations under similar conditions in
the field. The consistent differences in levels of DST found be-
tween blue mussels on one hand and cockles and oysters on the
other hand provided evidence for interspecific differences that are
not caused by differences in exposure to toxic algae. Thus the
pattern that was found indicated differences in either the ability lo
acciMiiulate or depurate DST among the species studied.

Spatial Difference in Content of Diarrhetic Shellfish Toxins

ioiy

TABLE 1.

Sampling depths, size characteristics, and content of OA, analyzed by PIA in C. edule, M. editlis, and O. edulis from the 4 study locations. %

d.g.: proportion digestive gland ('^d of total tissue wet weight. Values for size characteristics are means ± SD (n = 6). Concentration of OA

is expressed as (ig OA equiv. g"' digestive gland and fjg OA equiv. kg"' mussel meat, n.d.: no toxins detected (detection limit 5 ng OA equiv.

g~' digestive gland). Values for OA are means ± 95% confidence intervals (ii = 6).

Shell

Tissue wet

OA equiv.

OA equiv.

Species

Region

Location

Depth (m)

length (mm)

weight (g)

% d.g.

(Mgg"')

(Mgkg-')

C. edule

Ljungskile

Buvenas

0.5-0.7

40.0 ± 5.7

10.4 + 4.3

7.6± 1.2

n.d.

Sparreviken

0.5

44.5 ± 4.6

13.0 ±2.5

8,4 ± 1.8

n.d.

Tjamo

Kockholmen

0.5-0.7

40.5 ± 2.7

9.3 ±1.8

11.5±3.2

n.d.

Tenskar

0.6-1.0

26.8 ± 9.2

3.4 ± 2.7

11.4 ±1.4

n.d.

M. edulis

Ljungskile

Buvenas

0.5-0.7

63.2 ± 7.9

17.5 ±7.0

8.6 ±1.8

2.6+ 1.4

222 ± 119

Sparreviken

3.0

73.5 + 7.2

29.9 + 9.0

9.6 + 2.0

2.4 ± 2.0

242 ± 223

Tjamo

Kockholmen

1.7-2,2

74.5 ± 14.7

29.2 ± 14.3

14.8 ± 3.2

1.4 ±0.5

209 ± 92

Tenskar

0.6-1.0

57.3+ 14.3

16.6+ 11.2

13.4 + 2.9

1.5 ±0.5

210 ±105

O. edidis

Ljungskile

Buvenas

0.5-0.7

71.3 ±13.0

10.8 ±6.1

9.2 ±2.7

n.d.

Sparreviken

3.0

80.3 ±15.5

20.0 ±10.4

9.1 ±2.2

n.d.

Tjamo

Kockholmen

1.7-22

91.5 + 8.8

19.7 + 8.5

8.2+ 1.8

n.d.

Tenskar

0.6-1.0

54.3 ± 5.9

2.7 ± 1.2

11.3 ±3.6

n.d.

To explain the interspecific differences in content of DST.
some behavioral ancj physiological phenomena can be proposeci as
discussed below.

Interspecific variability in pre- and/or post-ingestive selection
may occur in order to increase or reduce the uptake and processing
of the DST-producing algae. O. edulis from the North American
east coast has been found to selectively clear both toxic (PST) and
non-toxic dinoflagellates from mixed cell suspensions in labora-
tory experiments (Shumway and Cucci 1987). Sidari et al. (1998)
observed that during an event of DSP in Italy. M. gallopmvincialis
seemed to feed selectively on Diiiophysis sp., comparing algal
contents in the stomachs to those of the water column. Although
there are no previous observations on selection against algae con-
taining DST. a possible explanation of our results is that the
sampled populations of O. edulis and C. edule may be rejecting
these species, either pre- or post-ingestion. thus rendering the non-
toxic results in the analysis.

Recent evidence suggests that DST can be biotransformed by
bivalves (Lee et al.. 1989, Suzuki et al. 1999, Fernandez et al.
1996). A group of low-polar acyl-ester derivatives of OA, DTX-1
(referred to as DTX-3), and DTX-2 has been found in shellfish, but
never in the dinoflagellates producing DST. Therefore, it has been
suggested that the acylated forms of DST are products of meta-
bolic activity in the digestive glands of the molluscs (Lee et al.
1989). Using modifications of the HPLC method. Suzuki et al.
(1999) reported that Japanese scallops, Pactiiwpeaen yessoensis,
rapidly converted DTX- 1 to DTX-3 with significantly higher con-
tent of the latter in the tissue. In contrast, Fernandez et al. (1996)
found that although mussels, M. galloproviucialis. contained de-
tectable amounts of acylated DST, the major content in the extracts
was always OA, implying only a slow rate of biotransformation of
OA for mussels. Due to the chemical nature of the acyl derivatives,
they are not detected using traditional clean-up procedures for
HPLC. Also, the sensitivity of the PIA method to DTX-3 are low
compared to OA and DTX-1 (Mountfort et al. 1999). Thus the
absence of OA and DTX-1 in oyster and cockle extracts in our
study could be explained by a rapid acylation of these compounds
in the digestive glands, which in that case, we were unable to
detect by PIA and HPLC. In future studies it should be possible to

test this hypothesis by using modifications of the HPLC method. It
is also possible that rates of depuration of the non-acylated com-
pounds vary among species, which could be evaluated by perform-
ing depuration experiments.

The interspecific differences in content of DST could also be an
effect of water temperature since sampling was performed during
a time of the year when temperatures were low (3 °C-5 "C). This
is close to the temperature when filtration is reduced or inhibited
in oysters (Child and Laing 1998). Mussels, on the other hand, are
active even at temperatures close to 0 °C (Loo 1992). Reduced
filtration and thus uptake of toxic algae in oysters and also cockles
may therefore explain the results. Also, in case that these species
did contain toxins earlier during the season, depuration may have
occurred after filtration (uptake of toxic algae) stopped.

Although the mechanisms causing the interspecific differences
in content of DST remain unknown, the observations made in this
study could have important implications for harvest and cultivation
bivalves. Currently within the EC, marketing of shellfish from
certain areas is regulated by content of algal toxins in blue mussels,
a species which generally accumulates high levels of toxins. Our
results suggest the possibility that oysters and cockles could be
harvested and marketed for human consumption during periods
when mussels contain significant levels of toxins. However, fur-
ther sampling and analysis of DST in oysters and cockles, includ-
ing temporal replication must be performed during periods of DST
in order to confirm the results found in this study. Also, biological
tests should be conducted in parallel with the chemical analyses to
test whether any toxicity due to unknown substances or biotrans-
formed products is present in the shellfish meat.

ACKNOWLEDGMENTS

We thank Prof Lars Edebo at the Department of Clinical Bac-
terilogy, Goteborg University for providing the facilities for toxin
analysis. This research was financed by grants from the Founda-
tion for Strategic Environmental Research (MISTRA, SuCoZoMa
project Dnr 95005), by the Regional EU Developmental Fund,
Tjamo Center of Exellence. and by the Interegional EU Develop-
mental Fund, INTERREG lie.

1020

SVENSSON ET AL.

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molluscs: occurrence, transfer kinetics and biotransformation. Rev
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Fernandez, M.L., A. Mi'guez, E. Cacho & A. Martinez. 1996. Detection of
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Lee. J.S.. T. Yanagi. R. Kennia & T. Yasunioto. 1987. Fluorometric de-
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Lee. J.S., T. Igarashi. S. Fraga, E. Dahl, P. Hovgaard & T. Yasumoto.
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CONTENTS CONTINUED

bhk ,^9

JOURNAL OF SHELLFISH RESEARCH

Vol. 19, No. 2 December 2000

CONTENTS

Kathleen M. Castro and Thomas E. Angell

Prevalence and progression of shell disease in American lobster, Homanis wnerkanus. from Rhode Island waters and

the offshore canyons 69 1

Fernando Luis Medina Mantelatto and Adilson Fransozo

Brachyuran community in Ubatuba Bay. northern coast of Sao Paulo State, Brazil 701

Enrique M. Dupre

Laboratory observations on the reproductive and molt cycles of the Robinson Crusoe Jsland lobster Jusus frontalis
(Milne-Edwards, 1 836) 711

Lixin Wu, Shuanglin Dong, Fang Wang, and Xiangli Tian

Compensatory growth response following periods of starvation in Chinese shrimp, Penaeus chinensis Ofhsck 717

Teena F. Middleton, Peter R. Ferket, Harry V. Daniels, Leon C. Boyd, Larry F. Stikeleather, and Robert J. Mines
The use of poultry mortalities as an alternative bait for the harvesting of blue crabs Callinectes sapidiis
(Rathbun, 1 885) 723

Cathy M. Dichmont, Mike C. L. Dredge, and Kate Yeomans

The first large-scale fishery-independent survey of the saucer scallop, Anmsiuin japonicum halloli

in Queensland. Australia 731

Tore Strohmeier, Arne Duinker, and 0yvind Lie

Seasonal variations in chemical composition of the female gonad and storage organs in Pecten maxiinus (L.)

suggesting that somatic and reproductive growth are separated in time 741

Marcelo Aguilar and Wolfgang B. Stotz

Settlement sites of juvenile .scallops Argopecten purpuratus (Lamarck, 1819) in the subtidal zone at Puerto Aldea,

Tongoy Bay. Chile 749

David B. Rudders, William D. DuPaul, and James E. Kirkley

A comparison of size selectivity and relative efficiency of sea scallop, Placopecten nuigellaniciis (Gmelin. 1791 ),

trawls and dredges 757

Alfonso M Maeda-Martinez, Maria Teresa Sicard, and Teodoro Reynoso-Granados

A shipment method for scallop seed 765

Chang-Keun Kang, Mi Seon Park, Pil-Yong Lee, Woo-Jeung Choi, and Won-Chan Lee

Seasonal variations in condition, reproductive activity, and biochemical composition of the Pacific oyster, Crassostrea

gigas (Thunberg), in suspended culture in two coastal bays of Korea 77 1

Daniel J. McGoldrick, Dennis Hedgecock, Louise J. English, Puttharat Baoprasertkul, and Robert D. Ward

The transmission of microsatellite alleles in Australian and North American stocks of the Pacific oyster (Crassostrea

gigas): selection and null alleles 779

Zaul Garcia-Esquivel, Marco A. Gonzalez-Gomez, Dahen L. Gomez-Togo, Manuel S. Galindo-Bect, and

Martin Herndndez-Ayon

Microgeographic differences in growth, mortality, and biochemical composition of cultured Pacific oysters

(Crassostrea nifias) fron San Quintin Bay, Mexico 789

Eva Marie Rodstrom and Per R. Jonsson

Survival and feeding activity of oyster spat (Ostrea edidis L) as a function of temperature and salinity with

implications for culture policies on the Swedish west coast 799

Imad G. Saoud, David B. Rouse, Richard K. Wallace, John E. Supan, and Scott Rikard

An ill silii study on the survival and growth of Crassoslrca vir)iiiuca juveniles in Bon Secour Bay. Alabama 809

Marcela Pascual

Dwarf males in the puelche oyster (Ostrea piielchana. d'orb): differential mortality or selective settlement? 815

Andrew C. Beer and Paul C. Southgate

Collection of pearl oyster (family Pteriidae) spat at Orpheus Island. Great Barrier Reef (Australia) 821

Aswani K. Volety and William S. Fisher

In vitro killing of Perkinsiis nuiriniis by hemocytes of oysters Crassostrea virfiinica 827

CONTENTS CONTINUED ON INSIDE BACK COVER

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